TRPM2 channel deficiency prevents delayed cytosolic Zn2+ accumulation and CA1 pyramidal neuronal death after transient global ischemia.

Transient ischemia is a leading cause of cognitive dysfunction. Postischemic ROS generation and an increase in the cytosolic Zn(2+) level ([Zn(2+)]c) are critical in delayed CA1 pyramidal neuronal death, but the underlying mechanisms are not fully understood. Here we investigated the role of ROS-sensitive TRPM2 (transient receptor potential melastatin-related 2) channel. Using in vivo and in vitro models of ischemia-reperfusion, we showed that genetic knockout of TRPM2 strongly prohibited the delayed increase in the [Zn(2+)]c, ROS generation, CA1 pyramidal neuronal death and postischemic memory impairment. Time-lapse imaging revealed that TRPM2 deficiency had no effect on the ischemia-induced increase in the [Zn(2+)]c but abolished the cytosolic Zn(2+) accumulation during reperfusion as well as ROS-elicited increases in the [Zn(2+)]c. These results provide the first evidence to show a critical role for TRPM2 channel activation during reperfusion in the delayed increase in the [Zn(2+)]c and CA1 pyramidal neuronal death and identify TRPM2 as a key molecule signaling ROS generation to postischemic brain injury.

Trafficking of ATP-sensitive potassium channels in health and disease.

K(ATP) channels (ATP-sensitive potassium channels), comprising four subunits each of Kir6.2 (inwardly rectifying potassium channel 6.2) and the SUR1 (sulfonylurea receptor 1), play a central role in glucose-stimulated insulin secretion by the pancreatic beta-cell. Changes in the number of channels at the cell surface are associated with genetic diseases of aberrant insulin secretion, including CHI (congenital hyperinsulinism) and NDM (neonatal diabetes mellitus). The present review summarizes advances in our understanding of the vesicular trafficking of normal K(ATP) channels and how genetic mutations in Kir6.2 interfere with such trafficking. A mutation, E282K, causing CHI, was found to disrupt a DXE [di-acidic ER (endoplasmic reticulum)-exit signal], thereby preventing its assembly into COPII (coatamer protein II)-coated vesicles and subsequent ER exit. The resultant decrease in the cell-surface density of the channel could explain the disease phenotype. Two mutations, Y330C and F333I, reported in patients with NDM, disrupted an endocytic traffic signal, thereby impairing CCV (clathrin-coated vesicle) formation and endocytosis. The consequent increase in the density of K(ATP) channels, together with an attenuated sensitivity to ATP reported previously, may account for the severe form of NDM.

Hyperinsulinemic hypoglycemia in Beckwith-Wiedemann syndrome due to defects in the function of pancreatic beta-cell adenosine triphosphate-sensitive potassium channels.

BACKGROUND: Beckwith-Wiedemann syndrome (BWS) is a congenital overgrowth syndrome that is clinically and genetically heterogeneous. Hyperinsulinemic hypoglycemia occurs in about 50% of children with BWS and, in the majority of infants, it resolves spontaneously. However, in a small group of patients the hypoglycemia can be persistent and may require pancreatectomy. The mechanism of persistent hyperinsulinemic hypoglycemia in this group of patients is unclear. PATIENTS AND METHODS: Using patch-clamp techniques on pancreatic tissue obtained at the time of surgery, we investigated the electrophysiological properties of ATP-sensitive K(+) (K(ATP)) channels in pancreatic beta-cells in a patient with BWS and severe medically-unresponsive hyperinsulinemic hypoglycemia. RESULTS: Persistent hyperinsulinism was found to be caused by abnormalities in K(ATP) channels of the pancreatic beta-cell. Immunofluorescence studies using a SUR1 antibody revealed perinuclear pattern of staining in the BWS cells, suggesting a trafficking defect of the SUR1 protein. No mutations were found in the genes ABCC8 and KCNJ11 encoding for the two subunits, SUR1 and KIR6.2, respectively, of the K(ATP) channel. Genetic analysis of this patients BWS showed evidence of mosaic paternal isodisomy. CONCLUSIONS: In this novel case of BWS with mosaic paternal uniparental disomy for 11p15, persistent hyperinsulinism was due to abnormalities in K(ATP) channels of the pancreatic beta-cell. The mechanism/s by which mosaic paternal uniparental disomy for 11p15 causes a trafficking defect in the SUR1 protein of the K(ATP) channel remains to be elucidated.

Functional up-regulation of KCNA gene family expression in murine mesenteric resistance artery smooth muscle.

This study focused on the hypothesis that KCNA genes (which encode K(V)alpha1 voltage-gated K(+) channels) have enhanced functional expression in smooth muscle cells of a primary determinant of peripheral resistance - the small mesenteric artery. Real-time PCR methodology was developed to measure cell type-specific in situ gene expression. Profiles were determined for arterial myocyte expression of RNA species encoding K(V)alpha1 subunits as well as K(V)beta1, K(V)alpha2.1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Ca)beta1. The seven major KCNA genes were expressed and more readily detected in endothelium-denuded mesenteric resistance artery compared with thoracic aorta; quantification revealed dramatic differential expression of one to two orders of magnitude. There was also four times more RNA encoding K(V)alpha2.1 but less or similar amounts encoding K(V)beta1, K(V)gamma9.3, BK(Ca)alpha1 and BK(Cabeta)1. Patch-clamp recordings from freshly isolated smooth muscle cells revealed dominant K(V)alpha1 K(+) current and current density twice as large in mesenteric cells. Therefore, we suggest the increased RNA production of the resistance artery impacts on physiological function, although there is quantitatively less K(+) current than might be expected. The mechanism conferring up-regulated expression of KCNA genes may be common to all the gene family and play a functional role in the physiological control of blood pressure.

Two pore residues mediate acidosis-induced enhancement of C-type inactivation of the Kv1.4 K(+) channel.

Acidosis inhibits current through the Kv1.4 K(+) channel, perhaps as a result of enhancement of C-type inactivation. The mechanism of action of acidosis on C-type inactivation has been studied. A mutant Kv1.4 channel that lacks N-type inactivation (fKv1.4 Delta2-146) was expressed in Xenopus oocytes, and currents were recorded using two-microelectrode voltage clamp. Acidosis increased fKv1.4 Delta2-146 C-type inactivation. Replacement of a pore histidine with cysteine (H508C) abolished the increase. Application of positively charged thiol-specific methanethiosulfonate to fKv1.4 Delta2-146 H508C increased C-type inactivation, mimicking the effect of acidosis. Replacement of a pore lysine with cysteine (K532C) abolished the acidosis-induced increase of C-type inactivation. A model of the Kv1.4 pore, based on the crystal structure of KcsA, shows that H508 and K532 lie close together. It is suggested that the acidosis-induced increase of C-type inactivation involves the charge on H508 and K532.

Identification and pharmacological correction of a membrane trafficking defect associated with a mutation in the sulfonylurea receptor causing familial hyperinsulinism.

Persistent hyperinsulinemic hypoglycemia of infancy (PHHI) is a genetic disorder characterized by excess secretion of insulin and hypoglycemia. In most patients, the disease is caused by mutations in sulfonylurea receptor-1 (SUR1), which, in association with Kir6.2, constitutes the functional ATP-sensitive potassium (K(ATP)) channel of the pancreatic beta-cell. Previous studies reported that coexpression of the PHHI mutant R1394H-SUR1 with Kir6.2 in COS cells produces no functional channels. To investigate if the loss of function could be due to impaired trafficking of mutant channels to the cell membrane, we have cotransfected wild-type and mutant SUR1 subunits with Kir6.2 into HEK293 cells and examined their cellular localization by immunofluorescent staining. Our results show that unlike the wild-type subunits, which showed fluorescence at the cell surface, the mutant subunits displayed fluorescence in punctate structures. Co-immunostaining with antibodies against organelle-specific marker proteins identified these structures as the trans-Golgi network. Limited localization in clathrin-positive, but transferrin receptor-negative vesicles was also observed. The post-endoplasmic reticulum localization suggests that the mutation does not impair the folding and assembly of the channels so as to cause its retention by the endoplasmic reticulum. Diazoxide, a K(ATP) channel opener drug that is used in the treatment of PHHI, restored the surface expression in a manner that could be prevented by the channel blocker glibenclamide. When expressed in Xenopus oocytes, R1394H-SUR1 formed functional channels with Kir6.2, indicating that the primary consequence of the mutation is impairment of trafficking rather than function. Thus, our data uncover a novel mechanism underlying the therapeutic action of diazoxide in the treatment of PHHI, i.e. its ability to recruit channels to the membrane. Furthermore, this is the first report to describe a trafficking disorder effecting retention of mutant proteins in the trans-Golgi network.

Immunolocalisation of adenosine A(1) receptors in the rat kidney.

The location of adenosine A(1) receptors in the rat kidney was investigated using immunolabelling with antibodies raised to a 15-amino-acid sequence near the C-terminus of the receptor (antibody I) and to a 14-amino-acid sequence in the second extracellular loop (antibody II). In the cortex, antibody I bound to adenosine A(1) receptors in mesangial cells and afferent arterioles, whilst antibody II bound to receptors in proximal convoluted tubules. In the medulla, both antibodies bound to receptors in collecting ducts and the papillary surface epithelium. These observations provide support for the diverse functional roles previously proposed for the adenosine A(1) receptor in the kidney. The labelling of distinct but different structures in the cortex by antibodies raised to different amino acid sequences on the A(1) receptor protein suggests that differing forms of the receptor are present in this region of the kidney.

Identification of molecular regions responsible for the membrane trafficking of Kir6.2.

The subunits of the pancreatic ATP-sensitive potassium channel Kir6.2 and the sulphonylurea receptor (SUR1) contain endoplasmic reticulum (ER) retention signals (RKR), which prevent their plasma membrane expression when expressed individually. When co-expressed, however, these signals are masked and the complex traffics to the plasma membrane. To investigate this further, we have expressed epitope-tagged chimaeras between Kir6.2 and Kir2.1 (which traffics to the membrane independently of SUR1) in Xenopus oocytes alone and together with SUR1. By staining sections of the oocytes, we show that, in addition to the ER retention signal present in the distal C-terminus, the M2 transmembrane and the proximal C-terminal regions also contribute to the inability of Kir6.2 to traffic to the membrane in the absence of SUR1. Furthermore, by staining the whole oocytes for the hexa-histidine tag attached to the N-terminus of SUR1, we provide direct experimental evidence that the N-terminus of SUR1 is extracellular.

Inhibition of the K+ channel kv1.4 by acidosis: protonation of an extracellular histidine slows the recovery from N-type inactivation.

1. Acidosis alters the transient outward current, ito, in the heart. We have studied the mechanism underlying the effect of acidosis on one of the K+ channels, Kv1.4 (heterologously expressed in Xenopus laevis oocytes), known to underlie ito. 2. At pH 6.5, wild-type Kv1.4 current was inhibited during repetitive pulsing, in part as a result of a slowing of recovery from N-type inactivation. 3. Acidosis still caused slowing of recovery after deletion of just one (either the first or second) of the N-terminal inactivation ball domains. However, deletion of both the N-terminal inactivation ball domains greatly reduced the inhibition. 4. As well as the N-terminus, other parts of the channel are also required for the effect of acidosis, because, whereas the transfer of the N-terminus of Kv1.4 to Kv1.2 conferred N-type inactivation, it did not confer acidosis sensitivity. 5. Replacement of an extracellular histidine with a glutamine residue (H508Q) abolished the slowing of recovery by acidosis. Reduction of C-type inactivation by raising the bathing K+ concentration or by the mutation K532Y also abolished the slowing. 6. It is concluded that binding of protons to H508 enhances C-type inactivation and this causes a slowing of recovery from N-type inactivation and, thus, an inhibition of current during repetitive pulsing.

Effect of cysteine substitutions on the topology of the S4 segment of the Shaker potassium channel: implications for molecular models of gating.

1. The gating properties of voltage-gated potassium channels are largely determined by the amino acid sequence of their S4 segments. To investigate the nature of S4 movement during gating, we introduced single cysteines into the S4 segment of the Shaker potassium channel and expressed the mutants in Xenopus oocytes. We then measured the conductance-voltage (g-V) relationships and the rate and the voltage dependence of movement of the engineered cysteines, using p-chloromercuribenzene sulphonate (pCMBS) as a probe. 2. Mutation of charged residues at positions 362, 365 and 368, but not the uncharged residues (positions 360, 361, 363, 364 and 366), to cysteines shifted the g-V relationships to more positive potentials. Mutant channels in which cysteines replaced the charged residues at positions 362 and 365 (R362C and R365C) reacted faster with pCMBS than those in which cysteines were introduced in place of uncharged residues at positions 360 and 361 (I360C and L361C). Furthermore, the R365C mutant channel reacted with pCMBS even at hyperpolarised (-120 mV) potentials. Currents expressed by the doubly mutated R365S/V367C and R368S/V367C channels, but not the singly mutated V367C channel, were inhibited by pCMBS. Moreover, the R368C mutant channel was also affected by pCMBS. 3. Voltage dependence of block by pCMBS (2 min exposure) was steeper for L366C than for L361C and V363C mutant channels (effective charge 2.19, 1. 41 and 1.45, respectively). The voltage dependence of the pCMBS effect was also shifted to more depolarising potentials the deeper in the membrane the position of the residue mutated to cysteine (voltages for half-maximal effect -107, -94 and -73 mV for positions 361, 363 and 366, respectively). 4. Our data show firstly that charge-neutralising mutations in S4 alter the topology of this region such that the membrane-spanning portion of S4 is reduced. Secondly, our data for the other mutant channels suggest that S4 might move in at least two sequential steps, and can move up to its maximal limit even at the resting potential of the cell.

The effects of oxidizing and cysteine-reactive reagents on the inward rectifier potassium channels Kir2.3 and Kir1.1.

The inwardly rectifying potassium channel Kir2.3 possesses extracellular cysteine residues at positions 113, 140, and 145, as well as at position 79 near the outer membrane boundary. In this study, we have investigated the roles of these extracellular cysteine residues in mediating inhibition of the Kir2.3 channel by the cysteine-reactive reagents para-chloromercuribenzenesulphonate (PCMBS) and thimerosal, and the oxidizing agent hydrogen peroxide (H2O2). We have also compared the effects of these reagents with those on Kir1.1 channels (which do not possess cysteine residues equivalent to 140 and 79 in Kir2.3 channels). Mutant channels were made in which cysteine residues were mutated to serine by site-directed mutagenesis. Wild-type or mutant cRNA was injected into Xenopus oocytes and voltage-clamp recordings made 1-2 days later. Wild-type Kir2.3 currents were significantly inhibited by PCMBS, thimerosal and H2O2. Currents for mutants Kir2.3 C79S and C140S were also inhibited by PCMBS, thimerosal and H2O2. These mutations affected the time course of inhibition by all three reagents. For PCMBS, a slow component of inhibition was absent for the C79S mutation, and a fast component was absent for C140S. For the double mutation C79S/C140S, PCMBS no longer had any effect. For thimerosal, there was a slower time course for C140S, a faster time course for C79S, and a delayed onset for C79S/C140S. For H2O2, the main effect was a delayed onset for the double mutant. The reducing agent dithiothreitol (DTT) reversed the inhibition by both PCMBS and thimerosal of wild-type and mutant currents, but not the inhibition due to H2O2. Finally, wild-type Kir1.1 currents were not significantly inhibited by the applications of either PCMBS or thimerosal, while H2O2 produced small inhibition. The results taken together indicate that inhibition by the cysteine-reactive reagent PCMBS is mediated through cysteine residues 79 and 140 in Kir2.3 channels, with C79 mediating a slow component of inhibition and C140 a faster component, and that both residues are extracellularly exposed. The data indicate that these two cysteine residues are also main sites for inhibition by thimerosal and H2O2 but, unlike for PCMBS, an additional non-extracellular inhibitory site(s) must also be involved.

Conserved extracellular cysteine residues in the inwardly rectifying potassium channel Kir2.3 are required for function but not expression in the membrane.

The mouse potassium channel Kir2.3 possesses conserved extracellular cysteine residues at positions 113 and 145. We have investigated the role of these cysteines in structure/function and membrane trafficking. Cysteine to serine mutations resulted in the absence of potassium currents in oocytes and co-expression of these mutants with wild-type channel showed a dominant negative inhibition of wild-type currents. FLAG-tagged channels expressed in oocytes were detected in the cell membrane by anti-FLAG antibody for wild-type and mutant channels. In vitro translation using the reticulocyte lysate system showed that mutation of these residues did not affect processing nor insertion into membranes. Cysteine residues at 113 and 145 are therefore required for function of the Kir2.3 channel but not for processing into the cell membrane; disulfide bonds between subunits are unlikely.

Block of human aorta Kir6.1 by the vascular KATP channel inhibitor U37883A.

1. A human aorta cDNA library was screened at low stringency with a rat pancreatic Kir6.1 cDNA probe and a homologue of Kir6.1 (hKir6.1) was isolated and sequenced. 2. Metabolic poisoning of Xenopus laevis oocytes with sodium azide and application of the K+ channel opener drug diazoxide induced K+ channel currents in oocytes co-injected with cRNA for hKir6.1 and hamster sulphonylurea receptor (SUR1), but not in oocytes injected with water or cRNA for hKir6.1 or SUR1 alone. 3. K+ channel currents due to hKir6.1+SUR1 or mouse Kir6.2+SUR1 were strongly inhibited by 1 microM glibenclamide. K+-current carried by hKir6.1+SUR1 was inhibited by the putative vascular-selective KATP channel inhibitor U37883A (IC50 32 microM) whereas current carried by Kir6.2+SUR1 or Shaker K+ channels was unaffected. 4. The data support the hypothesis that hKir6.1 is a component of the vascular KATP channel, although the lower sensitivity of hKir6.1+SUR1 to U37883A compared with native vascular tissues suggests the need for another factor or subunit. Furthermore, the data suggest that pharmacology of KATP channels can be determined by the pore-forming subunit as well as the sulphonylurea receptor and point to a molecular basis for the pharmacological distinction between vascular and pancreatic/cardiac KATP channels.

Expression, characterization and engineered specificity of rat epididymal retinoic acid-binding protein.

Epididymal retinoic acid-binding protein (ERABP) is the major androgen-dependent protein present in the lumen of the epididymis and is thought to be involved in sperm maturation. It displays a high degree of three-dimensional structural similarity to serum retinol-binding protein (RBP). Although both proteins interact with retinoids, RBP exhibits a broad specificity, binding retinol, retinoic acid and retinaldehyde with roughly equal affinities, whereas ERABP is specific for all-trans- and 9-cis-retinoic acids. Consistent with this, the binding pockets of the two proteins are different: in RBP it is predominantly hydrophobic, whereas that for ERABP is amphipathic, with a network of charged residues at the open end of the binding pocket. In order to investigate the roles of these charged residues, Arg-80 and Glu-63 have been mutated to isoleucine. The resultant double mutant, Glu-63-->Ile/Arg-80-->Ile, as well as the wild-type protein, were subsequently expressed in Escherichia coli as fusion proteins, with the streptavidin recognition sequence (Strep) tagged to their C-termini. The expressed proteins were purified in a single step by streptavidin-affinity chromatography and their ligand-binding properties were examined using fluorimetric titrations. Whereas the wild-type ERABP binds only retinoic acid, the double mutant is capable of binding retinol, retinoic acid and retinaldehyde with similar affinities. These observations provide experimental support for the proposition that the charged residues near the open end of the binding pocket are responsible for restricting the specificity of ERABP for retinoic acid. These studies demonstrate that changes in specificity can be engineered into lipocalins.

Adenosine receptor mRNA levels during postnatal renal maturation in the rat.

Adenosine may affect the pattern of intrarenal blood flow during renal development. It provides an angiogenic stimulus for the growth of new blood vessels and may be involved in compensatory renal growth. It is therefore of interest to investigate the expression of adenosine receptor genes during postnatal renal development. In the present study this was carried out by measuring adenosine receptor mRNA levels in rats aged between 2 and 60 days. The order of abundance of adenosine receptor mRNA levels in 60-day-old rats was A2A > A2B > or = A1 > A3. A1 receptor mRNA levels showed only small changes with increasing age although, by contrast, A3 receptor mRNA increased markedly with age with levels at 60 days twenty-fold greater than at 2 days. A2A receptor mRNA levels declined during renal maturation with transcript numbers four- to fivefold that at 12-18 days compared with numbers at 60 days. By contrast to the A2A receptor, there were no significant changes in the renal levels of A2B receptor mRNA during kidney maturation. During postnatal renal maturation, the levels of mRNA for A2A and A3 adenosine receptor subtypes undergo marked changes which may be related to functional maturation, morphological development, or both.

The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor.

The hypothesis that the cellular uptake of retinol involves the specific interaction of a plasma membrane receptor with serum retinol-binding protein (RBP) at the extracellular surface followed by ligand transfer to cytoplasmic cellular retinol-binding protein (CRBP) has been investigated. The experimental system consisted of the [3H]retinol-RBP complex, Escherichia coli-expressed recombinant apo-CRBP containing the 10 amino acid long streptavidin-binding peptide sequence at its C terminus (designated as CRBP-Strep) and permeabilized human placental membranes. [3H]Retinol transfer from RBP to CRBP-Strep was monitored by measuring the radioactivity associated with CRBP-Strep retained by an immobilized streptavidin resin. Using this assay system, we have demonstrated that optimal retinol uptake is achieved with holo-RBP, the membrane receptor and apo-CRBP. The effects are specific: other binding proteins, including beta-lactoglobulin and serum albumin, despite their ability to bind retinol, failed to substitute for either RBP or apo-CRBP. The process is facilitated by membranes containing the native receptor suggesting that this protein is an important component in the transfer mechanism. Taken together, the data suggest that the RBP receptor, through specific interactions with the binding proteins, participates (either directly or via associated proteins) in the mechanism which mediates the transfer of retinol from extracellular RBP to intracellular CRBP.

Renal adenosine A1 receptor binding characteristics and mRNA levels during the development of acute renal failure in the rat.

1. The binding characteristics and mRNA levels for renal adenosine A1 receptors were investigated in normal rats and rats with acute renal failure (ARF) induced by either glycerol or HgCl2. 2. Saturation isotherms determined from the binding of [3H]-1,3-dipropyl-8-cyclopentylxanthine ([3H]-DPCPX), a selective adenosine A1 antagonist, to renal membranes of untreated rats gave values of 0.62 nM for the equilibrium dissociation constant (Kd) and 19.9 fmol mg-1 protein for the density of binding sites (Bmax). No saturable binding was observed with [3H]-2-(p-(carboxylethyl)-phenylethylamino)-5'-N-ethylcar box amido adenosine ([3H]-CGS 21680), a selective adenosine A2a agonist. 3. By contrast to time-matched controls, renal membranes obtained from rats 16 and 48 h following the induction of ARF with glycerol, showed statistically significant increases (2-4 fold) in both Bmax and Kd for the binding of [3H]-DPCPX. No significant changes in the binding characteristics of [3H]-DPCPX were noted with membranes from rats 48 h following the production of ARF with HgCl2. 4. Adenosine A1 receptor mRNA levels were significantly elevated 0.5, 16 and 48 h following induction of ARF with glycerol, whilst no change was noted in mRNA levels for beta-actin at the same time points. No statistically significant changes in adenosine A1 receptor or beta-actin mRNA levels were noted 48 h after the induction of ARF with HgCl2. 5. This study indicates that glycerol-induced ARF in the rat is associated with an increase in renal adenosine A1 receptor density which appears to result from increased transcription of the gene for this receptor. An increase in adenosine A1 receptor density in renal resistance vessels may explain, at least in part, the enhanced renal vasoconstrictor response to adenosine in glycerol-induced ARF that was noted in a previous study.

Measurement of the movement of the S4 segment during the activation of a voltage-gated potassium channel.

Voltage-gated ion channels contain a positively charged transmembrane segment termed S4. Recent evidence suggests that depolarisation of the membrane potential causes this segment to undergo conformational changes that, in turn, lead to the opening of the channel pore. In order to define these conformational changes in structural terms, we have introduced single cysteine substitutions into the S4 segment of the prototypical Shaker K+ channel at various positions and expressed the mutants in Xenopus oocytes. The cells were depolarised to induce K+ currents and the effect of application of 100 microM parachloromercuribenzenesulphonate (PCMBS) on these currents was examined by the two-electrode voltage-clamp technique. PCMBS inhibited K+ currents elicited by mutants L358C, L361C, V363C and L366C, but not those by V367C and S376C. Since PCMBS is a membrane-impermeable cysteine-modifying reagent, the data suggest that depolarisation must have caused the S4 segment to move out of the lipid bilayer into the extracellular phase rendering the residues at positions 358, 361, 363 and 366 susceptible to PCMBS attack. The lack of effect of PCMBS on V367C suggests that the exposure of S4 terminates at L366. Detailed analysis of L361C mutant revealed that the S4 movement can occur even below the resting potential of the cell, at which potential voltage-gated K+ channels are normally in a non-conducting closed state.