V) channels are essential for the transmission of pain signals in humans making them prime targets for the development of new analgesics. Spider venoms are a rich source of peptide modulators useful to study ion channel structure and function. Here we describe β/δ-TRTX-Pre1a, a 35-residue tarantula peptide that selectively interacts with neuronal NaV channels inhibiting peak current of hNaV1.1, rNaV1.2, hNaV1.6, and hNaV1.7 while concurrently inhibiting fast inactivation of hNaV1.1 and rNaV1.3. The DII and DIV S3-S4 loops of NaV channel voltage sensors are important for the interaction of Pre1a with NaV channels but cannot account for its unique subtype selectivity. Through analysis of the binding regions we ascertained that the variability of the S1-S2 loops between NaV channels contributes substantially to the selectivity profile observed for Pre1a, particularly with regards to fast inactivation. A serine residue on the DIV S2 helix was found to be sufficient to explain Pre1a’s potent and selective inhibitory effect on the fast inactivation process of NaV1.1 and 1.3. This work highlights that interactions with both S1-S2 and S3-S4 of NaV channels may be necessary for functional modulation, and that targeting the diverse S1-S2 region within voltage-sensing domains provides an avenue to develop subtype selective tools.Voltage-gated sodium (Na Vs) are membrane proteins with four homologous domains (DI-DIV), each composed of six transmembrane segments (S1-S6) assembled into a single, functional α-subunit (~260 kDa). Each domain has two functionally distinct regions; the S1-S4 segments comprise the voltage-sensing domain (VSD) whereas the S5-S6 helices and extracellular ‘P-loop’ form the selectivity filter and ion-conducting pore. There are nine mammalian α-subunits (NaV1.1 to NaV1.9) with greater than 64% sequence identity between isoforms 1.1 to 1.7, whereas 1.8 and 1.9 share ~50–60% identity with other members2. The NaV channel family contains important therapeutic targets for local anaesthetics, anti-arrhythmics, analgesics, and anti-epileptics4. For example, the peripherally expressed neuronal NaV1.7 has been identified as a potential target for the treatment of chronic pain, largely from studies of human channelopathies5. Mutations of SCN9A that result in NaV1.7 gain-of-function underlie paroxysmal extreme pain disorder (PEPD) and primary erythromelalgia7, whereas mutations that result in the loss of NaV1.7 function underlie ‘congenital insensitivity to pain’ (CIP)8. The neuronal isoforms NaV1.3, NaV1.8 and NaV1.9 have also been implicated in various forms of chronic and acute pain12, whereas recent evidence suggests that NaV1.1 also plays a role in mechanical pain transmission13. However, the similarity between NaV channel α-subunits poses a challenge to discover and develop molecules that can selectively modify the function of each subtype.Voltage-gated sodium channels  and interact with one of four VSDs at partially defined receptor sites18.Natural toxins have been instrumental in defining Na17. Domain IV of the NaV channel α-subunit uniquely controls inactivation of the channel19, thus spider peptides that interact with and hinder the normal function of VSDIV slow or inhibit fast inactivation of the channel and may result in persistent current20. Conversely, spider venom peptides that interact with DI, DII, and DIII VSD modulate channel activation21. Most of those discovered to date cause a shift in the voltage-dependence of activation in the depolarising direction and inhibit activation23, whereas some such as β-HXTX-Mg1a (Magi 5) cause a hyperpolarising shift, thereby facilitating activation24. The VSDs of different voltage-gated ion channels exhibit higher sequence variability than the highly conserved pore region, thus offering the opportunity for subtype-selective interactions between NaV subtypes as well as the greater voltage-gated ion channel superfamily. Indeed, certain families of spider venom peptides demonstrate remarkable subtype selectivity profiles25.These VSD modulating peptides have been described to modify channel gating in three distinct ways depending on the domain they target and the effect on that domainV channels is helping efforts to design a new generation of pharmacological tools and potential therapeutic lead molecules28. To this end, we identified β/δ-TRTX-Pre1a (Pre1a) from the venom of the tarantula Psalmopoeus reduncus in a screen for NaV1.7 inhibitors. Pre1a exhibited a unique and complex pharmacological profile across neuronal NaV channel subtypes where it preferentially inhibits fast inactivation of NaV1.3, inhibits activation of NaV1.2, NaV1.6, and NaV1.7, and inhibits both activation and fast inactivation of NaV1.1, with no effect on NaV1.4 or NaV1.5 at sub-micromolar concentrations. This functional profile is dictated by classical interactions with the DII and DIV S3-S4 loops of the NaV channel VSDs. However, our evidence points to the S1-S2 loops as critical for imparting the isoform selectivity demonstrated by Pre1a. Pre1a thus represents a valuable tool to study the subtle differences in DII and DIV interaction sites between members of the NaV channel family.A deeper understanding of the molecular basis of the interactions between spider venom peptides with NaPsalmopoeus reduncus venom at 1:1000 dilution (~200 μg/ml) consistently and potently inhibited human (h) NaV1.7 expressed in Xenopus laevis oocytes. Activity-guided fractionation resulted in the identification of a major component exhibiting inhibitory activity at hNaV1.7 expressed in oocytes  NaV1.3  matched that of the theoretical M + H+ with a free acid C-terminus  that was confirmed using MSMS on tryptic digest fragments (data not shown). The peptide was named TRTX-Pre1a (Pre1a) according to the current rational nomenclature29.In a small screen of 14 spider venoms, tes Fig. . The pureak Fig.  and was 1.3 Fig. . The masdes Fig. , determiIn order to carry out functional characterisations, we produced Pre1a using Boc solid-phase peptide synthesis. The folded synthetic peptide co-eluted with the peptide isolated from the native source  for rPre1a were completed by analysis of the 3D HNCACB, CBCA(CO)NH, and HNCO spectra, and the side chain 1H and 13C chemical shifts from the 3D H(CC) (CO)NH-TOCSY and (H)CC(CO)NH-TOCSY spectra, respectively. Together we achieved 90% completion of all proton assignments. However, due to the structural heterogeneity, insufficient unique constraints were derived from the NOESY spectra to enable the structure calculations using CYANA30 to produce a adequately converged high-resolution structure. However, the chemical shifts determined for the backbone resonances allowed us to predict the secondary structure of Pre1a using the program TALOS+ 31, which suggests that residues around C3–L4 and Y21–K22 are likely to form β-strands . Additionally, a homology model of Pre1a was constructed using the structure of HwTx-IV as a template (PDB 2M4X)34 using SWISS-MODEL35. The Pre1a model shows a highly dense packing of five aromatic residues with W6 and F7 at the tip of Loop 1 falling between W29 and Y32/Y21 on one face of the peptide  and 114.0 nM (pIC50 6.94 ± 0.06), respectively . Similar to its effect on NaV1.3, 1 μM Pre1a only weakly inhibited the peak current amplitude of rNaV1.4 and hNaV1.5 by 16.47 ± 0.05% and 8.60 ± 0.05%, respectively  and the degree of persistent sodium current measured at 50 ms of depolarisation (NaV1.2 and 1.7) and 100 ms for rNaV1.3 due to the longer time required to reach complete inactivation in the oocyte expression system without β1. Consistent with results observed during assay-guided fractionation, Pre1a potently inhibited the inactivation of rNaV1.3 with an EC50 of 45.5 nM (pEC50 7.34 ± 0.27) , Pre1a also decreased the rate of current inactivation, reflected as a concentration-dependent increase in τinact  Fig. , a selecact Fig. . At 1 μM1.5 Fig. .V1.1 and NaV1.628, thus we assessed the activity of Pre1a on hNaV1.1 and hNaV1.6 stably expressed in HEK cells using the Qpatch16 automated patch-clamp platform , 221.6 nM (pIC50 6.65 ± 0.06), and 15.0 nM (pIC50 7.82 ± 0.04), respectively   and the cardiac (NaV1.5) isoforms. Using the IC50 for inhibition of NaV1.7 peak current as a reference point  results in a relative rank order potency for NaV modulation of rNaV1.3inact (0.4) > hNaV1.7act (1) > rNaV1.2act (1.7) > hNaV1.1inact (2.7) > hNaV1.1act (3.8) > hNaV1.6act (15) > rNaV1.3act (70). Thus the effect of Pre1a on NaV1.3 inactivation has the highest relative potency for all NaV activity (peak current or inactivation) on the channels tested.In addition to the effect of Pre1a on Na51) Fig. . Beyond ion Fig. . TogetheV1/2) of channel gating for rNaV1.2, rNaV1.3 and hNaV1.7 expressed in oocytes  had no effect on wild-type KV2.1 or the DI and DIII chimaeras, however it inhibited outward current carried by the NaV1.7 DII and DIV/KV2.1 chimaeras by 44.0 ± 4.6% and 27.1 ± 3.8% (n = 6), respectively  of hNaV1.1, rNaV1.7 and rNaV1.2, but not rNaV1.3, whereas it selectively inhibits the inactivation (DIV effect) of NaV1.1 and 1.3, but not 1.2 or 1.7. The striking differences in effects on NaV1.1, 1.2 and 1.3 are of particular significance for understanding the molecular basis of Pre1a’s subtype selectivity as these three channels have essentially identical S3-S4 linker regions in both DII and DIV . Thus, this region cannot account for the substantial differences in NaV channel subtype selectivity observed for Pre1a. The S1-S2 linker region has previously been shown to contribute to the interaction of HwTxIV with NaV1.7 via E753 a the top of S123. This residue is highly conserved in all NaV channels, thus it cannot account for the subtype selectivity that we observed. Figure V1.1, 1.2, 1.7 and 1.3 in the first half of S2 of DIV  to possibly explain our data. Previous mutagenesis studies on DII23 and DIV13 have shown that the divergent residues in region 1 have little effect on the activity of HwTxIV and δ-Hm1a, respectively. As region 3 of helix S2 is identical in both NaV1.1 and NaV1.3, but not 1.2 and 1.7, we assessed the potential role of these two residues (Ser/Arg) in the ability of Pre1a to inhibit inactivation. Using a chimaera-based approach we found that replacing the S3-S4 linker of rNaV1.4 (Pre1a insensitive) with corresponding linker from hNaV1.1 (Pre1a sensitive) was not sufficient to allow inhibition of inactivation by Pre1a (1 μM)  background, introduction of Ser1379 (S1574 in hNaV1.1), but not Arg1380 from NaV1.1 S1-S2 was sufficient for Pre1a to have a functional effect on both the extent  Fig. . Using tent Fig.  and rateent Fig.  of fast V1.2, NaV1.3 and NaV1.7 (but not NaV1.1 or NaV1.6) are robustly expressed in the human neuroblastoma cell line, SH-SY5Y42. Therefore, synthetic Pre1a was tested on SH-SY5Y cells using manual patch clamp to assess the effects on native human NaV channel currents. As shown in Fig. + current in a manner consistent with previously observed effects on rNaV1.2 and hNaV1.7, while concurrently slowing fast inactivation as was observed with rNaV1.3. This suggests that endogenously expressed hNaV1.3 exhibits similar sensitivity to Pre1a as compared to the rat isoform expressed in oocytes.As reported previously, Na+ current and slowed fast inactivation in a manner comparable to SH-SY5Y cells  also inhibits fast inactivation of NaV1.1 but not NaV1.6, thus the persistent current observed with Sham rat DRG neurons in the presence of Pre1a is likely due to effects on NaV1.1, which is present in adult neurons and has been reported to not undergo regulatory changes post-axotomy46.Additionally, small DRG neurons were excised from both sham and peripheral nerve ligated (PNL) adult rats, 7 days post operation. In both sets of DRG neurons, 300 nM synthetic Pre1a inhibited the peak Nalls Fig. . NaV1.3 ved Fig. . This reV channels and possesses a unique structural and pharmacological profile. Pre1a exhibits over 70% sequence similarity with several other spider venom peptides ; however, the toxin is unique among these peptides in its ability to potently and selectively inhibit fast inactivation of NaV1.1 and NaV1.3. Furthermore, Pre1a shows a marked structural heterogeneity/flexibility in both aqueous and organic solvents, which may be related to its unique activity profile when compared to its closest sequence relatives. Thus, Pre1a represents a valuable research tool to help determine the molecular basis for spider venom peptide interactions with multiple modulatory sites in sodium channels, particularly in respect to effects on fast inactivation.We have identified and characterised β/δ-TRTX-Pre1a, a tarantula venom peptide that potently modulates neuronal NaV channel fast inactivation is controlled by the movement of the DIV voltage-sensing domain17. Although several spider venom peptides (such as JzTxI and JzTxII) have previously been shown to preferentially inhibit NaV channel fast inactivation, they are either relatively non-selective48 or preferentially target NaV1.550. Two other spider peptides (ProTx-II and JzTxIV) have been found to inhibit both peak current and fast inactivation of NaV channels25, much like Pre1a. However, all these peptides share less than 50% similarity with Pre1a  and rNaV1.3 (S1510) is responsible for the selective modulation of fast inactivation by Pre1a. Our results are consistent with several reports suggesting that this serine residue is a major determinant for the impressively subtype selective effects of both small molecule drugs and another spider venom peptide not related to Pre1a. ICA-121431 inhibits hNaV1.3 and hNaV1.1 peak current with ~1000-fold selectivity and functions by trapping the DIV VSD in the activated state 51, whereas δ-TRTX-Hm1a (Hm1a) was recently characterised as a selective inhibitor of inactivation of NaV1.113, having been previously discovered as a low-affinity KV4.2 inhibitor52. Furthermore, a co-crystal structure of a NaV1.7 VSD IV chimaera with the small molecule NaV channel inhibitor GX-936 has been solved revealing the detailed molecular interactions responsible for its selectivity53. GX-936 has the same mechanism of action as ICA-121431 thus arrests the VSD in the inactivated state as opposed to the resting state targeted by Pre1a and Hm1a. Remarkably, and despite the different state stabilised by these peptides and small molecules, they all rely on the same region in S2 (S1574 in hNaV1.1 and neighbouring residues) for their functional selectivity. Together, these results illustrate that the S2 helix is an important locus for subtype selective modulation of NaV channel fast inactivation and that the key residues involved appear to be accessible to ligands in both the resting and inactivated states.NaSee Fig.  and haveV1.1 fast inactivation, they differ greatly in primary sequence . Interestingly, the small amount of similarity that does exist between them is a hydrophobic patch clustered in loop 1 of the peptide , the aromatic residues at the tip of Loop 1 are key residues for the interaction of these peptides with NaV channels, in particular for their inhibitory effects on channel activation. Whether the flexibility of loop 1 in Pre1a has any role in its ability to potently interact with both DII and/or DIV VSDs  remains to be elucidated.Although Hm1a and Pre1a share the ability to inhibit Naide Fig. . Co-inciV channel activation have been the focus of extensive chimaera and scanning mutagenesis studies to characterise the binding site on the channel. Bosmans et al. demonstrated that the S3-S4 linker region of any of DI to DIII can be a primary binding determinant for a number of venom peptides by transplanting this region in to a toxin insensitive background and transferring functional peptide binding18. Subsequently, a motif, which spans the DII S1-S2 and S3-S4 regions, that is critical for the inhibitory activity of HwTx-IV on hNaV1.7 was identified and consists of the residues E753 in S1, and E811, L814, D816, E818 in S3-S423  for the interaction of VSTX1 and the VSD of KVAp32. Our data showing a greater effect of Pre1a on the DII Kv2.1/hNav1.7 S3-S4 chimaera than that containing the DIV S3-S4 linker is in accord with the these previous studies. However, interaction with the residues identified in S1 and S3-S4 does not explain the substantial differences in Pre1a’s ability to inhibit NaV channel activation and despite the studies carried out to date, little insight into the basis of NaV channel subtype selectivity has been gained in regards to DII. Pre1a most potently inhibited the activation of NaV1.7 and NaV1.2 with ~70-fold selectivity over NaV1.3. The latter two channels have identical sequences in the S3-S4 linker, thus binding to this region cannot account for the observed selectivity. Interestingly hNaV1.1 and rNaV1.2 only differ by only two residues in the S2 helix, a conservative Thr (1.3) to Ser (1.2) substitution (at the position corresponding to the Ser1574 in Nav1.1 DIV) and a less conservative Gln (1.3) to Glu (1.2) substitution at the top of S2. This strongly suggests that residues in one or both of these positions make major contributions to the interaction of Pre1a with NaV VSD (driven primarily by interactions with S3-S4) that together result in functional inhibition of the channel. Thus, similar to what we showed for the molecular basis of sub-type selective inhibition of inactivation, subtype selective interactions at DII are also likely determined by the variability in the S2 helix.The interactions of spider venom peptides that inhibit NaV channel family can be used to gain insight into the molecular basis of subtype selectivity. It should be noted that few venom peptide modulators of NaV channels have been studied in the same detail as reported here, thus the structural heterogeneity and binding site promiscuity of Pre1a may indeed be a more common property than we realise. Pre1a has great value as a research tool for exploration of the selectivity profile of activation and inactivation on different NaV channel isoforms, as well as exploring the consequences and effects of conformational flexibility to activity in this family of channel modulators.In conclusion, we have identified a novel spider venom peptide and demonstrated how its so far unique activity on the NaPsalmopoeus reduncus venom was purchased from SpiderPharm (Yarnell). Venom was fractionated using a C18 218TP54 column  with solvent A  and B  over a gradient of solvents . Fractions were collected, dried and assayed for activity against hNaV1.7 expressed in Xenopus laevis oocytes. The active fractions were further separated on a PromixMP column  with a gradient of 10–35% B in 35 min at 1 ml/min, then a Prosphere C4 column  with a gradient of 20–45% B at 0.75 ml/min.Peptides were analysed using MALDI-TOF mass spectrometry  in reflector mode using α-cyano-4-hydroxy-cinnamic acid . Reduction/alkylation and sequencing of approximately 3 μg of the peptide was carried out by N-terminal Edman degradation.56. The side-chain protecting groups chosen were Asn (Xan), Arg (Tos), Asp (OcHex), Cys (4-MeBzl), Lys (ClZ), Ser (Bzl), Trp (CHO) and Tyr (BrZ). The crude reduced peptide was purified using preparative reversed-phase chromatography (Vydac C18 218TP1022), using a gradient of 0–80% B over 80 min). The peptide was oxidised at a concentration of 0.02 mM in either aqueous 0.33 M NH4OAc/0.5 M GnHCI or 2 M NH4OH/0.1 M NH4OAc at pH 7.8, 4 °C in the presence of both reduced and oxidised glutathione . Oxidised peptides were purified using preparative RP-HPLC.Synthetic Pre1a was assembled manually using Boc SPPS chemistry as described previouslyE. coli periplasmic expression system as described previously57. Briefly, a synthetic gene encoding Pre1a was codon optimised for bacterial expression and subcloned into a pLicC-His6-MBP periplasmic expression vector, where the peptide is expressed as a C-terminal fusion to His6-tagged maltose binding protein (MBP) separated by a tobacco etch virus (TEV) protease cleavage site, leaving an additional N-terminal Ser after cleavage. Fusion proteins were expressed in E. coli strain BL21(λDE3) and isolated from cell lysates using Ni-NTA Superflow resin (Qiagen). The His6-MBP tag was removed from the fusion protein using TEV protease, and recombinant Pre1a purified using RP-HPLC.Pre1a was produced recombinantly using an 13C/15N-labelled Pre1a, cultures were grown in minimal medium supplemented with 13C6-glucose and 15NH4Cl as the sole carbon and nitrogen sources, respectively. In order to facilitate comparisons between synthetic and recombinant Pre1a, residue numbers for the native toxin are used throughout the text even though the recombinant toxin contains an additional N-terminal serine residue that is a vestige of the TEV cleavage site.For production of uniformly Xenopus laevis oocytes and NaV channel recordings using two-electrode voltage-clamp (TEVC) were carried out as described previously58. Capped cRNA encoding rat NaV1.2, NaV1.3 and NaV1.4, and human NaV1.5 and NaV1.7 were injected at 20–40 ng of cRNA/oocyte and kept at 17–18 °C in ND96 solution containing (in mM) 96 NaCl, 2 KCl, 1 CaCl2, 2 MgCl2, 5 HEPES, 5 pyruvic acid, 50 µg/ml gentamicin (pH 7.4), and horse serum (2.5%). Membrane currents were recorded 2–5 days after injection. Oocytes were clamped at −90 mV and currents were elicited by a 50 ms depolarising step to −10 or 0 mV every 10 s for concentration response curves. Both peak and late current (50 ms post peak) were analysed. The concentration response curves were fitted to the Hill equation (GraphPad Prism 6). Current-voltage (I-V) relationships and voltage-dependence of fast inactivation were determined using a family of 100 ms conditioning pulses from −100 mV to +50 mV (+60 mV for rNaV1.3) in 5 mV steps, followed by a test depolarisation step to 0 mV with a 20 s sweep interval from a holding potential of −90 mV. To determine the voltage-dependence of activation non-normalised peak current was used from the I-V family to calculate channel conductance using the equation: G(V) = I/(V − Vrev), in which I, V, and Vrev represent inward NaV current, test potential, and reversal potential, respectively. The half-activation potential (V1/2) was determined using a Boltzmann fit (GraphPad Prism 6). All data points are shown as the pIC50 ± S.E.M., as well as the IC50. Replicates (n) represent separate experimental oocytes. Pre1a was resuspended in ND96 solution containing 0.1% bovine serum albumin for all activity analysis. Construction and recording of the KV2.1chimaeras containing hNaV1.7 DI-DIV S3-S4 linker regions was completed as described previously41. Design and construction of the hNaV1.1/rNaV1.4 DIV chimaeras is detailed in Osteen et al.13. Not all isoforms of NaVs from the same organism were available at the time of experimentation. Even though different species (rat and human) were used, careful analysis of the data in relation to the sequence differences of the isoforms used, still yields valuable insights in to the molecular basis of peptide:channel interaction.The preparation and injection of 13C/15N-labelled peptide in 20 mM potassium phosphate buffer, pH 5 containing 5% D2O was filtered using a low-protein-binding Ultrafree-MC centrifugal filter , then 300 μL of ~300 μM was added to a susceptibility-matched 5 mm outer-diameter microtube (Shigemi Inc.). NMR spectra were acquired at 298 K on a 900 MHz Bruker AVANCE II+ spectrometer (Bruker BioSpin) equipped with a cryogenically cooled probe. Data used for resonance assignment were acquired using non-uniform sampling (NUS); sampling schedules that approximated the rate of signal decay along the various indirect dimensions were generated using sched3D59. The decay rates used were 1 Hz for all constant-time 15N dimensions, 30 Hz for all 13C dimensions, and 15 Hz for the semi-constant indirect 1H dimension of the H(CC) (CO)NH/(H)CC(CO)NH-TOCSY experiments. 13C- and 15N-edited HSQC-NOESY experiments were acquired using linear sampling. Separate experiments were acquired for the aliphatic and aromatic regions of the 13C dimension.60. NMR spectra were analysed and assigned using the program XEASY61 or SPARKY62. 1HN, 15N, 13Cα, 13Cβ, and 13C′ resonance assignments were obtained from analysis of amide-proton strips in 3D HNCACB, CBCA(CO)NH, and HNCO spectra. Side-chain 1H and 13C chemical shifts were obtained primarily from 3D H(CC) (CO)NH-TOCSY and (H)CC(CO)NH-TOCSY spectra, respectively. The remaining side-chain assignments were derived from 3D 15N- and 13C-edited NOESY-HSQC spectra. The program TALOS+ 31 was used to predict the secondary structure of the peptide. Disulfide connectives were determined from NOESY patterns59. During the automated NOESY assignment/structure calculation process, CYANA assigned 90.1% of all NOESY cross-peaks (829 of 920).NUS data were processed using the Rowland NMR toolkit (www.rowland.org/rnmrtk/toolkit.html); maximum entropy parameters were selected automatically as describedV1.1, hNaV1.6, or hNaV1.7 α-subunit with the NaVβ1-subunit (SB Drug Discovery) were cultured following manufacturer guidelines. Cells were removed from culture at 70% confluency using Detachin (Genlantis) and resuspended to 5 × 106 cells/mL in Ex-Cell ACF CHO Medium (Life Technologies) supplemented with 25 mM HEPES (Sigma-Aldrich) and 1 × Glutamax (Life Technologies) before being transferred to the Q-Patch Q-Stirrer and allowed to recover for 30 min before assay.For patch clamp analysis, HEK293 cells stably co-expressing the hNa2, 1 MgCl2, 10 HEPES, 20 TEA-Cl, 10 glucose, pH 7.4 (with NaOH) and adjusted to 315 mOsm 0.05% BSA was added to prevent adsorptive loss of peptide. The intracellular solution contained (in mM): 140 CsF, 1/5 EGTA/CsOH, 10 HEPES and 10 NaCl, pH 7.4 (with CsOH) and adjusted to 320 mOsm. Whole-cell patch-clamp experiments were performed at room temperature on a QPatch-16 automated electrophysiology platform (Biolin Scientific) using 16-channel planar patch chip plates (Q-Plates) with a patch hole diameter of 1 µm and resistance of 2 ± 0.1 MΩ. Cell positioning and sealing parameters were set as follows: positioning pressure −60 mbar, minimum seal resistance 0.1 GΩ, holding potential −90 mV and holding pressure −20 mbar. Whole-cell currents were filtered at 5 kHz (8-pole Bessel) and digitised at 25 kHz. A P/6 online leak-subtraction protocol was used with non-leak-subtracted currents acquired in parallel. Cells were maintained with holding potentials of −80 (hNaV1.1) or −100 mV (hNaV1.6 and hNaV1.7) between protocols, respectively and depolarised to −10 mV with a sweep interval of 20 s to allow complete recovery from inactivation between sweeps. Pre1a concentrations were incubated for a fixed time of 5–10 min to allow steady-state activity, monitored by the I-T plot.The external solution contained (in mM): 140 NaCl, 4 KCl, 2 CaClSH-SY5Y cells as described previously42 or from small (<25 μm diameter) acutely isolated adult male rat DRG neurons as described previously63. DRG neurons were taken from rats that had undergone either partial ligation of the left sciatic nerve (PNL) to induce a state of neuropathic pain  or sham operated rats64. SH-SY5Y cells were used within 24–72 h, and DRG neurons were used within 6 hours of plating. Only cells with minimal or no processes were used. Whole-cell patch-clamp recordings were performed at room temperature with fire-polished patch electrodes prepared from borosilicate glass . Electrode resistance was 3.5–5 MΩ when filled with an internal solution containing (composition in mM): 120 CsCl, 5 MgATP, 5 NaCl, 2 CaCl2, 20 HEPES, 10 EGTA, pH 7.3 and adjusted to 283–286 mOsm. Cells were continuously bathed in a HEPES-buffered physiological saline : 155 NaCl, 2.5 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, 10 glucose, pH 7.4 (adjusted with NaOH) and adjusted to 328–331 mOsm. Currents were recorded using a HEKA EPC-9 amplifier and PULSE software . Data was filtered at 4 kHz and sampled at 20 kHz. Series resistance was compensated by 80%. Capacitance transients were compensated and leak subtraction was performed with a P/8 online protocol. Drugs were applied to cells using a gravity-fed superfusion system (250 μm diameter) positioned directly above the cell resulting in rapid solution exchange (<500 ms).Whole-cell patch clamp electrophysiology was carried out on th Ed. 2013) and great care was taken to minimise animal suffering at all times. Experiments involving rats were approved by the University of Sydney  or Royal North Shore Hospital/University of Technology Animal Ethics Committee . Experiments using X. laevis were approved by The University of Queensland Animal Ethics Committee . Oocytes were obtained via recovery surgery performed under tricaine methanesulfonate (MS-222) anaesthesia . Minimum time between surgeries on the same animal was six months.All animal experiments complied with the Australian code of practice for the care and use of animals for scientific purposes, (8Supplementary data


A universal sensing platform with significantly enhanced image contrast and apparent brightness in biological imaging applications. HN7, with large stokes shift of 73 nm and fluorescence quantum yield as high as 0.72 in ethanol, values superior to those of such traditional cyanine NIR dyes as Cy5. Using HN7, living cells, tissues and mice were imaged, and the results showed significantly enhanced contrast, improved spatial resolution, and satisfactory tissue imaging depth when compared to Cy5. Moreover, the nonfluorescent spirocyclic structure of rhodamine B is an inherent component of HN7; therefore, our strategy provided a universal platform for the design of efficient NIR turn-on bioimaging probes for various targets. As a proof-of-concept, two different NIR probes, HN7-N2 and HN7-S for NO and Hg2+, respectively, were designed, synthesized, and successfully applied for the imaging of NO and Hg2+ in living cells, tissues and mice, respectively, demonstrating the potential bioimaging applications of the new probes. In sum, this new type of dye may present new avenues for the development of efficient NIR fluorescent probes for contrast-enhanced imaging in biological applications.Near-infrared (NIR) fluorescent probes are attractive molecular tools for bioimaging because of their low autofluorescence interference, deep tissue penetration, and minimal damage to sample. However, most previously reported NIR probes exhibit small Stokes shift, typically less than 30 nm, and low fluorescence quantum yield, strictly limited contrast and spatial resolution for bioimaging. Herein, by expanding the π-conjugated system of rhodamine B, while, at the same time, keeping its rigid and planar structure, we reported an efficient NIR dye,  The application of fluorescence imaging through staining with a fluorescent probe is characterized by high sensitivity, real-time spatial imaging and detection of targets in living cells or tissues with minimal damaging effects. As such, fluorescence imaging has become a facile and powerful tool for the detection of biologically relevant species with the ability to visualize morphological details and monitor various physiological processes in living systems.via photoinduced electron transfer (PET).Cyanine dyes are classic NIR fluorophores with an odd number of carbons in a conjugated polymethine framework between two nitrogen-containing heterocycles.The classic rhodamines possess excellent photophysical properties, such as high molar extinction coefficients, high fluorescence quantum yields, typically larger than 0.5,HN1–7. Among these dyes, the rigid and planar dye HN7 exhibits excellent photophysical properties, such as fluorescence emission located in the NIR region (>670 nm), large Stokes shift (73 nm in PBS buffer), high fluorescence quantum yield , excellent stability and aqueous solubility. To understand the structure–optical properties of HN7, we carried out quantum chemical calculations with the B3LYP exchange functional employing 6-31G* basis sets using a suite of Gaussian 09 programs.HN7 was found to display an image contrast of 85-fold in living cells (10 μM) and 42-fold in living mice (5 nmol), values much greater than those of traditional rhodamine B and Cy5. The tissue penetration capability of HN7 was slightly superior to that of Cy5. It is also worth noting that HN7 showed obviously higher brightness and enhanced contrast over that of Cy5. Moreover, HN7 possesses a rhodamine-like spirolactam structure, which provides a universal platform for the design of NIR turn-on bioimaging probes. To demonstrate the feasibility of this probe design, two different NIR probes, HN7-N2 and HN7-S for NO and Hg2+, respectively, were designed, synthesized, and applied for imaging of NO and Hg2+ in living cells, tissues and mice.Therefore, in this work, we have expanded the π-conjugated system of rhodamine B beyond that of traditional rhodamine dyes, while keeping its rigid and planar structure, and developed a new series of far-red to NIR dyes named HN1, HN3, and HN5 with various amino substituents, were first synthesized -naphthalenone instead of 4-aminoacetophenone derivative as the starting reactant, we also designed and easily synthesized rigid dyes HN2, HN4, and HN6 with an alkyl-ring to limit molecular rotation and vibration -naphthalenone and dimethyl phthalate.HN1–7 were characterized by 1H NMR, 13C NMR and MS (ESI).In general, the emission wavelengths of organic dyes are highly dependent on intramolecular π-conjugated systems and substituent electronic effects, while rigid and planar molecular structures afford high fluorescence quantum yield for dyes. To test this hypothesis, several compounds possessing expanded π-conjugated systems and, thus, potential emission of red-shift fluorescence in comparison with traditional rhodamine dyes, were designed and synthesized. Compounds with a flexible structure, including thesized , using 4ibration .38 In orHN1–7, rhodamine B and Cy5 in various solvents, including buffered aqueous solution , EtOH, CH2Cl2, DMSO, and CH3CN. The absorption and emission profiles are illustrated in HN1–7 display broad and intense maximum absorption bands in the 571–600 nm region, which is assigned to the 0–0 band of the S0 → S1 transition. Broad and weak absorption bands were found around 420 nm, which could be attributed to the S0 → S3 transition. Notably, the absorption of HN7 exhibited an obvious blue-shift in comparison to Cy5, resulting in a larger Stokes shift in spite of its π-conjugated system which was expanded over that of rhodamine. On the other hand, the molar extinction coefficients (εmax) of all our new dyes are in the range of 120 000 M–1 cm–1 and thus superior to the classic rhodamines.HN1–7 solutions are all much deeper than the classic rhodamine B by their long absorption wavelengths and large molar extinction coefficients.We first tested the optical properties of HN1–6 display maximum fluorescence emission in the far-red region (615–638 nm) in PBS buffered solution. Remarkably, HN7 displays Cy5-like fluorescence emission with maximum at 673 nm in PBS buffer and covers a significant part of the NIR region. Furthermore, according to the absorption and emission data, the Stokes shift of HN7 is 73 nm in neutral buffered solution, which is superior to that of the classic rhodamine, fluorescein, BODIPY®, and cyanine dyes, all typically less than 30 nm. Such large Stokes shift could reduce background fluorescence for HN7 and avoid self-quenching and false-positive signals from excitation backscattering effect.Since their π-conjugated systems are expanded beyond those of the classic rhodamines, HN1, HN3, HN5 with their flexible structure showed fluorescence quantum yields less than 0.22 in EtOH and less than 0.06 in aqueous solution.HN2, HN4, HN6, and HN7 all possess rigid and planar molecular structures similar to rhodamine dyes, but they exhibit higher fluorescence quantum yields than the classic cyanine NIR dyes , ranging from 0.72 to 0.93 in EtOH and from 0.25 to 0.43 in aqueous solution.εmaxΦf) is another important parameter to test, as it is involves imaging contrast and spatial resolution. Accordingly, the brightness of HN7 was calculated to reach 34 325 M–1 cm–1 in buffered aqueous solution, which is superior to that of traditional rhodamine B and Cy5. All these results illustrate that our NIR dye, HN7, is suitable for bioimaging applications with enhanced brightness and contrast.Classic rhodamine dyes possess high fluorescence quantum yields, typically larger than 0.5, as a result of their rigid and planar molecular structures. As expected, compounds HN2, HN4, HN6, HN7 and Cy5 in PBS buffer. Exactly as anticipated and as shown in HN dyes was retained after 120 min irradiation, while only 62% of Cy5 was retained, indicating that our rigid HN dyes have sufficient photostability for prolonged biological imaging. On the other hand, it is well known that many organic NIR dyes are easily bleached by reactive oxygen species (ROS).HN dyes under these conditions, the fluorescence profiles of HN2, HN4, HN6, HN7 and Cy5 in the presence of various ROS  were also investigated. As shown in HN dyes with ROS in excess of 120 min, values much improved of those of Cy5. All these data suggest that our HN dyes have excellent photostability and the chemical stability required to resist photobleaching and common ROS in biological imaging applications.Still another important parameter is photostability. By using continuous irradiation, as described above, we investigated the photostability of HN dyes, the effects of pH on their fluorescence response and a representative spirolactam derivative were investigated. As shown in HN7. However, fluorescence did decrease from protonation reaction in strong acidic conditions or the development of nonfluorescent spirocyclization in alkaline conditions, with pKa at 2.7 and pKcycl reaching 12.0. Moreover, the spirolactam derivative HN7-N2 showed pKcycl of about 4.1 and predominately existed in the nonfluorescent spirocyclic form with a broad range of pH values over 6.0. Therefore, just like the classic rhodamines and their spiro-ring derivatives,HN dyes and their spiro-ring derivatives would seem to have unlimited potential applications based on the robust rhodamine sensing platform.The pH value around the rhodamine analogues and their spirolactam derivatives typically affects performance because of protonation reaction in acidic conditions or cyclization reaction in alkaline conditions. Therefore, to study the practical applicability of the newly designed HN7, we carried out DFT calculations with the B3LYP exchange functional employing 6-31G* basis sets using a suite of Gaussian 09 programs.37HN7. The C–C (N or O) bond lengths (in pm) of HN7, as determined by DFT calculations, are displayed in  PBM data was replaced with SVG by xgml2pxml:<glyph-data id="z.dbdfnccb" format="PBM" resolution="300" x-size="34" y-size="34" xml:space="preserve"> 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 1111111111111111111111111111111111 1111111111111111111111111111111111 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000 0000000000000000000000000000000000	</glyph-data><svg xmlns="http://www.w3.org/2000/svg" version="1.0" width="16.000000pt" height="16.000000pt" viewBox="0 0 16.000000 16.000000" preserveAspectRatio="xMidYMid meet"><metadata>Created by potrace 1.16, written by Peter Selinger 2001-2019</metadata><g transform="translate scale" fill="currentColor" stroke="none"><path d="M0 1440 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z M0 960 l0 -80 1360 0 1360 0 0 80 0 80 -1360 0 -1360 0 0 -80z"/></g></svg>N, a double bond around 128 pm. Obviously, these bond length calculations support the strong electronic delocalization along the π-conjugated system of HN7.To understand the optical properties of HN7  and HN7, Cy5 (650–720 nm) were also confirmed. As a proof-of-concept, HeLa cells were incubated with HN7, rhodamine B and Cy5  at 37 °C for 60 min, and the fluorescent imaging results were shown in HN7 not only showed brightness comparable to that of rhodamine B, but also considerable relative fluorescence intensity enhancement ratios as high as 85-fold, with a high enhancement ratio of 24.6-fold, even at the low concentration of 1 μM. While the relative fluorescence intensity enhancement ratios of Cy5 were tolerable, imaging brightness was very weak and not suitable for visual observation. Therefore, compared to classic rhodamine B and Cy5, our new HN7 bore excellent imaging properties, including NIR emission, high brightness and enhanced contrast. As such, these results demonstrate the potential in vivo imaging applications of HN7, especially for trace fluorescence imaging.To evaluate the imaging performance of HN7 in HeLa cells was also examined using the MTS -5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) assay. Fig. S5HN dyes exhibited low cytotoxicity after incubation in HeLa cells for 48 h. Such desirable property may also render the new HN dyes suitable for applications in living systems.The cytotoxicity of HN7 was further applied for tissue imaging of rat liver frozen slices, and the changes of fluorescence signal intensity with scan depth were then determined by confocal microscopy  in z-scan mode. As shown in HN7 possessed satisfactory tissue imaging capability at depths of 40–120 μm. Meanwhile, Cy5 was capable of tissue imaging at depths of 75–100 μm, indicating that the tissue penetration capability of HN7 was slightly superior to that of Cy5. It is worth noting that HN7 showed obviously enhanced brightness and contrast when compared to Cy5 for imaging frozen rat liver tissue slices. These data demonstrated the potential of HN7 in tissue imaging applications.To evaluate the imaging depth of the new NIR dye, in vivo imaging. Compared to the traditional NIR dye Cy5, our unique HN7 dye exhibits a similar fluorescence emission spectrum, but has additional advantages, such as larger Stokes shift, higher fluorescence quantum yield, greater photostability and satisfactory aqueous solubility, all of which could provide low background, high fluorescence signal and enhanced contrast for bioimaging applications in living animals. As confirmation, we further tested the analytical characteristics of Cy5 and HN7in vivo. Fluorescence images of four-week-old male BALB/c nude mice were first taken with two well-separated wavelengths of an excitation filter 605 nm and a Cy5.5 emission filter. Significantly, as shown in HN7 and Cy5 (5 nmol), compared to the blank group, a considerable enhanced fluorescence intensity of 42-fold was observed for the HN7-incubated group , HN7-N2 instantly exhibited blue color, as determined by the naked eye, accompanied by considerable red fluorescence . Similar results were also obtained when using the HN7-S probe for Hg2+ detection  for 30 min showed no observable fluorescence signal, indicating that the HN7-N2 probe maintained its spirocyclic form in cells. When HeLa cells preincubated with HN7-N2 were further treated with different concentrations of NO donor, diethylamine NONOate , a dramatic increase in fluorescence brightness was observed, suggesting that the new probe was cell permeable and could be used as an imaging tool to quantify the concentration of NO in living cells.The excellent photophysical properties of HN7-N2 for NO imaging in tissue of rat liver frozen slice. The changes of fluorescence intensity with scan depth were determined by confocal microscopy  in the z-scan mode. As shown in HN7-N2 probe was capable of tissue imaging at depths of 45–110 μm. These data showed that the HN7 probe had good tissue penetration and staining ability. HN7-N2 was also used for NO detection in vivo. For four-week-old male BALB/c nude mice, the control group was only given an intraperitoneal injection of HN7-N2 (20 nmol). The experimental group was first given an intraperitoneal injection of HN7-N2 probe (20 nmol), followed by an intraperitoneal injection with NO donor, diethylamine NONOate (50 nmol), after 30 min. The mice were then imaged using a Caliper VIS Lumina XR small animal optical in vivo imaging system with an excitation filter 605 nm and a Cy5.5 emission filter. As shown in in vivo. However, mice treated with both HN7-N2 and NO exhibited significantly stronger fluorescence intensity than the control group, indicating that HN7-N2 could be applied for imaging NO in vivo.We further used HN7-S for Hg2+ in living cells, tissue slices and mice were also performed, and results were similar to those of HN7-N2 for NO were obtained (Fig. S13–15HN7 is an efficient and universal platform for the design of NIR bioimaging probes for various analytes with enhanced brightness and contrast.Fluorescence imaging experiments of HN7, with excellent photophysical properties, such as large Stokes shift and high fluorescence quantum yield, which could afford significantly enhanced image contrast and apparent brightness in biological imaging applications when compared to traditional rhodamine B and Cy5. Furthermore, the spirocyclic structure of rhodamine B is inherent in HN7; as such, our strategy provided a universal platform for the design of efficient NIR fluorescent turn-on bioimaging probes for various targets. As a proof-of-concept, two different NIR probes, HN7-N2 and HN7-S for NO and Hg2+, were designed, synthesized, and successfully applied for the imaging of NO and Hg2+ in living cells, tissues and mice, respectively, demonstrating the potential bioimaging applications of the new probes. Thus, the new type of dye presented herein may open up new avenues for the development of efficient NIR fluorescent probes for contrast-enhanced biological imaging applications.In summary, by introducing a practical strategy of constructing a rigid molecular structure, we have successfully developed a novel NIR functional fluorescent dye, Supplementary informationClick here for additional data file.


For reprogramming fibroblasts, carrying the novel homozygous mutation c.1180T>C and the prevalent homozygous mutation c.3182T>C, were used. Reprogramming into patient-specific iPSCs was induced by retroviral transduction of the transcription factors Sox2, Klf4, Oct4 and c-Myc, and confirmed according to their pluripotency. The iPSCs were subsequently differentiated into neural progenitor cells, which were terminally differentiated into functional neurons and glial cells. The generation of these cell lines provides further valuable tools to investigate pathogenic mechanism of NPC1 in human neuronal cells carrying different NPC1 mutations.Data presented in this article demonstrate the generation and characterization of two novel Niemann-Pick disease Type C1 (NPC1) patient-specific induced pluripotent stem cell (iPSC) lines, related to the research article Trilck et al. (Diversity of Glycosphingolipid GM2 and Cholesterol Accumulation in NPC1 Patient-Specific iPSC-Derived Neurons; Brain Res.; 2017; 1657:52-61. doi:  Specifications TableValue of the data•Data in support of the generation of patient-specific iPSCs carrying novel NPC1 mutation.•Data demonstrating the pluripotency of generated iPSCs and derived neural progenitors.•Data showing differentiation of neural progenitors into neurons and glial cells.•Data describing the establishment of human cell model applicable in NPC1 disease modeling and drug discovery.1NPC1 mutation c.1180T>C or c.3182T>C, were used for reprograming. The c.1180T>C is a novel unpublished mutation while mutation c.3182T>C is the most prevalent mutation of the NPC1 gene found in patients NPC1 mutation. Data demonstrate the pluripotency of the generated iPSCs by immunocytochemical detection of a panel of specific pluripotency marker (These data show the generation of NPC1 patient-specific induced pluripotent stem cells (iPSCs) using a standardized protocol y marker . Pluripoy marker . Data pry marker . Terminay marker  and the y marker . Data oby marker .22.12.Human dermal fibroblast cell lines from male donors were obtained from Coriell Institute for Medical Research, Camden, USA  and Centogene AG, Rostock, Germany (control and NPC1 homozygous mutated c.1180T>C [p.Y395H]), respectively. Cells were cultivated in fibroblast medium containing DMEM high glucose, 10% FBS and 1% penicillin/streptomycin. Mitotically inactivated mouse embryonic fibroblasts  were used as the feeder cell layer for iPSCs and were cultivated in fibroblast medium. HEK293FT cells  were also cultivated in fibroblast medium but without antibiotics. Human iPSCs were cultured on a feeder cell layer in iPSC medium containing DMEM/F12, 20% knockout serum replacement, 1% penicillin/streptomycin, 1% GlutaMAX, 1% MEM non-essential amino acids, 0.2% 2-mercaptoethanol, and 10 to 15 ng/ml FGF2  or on Matrigel  cultured in mTESR1 medium . Medium was changed daily and cells were passaged weekly. All cells were cultivated at 37 °C in a saturated humidity atmosphere containing 5% CO2.22 until a stable cell line was expanded.Human iPSCs were generated as described earlier 2.3g for 10 min and the pellet was resuspended in 4 ml KCl solution (5.62%). Cells were incubated for 5 min at 37 °C and centrifuged at 300g for 10 min. After resuspension cells were fixed in 5 ml glacial acetic acid and methanol (1:3) and subsequently centrifuged for 7 min at 350g. This step was repeated once. Next, supernatant was removed and cells were resuspended. Cell suspension was dropped onto cold slides and dried at 100 °C for 1 h. Giemsa solution (5%) was added and incubated for 5 min. Slides were washed in distilled water two times, dried at room temperature and sealed with cover slips.Karyotyping was performed by Giemsa Trypsin banding. Colonies were incubated with colcemid solution (10 µg/ml in HBSS) overnight for metaphase arrest. IPSCs were harvested by trypsin treatment (0.25%), wherein the reaction was stopped with Amniomax solution , centrifuged at 3002.4Alkaline phosphatase (AP) staining used to prove pluripotency of generated iPSCs. Colonies of iPSCs, cultivated on feeder cells, were fixed with ice-cold methanol (100%) for 10 min and incubated at room temperature for 15 min with the staining solution, containing 75% distilled water, 10% sodium chloride solution (1 M), 10% Tris solution , 5% magnesium chloride solution (1 M), and NBT/BCIP solution . Microphotographs were taken using a Nikon Eclipse TS100 microscope system .2.53 at 4 °C. Immunocytochemistry was performed for Nanog , Tra-1–60 , Tra-1–81 , SSEA3 , SSEA4 . Blocking and permeabilization was carried out using 0.3% Triton X-100 and 5% normal goat serum  for 30 min at room temperature. Cells were incubated with primary antibodies for 2 h at room temperature in 1% normal goat serum, followed by three washing steps with PBS. Alexa Fluor 568  and Alexa Fluor 488  were used as secondary antibodies, incubated 1 h at room temperature in PBS. Cells were washed three times with PBS and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system .Cells were fixed at room temperature for 15 minutes in 4% paraformaldehyde, washed with PBS and stored in 0.02% NaN2.6in vitro embryoid body formation assay is discussed by Sheridan and colleagues Embroid body (EB) formation was used for proving pluripotency of iPSCs by using marker for the three germ layers. Whole iPSC colonies were mechanically lifted off the feeder cell layer and transferred into the cavity of a low attachment 6-well plate in 5 ml of differentiation medium . EBs were formed after five to seven days and reseeded onto gelatin-coated glass cover slips. After 10 days of random differentiation, spread cells were fixed with 4% PFA for 15 min and immunocytochemically stained for muscle actin , alpha fetoprotein  and nestin . Alexa Fluor 488  was used as secondary antibody, incubated 1 h at room temperature in PBS. Cells were washed three times with PBS and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system . Teratoma formation assay in immune deprived mice was not performed any more, following the 3Rs principle, to replace, refine or reduce, the use of animals, mandated by the German Protection of Animal Act. In regards of the teratoma formation assay in stem cell research the applicability of the 2.72 on poly-L-ornithine /laminin -coated dishes in proliferation medium containing 60% DMEM, 40% DMEM/F-12, 1X B27, 0.5% penicillin/streptomycin, 20 ng/ml FGF2 , 20 ng/ml EGF . Pluripotency of neural progenitor cells was proven by stainings for Sox2 , nestin , Pax6 . Alexa Fluor 568  and Alexa Fluor 488  were used as secondary antibodies, incubated 1 h at room temperature with 1% normal goat serum in PBS. Cells were washed three times with PBS and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system .Neural differentiation of iPSCs was induced by density-dependent growing of the iPSCs on Matrigel . After neural rosettes had been formed spontaneously, cells were washed with DMEM/F12, singled using Accutase® and then magnetically sorted using magnetic beads against the surface marker PSA-NCAM  which is a marker of the neural lineage. The generated neural progenitor cells were used for 25 passages and seeded at an expansion density of 100,000 cells/cm2 in differentiation medium, containing 60% DMEM, 40% DMEM/F-12, 1X B27, 0.5% penicillin/streptomycin, which was changed every 4 days over a period of 6 weeks. Cells were stained for the neuronal marker beta III-tubulin , GFAP  and synaptophysin . Blocking and permeabilization was carried out using 0.3% Triton X-100 and 5% normal goat serum  for 30 min at room temperature. Cells were incubated with primary antibodies for 2 hours at room temperature in 1% normal goat serum, followed by three washing steps with PBS. Alexa Fluor 568  and Alexa Fluor 488  were used as secondary antibodies, incubated 1 h at room temperature with 1% normal goat serum in PBS. After washing with PBS, cells were stained with DAPI , washed three times and mounted with Mowiol-DABCO mounting medium. Pictures were taken with a Biozero 8000 microscope system .For terminal neuronal differentiation cells were plated at a density of 45,000 cells/cm2.82x6H2O 1, CaCl2xH2O 2, Mg-ATP 2. pH was adjusted to 7.2. When filled, electrodes had a resistance of 6–8 MΩ. Cell cultures were continuously superfused with an extracellular solution consisting of (mM): NaCl 125, KCl 2.5, CaCl2xH2O 2, MgCl2x6H2O 1, NaHCO3 26, NaH2PO4xH2O 1.25, glucosexH2O 25. Solution was continuously bubbled with carbogen to maintain a pH of 7.4. Recordings were made in the whole cell configuration with holding potentials (VH) of −60 or −80 mV. Current voltage responses were evoked by applying 100 ms voltage steps from −60 mV to +50 mV in 10 mV increments. Current clamp mode was used to apply current steps to induce action potentials or to measure spontaneous action potentials. Postsynaptic currents were measured in the voltage clamp mode at a VH of −60 mV. Mini Analysis 6  was used to analyze recordings of post-synaptic currents.Patch clamp recordings were performed using an EPC-10 amplifier . Patch pipettes were pulled from borosilicate glass tubing . The internal solution contained (mM): KCl 130, NaCl 10, HEPES 10, EGTA 11, MgCl


UGT1A and DPYD polymorphism panel to better predict irinotecan-induced toxicity and the clinical response in Chinese patients with metastatic colorectal cancer (mCRC).To evaluate a new UGT1A  and DPYD  were examined by direct sequencing in 661 mCRC patients receiving irinotecan-based chemotherapy. The influences of UGT1A and DPYD polymorphisms on severe irinotecan-induced toxicities and clinical outcomes were assessed.The genotypes of UGT1A1*6, UGT1A1*28, UGT1A7*2, UGT1A7*3, UGT1A9*22, DPYD*5, and DPYD c.1896 T > C variants were 34.8%, 24.2%, 34.3%, 39.4%, 81.8%, 48.4% and 20.4%, respectively. UGT1A1*27 and DPYD*2A had low frequencies and UGT1A7*4 was not found. A total of 59 patients (8.9%) suffered severe diarrhea and 136 patients (20.6%) suffered severe neutropenia. UGT1A1*28 heterozygotes , UGT1A1*28 homozygotes  and UGT1A1*6 homozygotes  were independent risk factors for severe neutropenia. UGT1A polymorphisms were not found to relate to severe diarrhea. DPYD*5 was determined to be an independent risk factor for severe diarrhea . Neither DPYD*5 nor DPYD c.1896 T > C was found to relate to severe neutropenia. In the first-line irinotecan-based treatment, UGT1A1*28 and DPYD*5 contributed to higher response rates , while DPYD*5 was found to associate with better progression-free survival (P = 0.015). UGT1A1*27 contributed to worse overall survival (P < 0.001).In the cohort studied here, the incidence of UGT1A1*6 and UGT1A1*28 to be partially associated with irinotecan-induced toxicity and clinical response. An examination of more UGT1A loci, except for UGT1A1*6 and UGT1A1*28, was not helpful to improve the predictive value of irinotecan-based toxicity and efficacy. An examination of DPYD*5 assisted in the prediction of severe diarrhea.Results still showed The online version of this article (doi:10.1186/s12885-017-3406-2) contains supplementary material, which is available to authorized users. UGT1A polymorphisms, especially UGT1A1*6 and UGT1A1*28, were previously noted to predict irinotecan-induced toxicity, but the results were inconstant [UGT1A1*6 and UGT1A1*28 were found to be related solely to irinotecan-induced severe neutropenia, and not to diarrhea, as most studies in Asia indicated [UGT1A loci improved the predictive sensitivity and specificity to irinotecan-induced toxicity, it still focused on predictability for severe neutropenia [DPYD polymorphisms, which are associated with fluorouracil levels in vivo, are associated with the occurrence of fluorouracil-induced toxicity [UGT1A with an examination of DPYD polymorphisms. This is the first large sample analysis of a combined examination of both UGT1A and DPYD polymorphisms to predict irinotecan-based chemotherapy-induced toxicity and the clinical response in Chinese patients.Irinotecan is currently one of most important drugs in the management of metastatic colorectal cancer (mCRC) , 2. Althconstant , 4. Baseconstant , UGT1A1*tropenia . The restropenia , 10. Mortoxicity , 12. In UGT1A and DPYD polymorphisms in predicting the occurrence of treatment-induced toxicity, clinical response and survival in China. Because of regional ethnic diversity, the genotype distribution differs in various parts of China. Based on the genotype frequency distribution in Chinese and other Asian patients from previous studies [UGT1A1*6, UGT1A1*27 (c.686C > A), UGT1A1*28, UGT1A7*2 (c.387 T > G), UGT1A7*3 , UGT1A7*4 (c.622 T > C), UGT1A9*22 (−118 T9 > T10), DPYD*5 (c.1627A > G), DPYD*2A (c.1905 + 1G > A), and DPYD c.1896 T > C. The relationship of each genotype to the risk of treatment-induced toxicities, response rate and overall survival are explored here. These findings may be used to establish a new panel, that would be more efficient in predicting treatment-induced toxicity or efficacy in China.This study was designed to evaluate the combinations of  studies –16, the A total of 2783 colorectal cancer patients who received chemotherapy at Peking University Cancer Hospital between January 2007 and June 2016 were screened for this retrospective study. Patients eligible for the study met the following criteria: histologically confirmed adenocarcinoma of the colorectum, stage IV disease, they received at least 2 cycles of irinotecan-based chemotherapy unless intolerable toxicity or disease progression occurred, they had peripheral blood samples taken, and complete clinical information was available for toxicity and efficacy evaluation. Patients were excluded from the study based on the following criteria: they received irinotecan-based chemotherapy for adjuvant treatment, and they did not have toxicity and efficacy information available for evaluation. The screening process is shown in Fig. All patients provided written informed consent for their peripheral blood to be used in this research. This study was approved by the Medical Ethics Committee of Peking University Cancer Hospital and was performed according to the principles of the Declaration of Helsinki.n = 71, irinotecan dosage, 180 mg/m2), irinotecan combined with fluorouracil  or plus target treatment  and FOLFOXIRI . Each patient received at least 2 cycles of irinotecan-based chemotherapy, unless the patients suffered disease progression or intolerable toxicity. Routine blood tests and an evaluation of adverse events were performed after each administration of irinotecan or before the initiation of the next chemotherapy.Before patients received the irinotecan-based chemotherapy, routine blood tests of hepatic and renal function and performance status evaluation of each patient were performed and considered to be essential. The regimens in this study included irinotecanalone or combined with target treatment . Grade 3 or 4 neutropenia and diarrhea were defined as severe toxicity.Toxicity was evaluated based on the medical records according to the National Cancer Institute Common Toxicity Criteria for Adverse Events, Version 4.0  according to the response evaluation criteria in solid tumors (RECIST) . All of UGT1A  and DPYD  were amplified by polymerase chain reaction (PCR). All primers are shown in Table UGT1A1*27 and DPYD*5 were 95 °C for 5 min, 45 cycles of 95 °C for 10 s, 56 °C for 45 s and 72 °C for 20s, and a final extension at 72 °C for 10 min, and a final 4 °C for 10 min. The PCR products were indentified by 2% agarose gel electrophoresis and sequenced using an Invitrogen 3730XL genetic analyzer. The sequencing results were analyzed using Chromas software.Two-milliliter peripheral blood samples were acquired from metastasis colorectal cancer patients before receiving treatment and stored at −80 °C. The genomic DNA samples were extracted from these blood samples using QLAamp Blood Kit . The fragments of P values <0.05 were considered to be statistically significant.Differences between UGT1A and DPYD variants and severe irinotecan-induced toxicity were analyzed using the chi-square and Fisher’s exact tests. The association of genotypes with risk of severe irinotecan induced adverse events was assessed using logistic models. The Back-wald method of multivariate analysis model was used to avoid possible interactions. Survival curves were analyzed using the Kaplan-Meier method and compared by the log-rank test. All analyses were carried out using SPSS version 22.0 . The predictive powers of genotypes were recorded using Odds Ratios(ORs) and 95% confidence internals (CIs). All statistical analyses were two-sided testsand UGT1A and DPYD are shown in the Additional file n = 59) and 20.6% (n = 136), respectively. During the follow-up, 512 patients had disease progression and 346 patients were dead.There were 661 mCRC patients who were finally enrolled in this study  only finished an examination of UGT1A1*6 and UGT1A1*28, because of examination failure and sample depletion. The genotypes are shown in Table Among all of the patients, 49 of 71 patients who received single irinotecan-based chemotherapy had all of the UGT1A and DPYD polymorphisms were included in the analysis . UGT1A1*28 heterozygotes , UGT1A1*28 homozygotes  and UGT1A1*6 homozygotes  were the independent predictive factors of severe neutropenia.In this retrospective study, sex, age, primary tumor location , chemothis Table . Two locUGT1A1*6 and UGT1A1*28 were found to be more likely to suffered severe toxicity (P = 0.001), especially severe neutropenia (P < 0.001). The predictive sensitivity and specificity of UGT1A polymorphisms were 32.4% and 53.1%, respectively. Of the patients who received irinotecan plus fluorouracil-based chemotherapy, we analyzed the severe toxicity risk based on UGT1A1*6/*28 and DPYD*5 panels. More mutational alleles of UGT1A1*6/*28 and DPYD*5 were also revealed to had increased incidence of severe neutropenia (P = 0.008). And patients with ≧3 mutation alleles had higher risk of suffering severe diarrhea, with the incidence of 15.9%, but without significant P value. The predictive sensitivity and specificity of UGT1A*6/*28 and DPYD*5 panels were 33.1% and 85.3%, respectively  nor UGT1A/DPYD polymorphisms were related to the disease control rate (DCR) in any line of treatment  was 32.3% (30/93). For the second-line treatment or further, 22 patients could not evaluate efficacy due to stopping chemotherapy for intolerable toxicity. The objective rate was 12.2% (66/541). Of the patients who received irinotecan plus fluorouracil-based chemotherapy as the first-line treatment, DPYD*5 mutation contributed to better PFS than wild type .Of the patients who received the first-line irinotecan-based chemotherapy, the median PFS was 7.00 months . In this cohort, the incidence of severe diarrhea and neutropenia was 8.9% and 20.8%. These were consistent with the previously reported results at the same center . ClinicaUGT1A genotype frequency and the effect on treatment-induced toxicity varied across ethnic groups. Early in 2005, UGT1A1*28 was recognized as a risk factor for irinotecan induced toxicities by the U.S Food and Drug Administration (FDA). In Asia, however, UGT1A1*28 were not applicable to the prediction of irinotecan-induced toxicity because of its low frequency. In this study, the genotype frequency of UGT1A1*6 and UGT1A1*28 were similar to previous reports in Asia [UGT1A1*6 and UGT1A1*28 related to G3–4 neutropenia, rather than delayed diarrhea, which was consistent with several large-sample analysis in Asia [UGT1A1*28 and UGT1A1*6 could predict severe irinotecan-induced severe diarrhea [UGT1A1*6 and UGT1A1*28 were included in this analysis. Although individual UGT1A1*6 and UGT1A1*28 did not show a relationship with severe diarrhea neutropenia, the correlation of UGT1A1*6 and UGT1A1*28 revealed a significant association with severe neutropenia. Correlation of UGT1A1*6 and UGT1A1*28 also showed the same results in this study. In Thai patients, the UGT1A1*28 mutation frequency was nearly the same with Chinese patients (22.8% vs. 24.2%), while the UGT1A1*6 mutation frequency was lower than in Chinese patients (15.9% vs. 34.8%) [UGT1A1*27 is a genotype only in Asians with lower UGT enzyme activity and low frequency [UGT1A1*27 heterozygotes in this cohort, but only 2 patients suffered severe neutropenia. The incidence of severe toxicity was much lower than in previous reports [UGT1A9*22 was 81.8% in this study, which was similar to findings reported in Japan. However, the UGT1A9*22 homozygotes in China were much rarer than in Japan (0.7% vs. 34.7%) [UGT1A9*22 did not show an association with irinotecan-induced toxicity in this study. It has previously been reported that UGT1A9*22 variants have a lower risk of suffering irinotecan-induced severe neutropenia [UGT1A7*2/*3 frequency to Japanese patients, but a lower frequency than Greeks [UGT1A7*3 is associated with higher risk of suffering severe neutropenia [UGT1A7*3 to be related to severe diarrhea [UGT1A7*3 had a significant ability to predict severe neutropenia in univariate analysis, but the relationship did not appear to be significant in multivariate analysis. UGT1A7*3 was not an independent biomarker in the prediction of irinotecan-induced toxicity for Chinese patients. Among the patients who received targeted drugs, only UGT1A7*3 was found to be associated with a higher risk of G3–4 neutropenia incidence. Targeted drug treatment might affect the predictability of the toxicity of UGT1A polymorphisms. Regimens with different targeted drugs might also affect evaluation of toxicity. The influence of targeted drugs on the relationship between UGT1A polymorphisms and toxicity should be further studied. Finally, the results showed that UGT1A1*6 and UGT1A1*28 had an association with irinotecan-induced severe neutropenia. Patients with more mutant variants had a higher risk of suffering severe neutropenia; however, no other significant loci of UGT1A polymorphisms were found to set up a new panel to better indicate irinotecan-induced toxicity. in Asia . Severaldiarrhea , 22, whi. 34.8%) . The dif reports . The gen. 34.7%) . UGT1A9*tropenia , 26. Chitropenia . TziotouDPYD polymorphisms influenced the activity of dihydropyrimidine dehydrogenase (DPD) considerably, which was associated with fluorouracil’s metabolism and ethnic variation also appeared in DPYD polymorphisms [DPYD*2A mutant variants contribute to a higher risk of severe toxicity [DPYD*2A is rarely found. This was consistent with the findings of this study. Only 1 DPYD*2A heterozygote (0.2%) was found in this analysis. The ability of DPYD*2A to indicate fluorouracil-induced toxicity in China could not be assessed. DPYD*5 and DPYD c.1896 T > C had allele frequencies of 28.4% and 10.7%, respectively, which was consistent with previous reports [DPYD*5 associated with higher risk of severe diarrhea. However, in Zhang XP et al. and Yamauchi et al.’s study, DPYD*5 related to the incidence of severe neutropenia [DPYD c.1896 T > C independently predicted severe fluorouracil-induced toxicity, which did not happen in this analysis [UGT1A1*6, UGT1A1*28 and DPYD*5 was found to improve the predictive specificity for toxicity compared with an examination of UGT1A1*6 and UGT1A1*28 (53.1% vs. 85.6%) among patients receiving irinotecan plus fluorouracil-based chemotherapy.Fluorouracil is generally combined with irinotecan. orphisms , 16, 30. reports . In thistropenia , 32. In UGT1A and DPYD polymorphisms and clinical outcomes were analyzed, as well as the toxicity. The response rate and survival varied across different treatment lines. Among patients who received irinotecan-based chemotherapy as a first-line treatment, this analysis first noted that UGT1A1*27 contributed to worse OS than wild type variants, although the number of analyzed samples was small. Moreover, UGT1A1*28 contributed to a higher objective response rate, which was consistent with studies reported by Fujita and Toffoli G’s team [UGT1A1*28 led to bad clinical outcomes [UGT1A gene polymorphisms were found to have only a limited ability to predict survival, and multiple chemotherapy regimens might also be involved. Unlike UGT1A, there have only been limited studies assessing the relationship between DPYD polymorphisms and survival. In this analysis, DPYD*5 mutant variants predicted better PFS in the first-line treatment of irinotecan plus fluorouracil-based regimens, and DPYD polymorphisms were not found to associate with overall survival.The association between G’s team , 33. Whioutcomes . This miUGT1A and DPYD polymorphisms. Although it was not possible to establish a new panel to improve the predictability of toxicity in this study, the analysis showed that more attention should be paid to homozygote of UGT1A1*6in the context of irinotecan-induced severe neutropenia, such as constantly monitoring the levels of neutrophile granulocytes and preventive treatment for neutropenia. For this reason, further studies should focus on polymorphisms of other genes related to irinotecan metabolism.Because this study was a retrospective analysis, bias was unavoidable. The value of this study relies on the large samples’ combined examination for UGT1A1*6 and UGT1A1*28 variants were associated with irinotecan-induced neutropenia, but not with diarrhea. A combined examination of UGT1A1*6, UGT1A1*28 and DPYD*5 were found to improve the predictive specificity of toxicity. UGT1A and DPYD polymorphisms were still limited to the prediction of clinical response. A combined examination of more UGT1A polymorphisms will not be helpful in improving predictive value of irinotecan-induced toxicity.In brief, only


ATP) functions as a metabo-electric transducer in regulating insulin secretion from pancreatic β-cells. The pancreatic KATP channel is composed of a pore-forming inwardly-rectifying potassium channel, Kir6.2, and a regulatory subunit, sulphonylurea receptor 1 (SUR1). Loss-of-function mutations in either subunit often lead to the development of persistent hyperinsulinemic hypoglycemia of infancy (PHHI). PHHI is a rare genetic disease and most patients present with immediate onset within the first few days after birth. In this study, we report an unusual form of PHHI, in which the index patient developed hyperinsulinemic hypoglycemia after 1 year of age. The patient failed to respond to routine medication for PHHI and underwent a complete pancreatectomy. Genotyping of the index patient and his immediate family members showed that the patient and other family members with hypoglycemic episodes carried a heterozygous novel mutation in KCNJ11 (C83T), which encodes Kir6.2 (A28V). Electrophysiological and cell biological experiments revealed that A28V hKir6.2 is a dominant-negative, loss-of-function mutation and that KATP channels carrying this mutation failed to reach the cell surface. De novo protein structure prediction indicated that this A28V mutation reoriented the ER retention motif located at the C-terminal of the hKir6.2, and this result may explain the trafficking defect caused by this point mutation. Our study is the first report of a novel form of late-onset PHHI that is caused by a dominant mutation in KCNJ11 and exhibits a defect in proper surface expression of Kir6.2.The ATP-sensitive potassium channel (K KCNJ11, ABCC8, GLUD1, and GCK are the most common factors that lead to the development of PHHI.Persistent hyperinsulinemic hypoglycemia of infancy, or PHHI, is the most common cause of severe neonatal hypoglycemia that lasts beyond the first a few hours of life.KCNJ11 and ABCC8 genes encode the inwardly-rectifying potassium channel 6.2 (Kir6.2) and the sulphonylurea receptor 1 (SUR1), respectively, and co-assembly of four Kir6.2 with four SUR1 subunits forms a functional ATP-sensitive potassium channel (KATP channel).ATP channel via direct interaction with Kir6.2, the pore-forming subunit of the channel. While most excitable cells, including pancreatic β-cells, neurons, cardiac myocytes and skeletal muscles, have Kir6.2 as the KATP channel pore forming subunit, vascular smooth muscle cells have Kir6.1 instead.ATP channel openers.ATP channel functions as a metabo-electric transducer, since its gating is regulated by the intracellular metabolites such as ATP, long-chain fatty acid-CoA, and phosphatidylinositol-4,5-bisphosphate (PIP2).ATP channels are involved in multiple physiological processes, it is not surprising that mutations in either KCNJ11 or ABCC8 can cause a variety of diseases, ranging from diabetes and PHHI to epilepsy, mental retardation (DEND syndrome) and cardiac myopathies.i). Elevated [ATP]i closes the KATP channels, which depolarizes the β-cell membrane potentials and subsequently opens the voltage-gated Ca2+ channels. The resulting influx of Ca2+ into the cytosol ultimately triggers insulin secretion via exocytosis.ATP maintains the resting membrane potentials of the pancreatic β-cells, loss-of-function in either of the KATP channel subunits may lead to aberrant depolarization of β-cells and excessive insulin release.ABCC8, most of the PHHI causing mutations in KCNJ11 are recessive, requiring both parents to be carriers.KCNJ11 that codes for a Val substitution for Ala at position 28 of the Kir6.2 peptide chain (A28V hKir6.2). The index patient, carrying a maternally inherited A28V hKir6.2, developed diazoxide-nonresponsive, late-onset PHHI requiring a total pancreatectomy. Immediate family members who carry the same heterozygous mutation have also experienced various degrees of hyperinsulinemic hypoglycemic symptomology. Moreover, both the patient and one of his affected siblings developed primary hypopituitarism. Electrophysiological and cell biological studies reveal that the A28V hKir6.2 mutation produces only minuscule KATP currents due to a trafficking defect that prevents surface expression. Our results identify a novel KATP channel defect that causes PHHI and provides additional evidence that the N-terminus of Kir6.2 is involved in KATP channel trafficking.Metabolism of glucose by the pancreatic β-cells rapidly increase intracellular ATP  that encodes an Ala to Val substitution at amino acid 28 in the Kir6.2 protein in the proband. The patient's mother and sister carry the same A28V Kir6.2 mutation  subunit. Prior to the trafficking from the endoplasmic reticulum (ER) to the plasma membrane via the Golgi complex, functional KATP channels must be co-assembled to mask strong ER-retention signals in Kir6.2 and SUR1 subunits.ATP channels in the presence of Kir6.2 mutants. In the HEK cells transfected with wild-type hKir6.2 and HA-rSUR1, a very strong surface staining pattern was detected  mutation, suggesting that A28V hKir6.2 is a dominant-negative mutation that can suppress normal KATP channel trafficking. To control the expression ratio of wild-type and hKir6.2 (A28V) in the HEK293 cells, we generated a construct to express wide-type and A28V hKir6.2 in tandem, with the two sequences linked by a self-cleaving P2A peptide linker. P2A encodes an RNA skipping signal, so during translation, the ribosome produces a gap in the nascent polypeptide. Thus the two peptide sequences that are linked by the P2A are “self-cleaved” at the P2A sequence and yield two physically separate peptides.ATP channel trafficking to the plasma membrane. Our results showed that cells transfected with the wt-P2A-wt hKir6.2 tandem construct exhibited strong surface signals in HEK293 cells, while those transfected with the A28V-P2A-A28V hKir6.2 construct showed greatly reduced surface signals  ER-retention motifructures .18 As ilructures . Alanineructures . Howeverructures  revealedructures , E and Hructures , J and Kructures .Figure KCNJ11, the gene that encodes the pore-forming subunit of the KATP channels, hKir6.2. Patients that carry one copy of this KCNJ11(C83T) mutation exhibited various symptoms, ranging from a mild PHHI to hypothalamic deficiency.In this study, we have identified the first dominant-negative mutation in ATP channel in the closed conformation provide detailed structural and mechanistic insights into KATP channel assembly and gating.ATP channel trafficking have yet to be determined, as these trafficking domains are highly dynamic structures. The predicted full-length hKir6.2 structure indicated that the A28 is located on the loosely-packed N-terminal loop; Ala to Val not only re-organizes the N-terminal loop but also distortes the entire C-terminal domain  with many of these strongly affect glucose homeostasis.KCNJ11 can lead to either hyper- or hypo-insulin secretion, depending on their nature.ABCC8 mostly result in hyperinsulinemia.i.and Kir6.2 mutations located near the ATP binding pocket often render the KATP channel less sensitive to [ATP]i.ATP channels is a critical link in the pathway by which glucose stimulates insulin secretion, ATP-insensitive KATP channels that remain open keep pancreatic β-cells hyperpolarized even at elevated blood glucose levels. Consequently, glucose fails to stimulate insulin secretion, leading to the development of diabetes.Multiple mutations have been identified in both genes encoding KATP channels are depolarized, and continue to secrete insulin at low glucose concentration.ATP channels in the open state, even in the presence of physiological [ATP]i that are up to an order of magnitude higher than the normal IC50 of [ATP]i.ATP channelopathy: (1) A28V hKir6.2 is a maternally-inherited dominant negative mutation. The index patient and his immediate family members who carry one copy of the same A28V mutation all developed hypoglycemia. (2) KATP channels containing A28V hKir6.2 remain functional if the channel reaches the cell membrane .ATP channel trafficking. Patients carrying this mutation developed a novel form of PHHI and hypopituitarism. With the rapid improvement of personalized medicine, we expect novel therapeutic strategies can be developed to treat patients with this KATP channel trafficking defect.In summary, we have identified a novel form of late-onset, dominant-negative A28V hKir6.2 mutant that impairs K1272LHRELSAGLVYPYDVPDYAHRELSAGLVGLG1284at the site of the epitope insertion.2 at 37°C. Cells were plated on poly L-ornithine-coated glass coverslips and transiently transfected with 0.2 μg of the pcDNA3 containing hKir6.2 construct and 0.8 μg of pcDNA3 containing SUR1 construct by using FuGENE 6 . Cells were used 2–4 days after transfection. KATP currents were recorded using the whole-cell patch-clamp configuration by an Axopatch 700B amplifier  in a standard extracellular solution containing: 150 mM NaCl, 10 mM HEPES, 5 mM KCl, 2 mM CaCl2, 1 mM MgCl2 and pH 7.2, adjusted with NaOH.2ATP, 1 mM Na3GTP, 10 mM sodium phosphocreatine, 0.05 mM EGTA and pH 7.2, adjusted with KOH. The access resistances of whole-cell recording ranged between 5 and 20 MΩ and were compensated by ∼80%. All experiments were performed at room temperature (∼25°C). For all studies, all chemicals were purchased from Sigma-Aldrich (USA) if not stated otherwise.Human Kir6.2 (hKir6.2), SUR1 (hSUR1)  and rodent (hamster) SUR1 (rSUR1) cDNAs were cloned into the pcDNA3 plasmid. The HA-tagged rSUR1 was generated previously and was kindly provided as a gift from Dr. Lily Jan at the University of California, San Francisco, USA. The HA-epitope was inserted between the sixteenth and seventeenth transmembrane domains of the SUR1 subunit and the sequence of the HA-tag reads asXenopus laevis oocytes were harvested, injected with 30 ng of each cRNA, and incubated at 16°C for 2–4 days before recording. hKir6.2 and hSUR1 were cloned into the pGEM vector. hKir6.2 and hSUR1 cDNAs were first linearized by restriction enzyme digestion, and cRNA was then synthesized by using the mMESSAGE mMACHINE SP6 Transcription kit . Macroscopic currents were recorded from oocytes with two-electrode voltage clamp . Electrodes were filled with 3 M KCl and had a resistance between 0.4–1 MΩ. A small chamber with a fast perfusion system  was used to change extracellular ND96 solution containing: 96 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES and pH 7.4, adjusted with NaOH .Animal protocols used in this study were approved by the IACUC at Academia Sinica or University of California, San Francisco, and Xenopus oocytes were prepared as described previously.ATP channels, HEK cells were first transfected with hKir6.2 and an HA-tagged rSUR1. Three days post transfection, cells were incubated in the blocking solution  for 10 min at 4°C and then incubated with rat anti-HA antibody  in the blocking solution for 30 min at 4°C to minimize endocytosis. After washing four times with PBS for 5 min each at 4°C, cells were incubated for 30 min with goat anti-rat Alexa488 secondary antibody  then washed three times with PBS for 5 min each at 4°C. Next, the cells were fixed in the 4% paraformaldehyde/4% sucrose in PBS for 10 min at room temperature then washed three times with PBS for 5 min each. Fixed cells were permeabilized by incubating in blocking solution that contained 0.1% Triton-X-100 for 15 min. Then cells were incubated with the same rat anti-HA antibody (1:200) in the blocking solution for 30 min at room temperature. After washing four times with PBS for 5 min each at room temperature, cells were incubated for 30 min with goat anti-rat Alexa555 secondary antibody , then washed three times with PBS for 5 min each at room temperature. The coverslips were mounted using Fluoromount G mounting medium containing DAPI  and images were acquired using a confocal microscope .For non-permeabilized immunostaining of surface Kde novo structure prediction. TM-Align was used to align wild-type and A28V hKir6.2.The structures of the wild-type and A28V hKir6.2 were predicted by I-TASSERp < 0.05 .Imaging data from the immunostained cells were analyzed using ZEN software . Electrophysiology Data were analyzed with pClamp10 software . Results are reported as mean ± SEM. Statistical analysis was performed using Prism 5 , with differences considered significant at