Mechanism of blocking K+-channels with tetraethylammonium

Using the patch-voltage-clamp method action of tetraethylammonium on the fast (30 pS) and slow K+ channels was investigated. The slow K+ channels were presented by two types: with whole (30 pS) and decreased (20 pS) conductance. In all cases tetraethylammonium decreased the current magnitude and modified the channel kinetic parameters. Apparent blocking constants determined from the current decreasing are as 8-50 and 4-12 mM for the slow K+ channels with whole and decreased conductance, respectively, and 0.05-0.08 mM--for the fast K+ channel. The potential dependency of the blocking constants correlates with that of the channel conductance. Probability of the channel open state for the slow K+ channels decreases, and that for the fast K+ channel increases under application of tetraethylammonium. It is concluded that there are two sites of tetraethylammonium binding: the first site is into the channel pore, and the second one--into the regulatory centre responsible for the channel kinetic behaviour. Blocking of general conductance of the slow channels is accompanied by proportional decrease of the channel substate conductances without change of their number and cooperatively. Block of the fast K+ channel occurs without change of the channel elementary conductance but with decrease of the number of the channel substates and reversible violation of the channel transition cooperativity. The data are discussed from the point of the hypothesis on the channel clustery organization.     

Inhibition of ATP-regulated K+-channels by a photoactivatable ATP-analogue in mouse pancreatic β-cells

The effects of a photoactivatable (DMNPE-caged) ATP-analogue on ATP-regulated K⁺-channels (K_ATP-channel) in mouse pancreatic β-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a K_i of 17 μM; similar to the 11 μM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the K_ATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced K_ATP-channel inhibition.     

Single voltage-dependent and outward rectifying K+-channels in isolated rat heart cells

Studies on single K⁺-channel currents recorded from isolated rat heart muscle cells, in which early repolarization is known to be exceptionally fast, are reported here. A K⁺-channel which is blocked by TEA (tetraethylammonium) from the inside only has been found.The total open time of the channel, measured in steady-state after activation, indicated outward rectifying properties. The single channel conductance increases with depolarization from 25 pS at-70 mV to 75 pS at+70 mV.Selectivity of the channel has also been measured and it was found that only Rb⁺ and K⁺ can permeate the channel, whereas the permeability (P) for Li⁺, Na⁺, Cl^-, Mg²⁺, and Ca²⁺ is less than 0.05 times .Ba²⁺ and Cs⁺ block the channel activity.These results clearly demonstrate the existence of K⁺-selective outward rectifying conductance pathways in rat ventricular myocytes.     

Proton-activated K+-channels of frog taste receptor cells

It is conventionally accepted that sour transduction does not require a receptor mechanism and is based on a direct interaction of acid stimuli with apical ion channels. At the same time, it has been shown that a number of neuronal cells express H(+)-gated cation channels. We studied the effect of acid stimuli on ion currents recorded from frog Rana temporaria taste receptor cells and found that a substantial subpopulation of them exhibited K+ currents activated by extracellular protons. To our knowledge, this is the first demonstration of H(+)-gated K+ channels in cells of any type including taste receptor cells. These channels are presumably involved in sour transduction and/or contribute to intercellular communications between discoid cells.     

Protein-tyrosine phosphatase reduces the number of apical small conductance K+ channels in the rat cortical collecting duct

Previous studies have demonstrated that an increase in the activity of protein-tyrosine kinase (PTK) is involved in the down-regulation of the activity of apical small conductance K(+) (SK) channels in the cortical collecting duct (CCD) from rats on a K(+)-deficient diet (). We used the patch clamp technique to investigate the role of protein-tyrosine phosphatase (PTP) in the regulation of the activity of SK channels in the CCD from rats on a high K(+) diet. Western blot analysis indicated that PTP-1D is expressed in the renal cortex. Application of 1 microm phenylarsine oxide (PAO) or 1 mm benzylphosphonic acid, agents that inhibit PTP, reversibly reduced channel activity by 95%. Pretreatment of CCDs with PAO for 30 min decreased the mean NP(o) reversibly from control value 3.20 to 0.40. Addition of 1 microm herbimycin A, an inhibitor of PTK, had no significant effect on channel activity in the CCDs from rats on a high K(+) diet. However, herbimycin A abolished the inhibitory effect of PAO, indicating that the effect of PAO is the result of interaction between PTK and PTP. Addition of brefeldin A, an agent that blocks protein trafficking from Golgi complex to the membrane, had no effect on channel activity. Moreover, application of colchicine, a microtubule inhibitor, or paclitaxel, a microtubule stabilizer, had no effect on channel activity. In contrast, PAO still reduced channel activity in the presence of brefeldin A, colchicine, or paclitaxel. Furthermore, the effect of PAO on channel activity was absent when the tubules were bathed in 16% sucrose-containing bath solution or treated with concanavalin A. We conclude that PTP is involved in the regulation of the activity of SK channels and that inhibition of PTP may facilitate the internalization of the SK channels.     

Dual effect of adenosine triphosphate on the apical small conductance K+ channel of the rat cortical collecting duct

We used the patch-clamp technique to study the effects of ATP on the small-conductance potassium channel in the apical membrane of rat cortical collecting duct (CCD). This channel has a high open probability (0.96) in the cell-attached mode but activity frequently disappeared progressively within 1-10 min after channel excision (channel "run-down"). Two effects of ATP were observed. Using inside-out patches, low concentrations of ATP (0.05-0.1 mM) restored channel activity in the presence of cAMP-dependent protein kinase A (PKA). In contrast, high concentrations (1 mM) of adenosine triphosphate (ATP) reduced the open probability (Po) of the channel in inside-out patches from 0.96 to 0. 1.2 mM adenosine diphosphate (ADP) also blocked channel activity completely, but 2 mM adenosine 5'-[beta,gamma-imido]triphosphate (AMP-PNP), a nonhydrolyzable ATP analogue, reduced Po only from 0.96 to 0.87. The half-maximal inhibition (Ki) of ATP and ADP was 0.5 and 0.6 mM, respectively, and the Hill coefficient of both ATP and ADP was close to 3. Addition of 0.2 or 0.4 mM ADP shifted the Ki of ATP to 1.0 and 2.0 mM, respectively. ADP did not alter the Hill coefficient. Reduction of the bath pH from 7.4 to 7.2 reduced the Ki of ATP to 0.3 mM. In contrast, a decrease of the free Mg2+ concentration from 1.6 mM to 20 microM increased the Ki of ATP to 1.6 mM without changing the Hill coefficient; ADP was still able to relieve the ATP-induced inhibition of channel activity over this low range of free Mg2+ concentrations. The blocking effect of ATP on channel activity in inside-out patches could be attenuated by adding exogenous PKA catalytic subunit to the bath. The dual effects of ATP on the potassium channel can be explained by assuming that (a) ATP is a substrate for PKA that phosphorylates the potassium channel to maintain normal function. (b) High concentrations of ATP inhibit the channel activity; we propose that the ATP-induced blockade results from inhibition of PKA-induced channel phosphorylation.     

Studies on K+ permeability of rat gastric microsomes

A population of gastric membrane vesicles of high K+ permeability and of lower density than endoplasmic tubulovesicles containing (H+-K+)-ATPase was detected in gastric mucosal microsomes from the rat fasted overnight. The K+-transport activity as measured with 86RbCl uptake had a Km for Rb+ of 0.58 +/- 0.11 mM and a Vmax of 13.7 +/- 1.9 nmol/min X mg of protein. The 86Rb uptake was reduced by 40% upon substituting Cl- with SO2-4 and inhibited noncompetitively by ATP and vanadate with a Ki of 3 and 30 microM, respectively; vanadate also inhibited rat gastric (H+-K+)-ATPase but with a Ki of 0.03 microM. Carbachol or histamine stimulation decreased the population of the K+-permeable light membrane vesicles, at the same time increased K+-transport activity in the heavy, presumably apical membranes of gastric parietal cells, and enabled the heavy microsomes to accumulate H+ ions in the presence of ATP and KCl without valinomycin. The secretagogue-induced shift of K+ permeability was blocked by cimetidine, a H2-receptor antagonist. Four characteristics of the K+ permeability as measured with 86RbCl were common in the resting light and the carbachol-stimulated heavy microsomes; (a) Km for +Rb, (b) anion sensitivity (Cl- greater than SO2-4), (c) potency of various divalent cations (Hg2+, Cu2+, Cd2+, and Zn2+) to inhibit Rb+ uptake, and (d) inhibitory effect of ATP, although the nucleotide sensitivity was latent in the stimulated heavy microsomes. The Vmax for 86RbCl uptake was about 10 times greater in the resting light than the stimulated heavy microsomes. These observations led us to propose that secretagogue stimulation induces the insertion of not only the tubulovesicles containing (H+-K+)-ATPase, but also the light membrane vesicles containing KCl transporter into the heavy apical membranes of gastric parietal cells.     

Inhibition of the collapse of the Shaker K+ conductance by specific scorpion toxins

The Shaker B K(+) conductance (G(K)) collapses when the channels are closed (deactivated) in Na(+) solutions that lack K(+) ions. Also, it is known that external TEA (TEA(o)) impedes the collapse of G(K), and that channel block by TEA(o) and scorpion toxins are two mutually exclusive events. Therefore, we tested the ability of scorpion toxins to inhibit the collapse of G(K) in 0 K(+). We have found that these toxins are not uniform regarding the capacity to protect G(K). Those toxins, whose binding to the channels is destabilized by external K(+), are also effective inhibitors of the collapse of G(K). In addition to K(+), other externally added cations also destabilize toxin block, with an effectiveness that does not match the selectivity sequence of K(+) channels. The inhibition of the drop of G(K) follows a saturation relationship with [toxin], which is fitted well by the Michaelis-Menten equation, with an apparent Kd bigger than that of block of the K(+) current. However, another plausible model is also presented and compared with the Michaelis-Menten model. The observations suggest that those toxins that protect G(K) in 0 K(+) do so by interacting either with the most external K(+) binding site of the selectivity filter (suggesting that the K(+) occupancy of only that site of the pore may be enough to preserve G(K)) or with sites capable of binding K(+) located in the outer vestibule of the pore, above the selectivity filter.     

Myometrial expression of small conductance Ca2+-activated K+ channels depresses phasic uterine contraction

Mechanisms regulating uterine contractility are poorly understood. We hypothesized that a specific isoform of small conductance Ca²⁺-activated K⁺ (SK) channel, SK3, promotes feedback regulation of myometrial Ca²⁺ and hence relaxation of the uterus. To determine the specific functional impact of SK3 channels, we assessed isometric contractions of uterine strips from genetically altered mice (SK3^T/T), in which SK3 is overexpressed and can be suppressed by oral administration of doxycycline (SK3^T/T+Dox). We found SK3 protein in mouse myometrium, and this expression was substantially higher in SK3^T/T mice and lower in SK3^T/T+Dox mice compared with wild-type (WT) controls. Sustained contractions elicited by 60 mM KCl were not different among SK3^T/T, SK3^T/T+Dox, and WT mice. However, the rate of onset and magnitude of spontaneously occurring phasic contractions was muted significantly in isolated uterine strips from SK3^T/T mice compared with those from WT mice. These spontaneous contractions were augmented greatly by blockade of SK channels with apamin or by suppression of SK3 expression. Phasic but not tonic contraction in response to oxytocin was depressed in uterine strips from SK3^T/T mice, whereas suppression of SK3 channel expression or treatment with apamin promoted the predominance of large coordinated phasic events over tone. Spontaneous contractions and the phasic component of oxytocin contractions were blocked by nifedipine but not by cyclopiazonic acid. Our findings suggest that SK3 channels play an important role in regulating uterine function by limiting influx through L-type Ca²⁺ channels and disrupting the development of concerted phasic contractile events. uterus; Ca²⁺-activated K⁺ channel; doxycycline; mouse     

Selectivity and permeation of alkali metal ions in K+-channels

Ion conduction in K⁺-channels is usually described in terms of concerted movements of K⁺ progressing in a single file through a narrow pore. Permeation is driven by an incoming ion knocking on those ions already inside the protein. A fine-tuned balance between high-affinity binding and electrostatic repulsive forces between permeant ions is needed to achieve efficient conduction. While K⁺-channels are known to be highly selective for K⁺ over Na⁺, some K⁺ channels conduct Na⁺ in the absence of K⁺. Other ions are known to permeate K⁺-channels with a more moderate preference and unusual conduction features. We describe an extensive computational study on ion conduction in K⁺-channels rendering free energy profiles for the translocation of three different alkali ions and some of their mixtures. The free energy maps for Rb⁺ translocation show at atomic level why experimental Rb⁺ conductance is slightly lower than that of K⁺. In contrast to K⁺ or Rb⁺, external Na⁺ block K⁺ currents, and the sites where Na⁺ transport is hindered are characterized. Translocation of K⁺/Na⁺ mixtures is energetically unfavorable owing to the absence of equally spaced ion-binding sites for Na⁺, excluding Na⁺ from a channel already loaded with K⁺.     

Calcium-activated K+ channels increase cell proliferation independent of K+ conductance

The intermediate-conductance calcium-activated potassium channel (IK1) promotes cell proliferation of numerous cell types including endothelial cells, T lymphocytes, and several cancer cell lines. The mechanism underlying IK1-mediated cell proliferation was examined in human embryonic kidney 293 (HEK293) cells expressing recombinant human IK1 (hIK1) channels. Inhibition of hIK1 with TRAM-34 reduced cell proliferation, while expression of hIK1 in HEK293 cells increased proliferation. When HEK293 cells were transfected with a mutant (GYG/AAA) hIK1 channel, which neither conducts K(+) ions nor promotes Ca(2+) entry, proliferation was increased relative to mock-transfected cells. Furthermore, when HEK293 cells were transfected with a trafficking mutant (L18A/L25A) hIK1 channel, proliferation was also increased relative to control cells. The lack of functional activity of hIK1 mutants at the cell membrane was confirmed by a combination of whole cell patch-clamp electrophysiology and fura-2 imaging to assess store-operated Ca(2+) entry and cell surface immunoprecipitation assays. Moreover, in cells expressing hIK1, inhibition of ERK1/2 and JNK kinases, but not of p38 MAP kinase, reduced cell proliferation. We conclude that functional K(+) efflux at the plasma membrane and the consequent hyperpolarization and enhanced Ca(2+) entry are not necessary for hIK1-induced HEK293 cell proliferation. Rather, our data suggest that hIK1-induced proliferation occurs by a direct interaction with ERK1/2 and JNK signaling pathways.     

Activation of ATP-sensitive K+-channels of cardiomyocytes by endogenous cardiopeptides]

The "patch-clamp" method was applied to rat ventricular cardiomyocytes to study the effect of endogenous cardiopeptides (a component of a cardiologic medicine "cardialin") on single K(+)-channel currents. It was noted that ATP-sensitive K(+)-channel activity increased only in the presence of 10(-8)-10(-6) mg.ml-1 peptide solution in the patch-clamp micropipette. To check up the suggestion that G-protein takes part in a direct reaction of K(+)-channel by external ligand we studied the effect of GTP-gamma-S (0.1 mM) in the presence of Mg2+ (1 mM) on the K(+)-channel in "run-down" state in the "inside-out" configuration. It was demonstrated experimentally that reactivation of the ATP-sensitive K(+)-channel develops in the presence of GTP-analogue.     

The ontogeny of rat gastric H+/K+-ATPase

The ontogeny of rat H+/K+-ATPase was studied between foetal day 18 and neonatal day 18, using a specific monoclonal antibody (95-111 mAb). The H+/K+-ATPase content of gastric subcellular membranes was assayed and the ATPase subunits were characterized by Western blot. The epithelium density in parietal cells was measured by immunohistochemistry. H+/K+-ATPase was present in the 18-day-old foetuses and parietal cells were detected on foetal day 19. The H+/K+-ATPase concentration remained stable from foetal day 18 to neonatal day 1, while the parietal cell density increased 2.5-fold. The H+/K+-ATPase concentration increased by 2.5-fold on day 6, then remained constant up to day 18. The parietal cell density remained unchanged during this period, suggesting that the concentration increase on day 6 was due to an increase in parietal cell ATPase content. The 95-111 mAb recognized a 95 kDa single band on foetal day 18 and a doublet at all the other stages of development. Previous studies had demonstrated that acid secretion drops critically at day 12 post partum in the rat and that H+/K+-ATPase activity is lost. The present study demonstrates that the H+/K+-ATPase is, however, present on day 12.     

Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.     

Evidence for a deep pore activation gate in small conductance Ca2+-activated K+ channels

Small conductance calcium-gated potassium (SK) channels share an overall topology with voltage-gated potassium (K(v)) channels, but are distinct in that they are gated solely by calcium (Ca(2+)), not voltage. For K(v) channels there is strong evidence for an activation gate at the intracellular end of the pore, which was not revealed by substituted cysteine accessibility of the homologous region in SK2 channels. In this study, the divalent ions cadmium (Cd(2+)) and barium (Ba(2+)), and 2-aminoethyl methanethiosulfonate (MTSEA) were used to probe three sites in the SK2 channel pore, each intracellular to (on the selectivity filter side of) the region that forms the intracellular activation gate of voltage-gated ion channels. We report that Cd(2+) applied to the intracellular side of the membrane can modify a cysteine introduced to a site (V391C) just intracellular to the putative activation gate whether channels are open or closed. Similarly, MTSEA applied to the intracellular side of the membrane can access a cysteine residue (A384C) that, based on homology to potassium (K) channel crystal structures (i.e., the KcsA/MthK model), resides one amino acid intracellular to the glycine gating hinge. Cd(2+) and MTSEA modify with similar rates whether the channels are open or closed. In contrast, Ba(2+) applied to the intracellular side of the membrane, which is believed to block at the intracellular end of the selectivity filter, blocks open but not closed channels when applied to the cytoplasmic face of rSK2 channels. Moreover, Ba(2+) is trapped in SK2 channels when applied to open channels that are subsequently closed. Ba(2+) pre-block slows MTSEA modification of A384C in open but not in closed (Ba(2+)-trapped) channels. The findings suggest that the SK channel activation gate resides deep in the vestibule of the channel, perhaps in the selectivity filter itself.     

Inhibition of rat brain microsomal (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase by periodic acid

The effects of mild periodate exposure on the kinetics of (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase were studied using rat cerebral microsome preparations. Fifty percent inhibition of both enzyme activities was attained near 3 microM periodate concentrations. This inhibition was biphasic with time. Mg2+-ATPase and Mg2+-p-nitrophenylphosphatase activities were much less inhibited by periodate. Periodate inhibition was partially reversed by dimercaprol and dithiothreitol but not by diffusion. The possible reaction products formic acid, formaldehyde, glyceraldehyde, and acetaldehyde had no inhibitory effects in similar concentrations. Periodate exposure produced no detectable changes in the activation of (Na+ + K+)-ATPase by Na+, K+, Mg2+, or ATP. Residues shared by both (Na+ + K+)-ATPase and K+-p-nitrophenylphosphatase are both critical to hydrolytic function and sensitive to mild oxidation by periodate.     

Modulation of K+-channels in p-neurones of the leech CNS by phosphorylation

Two types of potassium channels of identified (p-) neurones of the leech (Hirudo medicinalis) were investigated by using the patch-clamp technique. The openstate probability of these channels in cell-attached patches can be reduced by addition of 5-hydroxytryptamine to the bath solution. After excising the patches the application of alkaline phosphatase to the cytosolic face of the patch increases the open probability. The 5-HT_1A-receptor agonist buspirone mimics the effect of 5-HT. Our experiments show that the effect of 5-HT might be due to a channel phosphorylation via a 5-HT_1A-receptor subtype.     

Factors controlling the K+ conductance inChara

Summary Previous current/voltage (I/V) investigations of theChara K⁺ state have been extended by increasing the voltage range (up to +200 mV) through blocking the action potential with La³⁺. A region of negative slope was found in theI/V characteristics at positive PD's, similar to that already observed at PD's more negative than the resting level. These decreases in membrane currents at PD's more negative than –150 mV and at PD's close to 0 or positive are thought to arise from the K⁺ channel closure. Both the negative slope regions could be reversibly abolished by 0.1mm K⁺, 20mm Na⁺, more than 10mm Ca²⁺ or 5mm tetraethylammonium (TEA). The K⁺ channels are therefore blocked by TEA, closed by low [K⁺] _o or high [Ca²⁺] _o and are highly selective to K⁺ over Na⁺. With the K⁺ channels closed, the remainingI/V profile was approximately linear over the interval of 400 mV (suggesting a leakage current), but large rectifying currents were observed at PD's more positive than +50 mV. These currents showed a substantial decrease in high [Ca²⁺] _o , sometimes displayed a slight shift to more positive PD's with increasing [K⁺] _o and were unaffected by TEA or changes in [Na⁺] _o . The slope of the linear part of theI/V profile was steeper in low [K⁺] _o than in TEA or high [Na⁺] _o (indicating participation of K⁺, but not Na⁺, in the leak current). Diethylstilbestrol (DES) was employed to inhibit the proton pump, but it was found that the leakage current and later the K⁺ channels were also strongly affected.     

Nitrergic relaxation in rat gastric fundus: influence of mechanism of induced tone and possible role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase

The aim of this study was to investigate the influence of the mechanism of induced tone and the role of sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCA) in nitrergic relaxation of rat gastric fundus. Prostaglandin F(2alpha) (PGF(2alpha)), thapsigargin (TSG) and cyclopiazonic acid (CPA) were used in concentrations that induced a similar contraction (20 g force/g tissue). Nifedipine (3 x 10(-7) M) completely relaxed PGF(2alpha)-contracted tissues and relaxed tissues contracted by TSG and CPA by 20 +/- 6% and 56 +/- 12% respectively; contraction induced by the three contractile agents was fully reversed by a general Ca2+ entry blocker 1-[2-(4-methoxyphenyl)-2-[3-(4-metoxyphenyl)propoxy]ethyl-1H-imidazole HCl (SKF 96365; 10(-5) M). In the presence of nifedipine (3 x 10(-7) M) or verapamil (10(-5) M), PGF(2alpha) and CPA-induced contractions were still approximately 50% relaxed by SKF 96365. This suggests that contractions induced by PGF(2alpha) are related to Ca2+ entry through L-type voltage-operated Ca2+ channels and that contractions by TSG are mainly related to Ca2+ entry through store-operated Ca2+ channels. Relaxant responses to exogenous nitric oxide (NO), to endogenous NO released by electrical field stimulation, and to vasoactive intestinal polypeptide (VIP) were studied in tissues contracted by TSG and CPA and compared to responses in tissues contracted by PGF(2alpha). Responses to exogenous and endogenous NO were greatly attenuated in TSG-contracted tissues, but not in CPA-contracted tissues. When contraction was induced by CPA in the presence of nifedipine or verapamil, relaxations to exogenous and endogenous NO were also significantly reduced. Relaxation induced by VIP was reduced in tissues contracted by either TSG or CPA in the presence of nifedipine or verapamil. These results suggest that the ability of the nitrergic neurotransmitter to induce relaxation of rat gastric fundus is influenced by the mechanism used to induce tone and are indicative for a role for SERCA in nitrergic relaxation. However, activation of SERCA appears to not be unique for nitrergic relaxation, but might also be used by VIP, a co-transmitter of NO in this tissue.     

A possible molecular mechanism of hanatoxin binding-modified gating in voltage-gated K+-channels

While S4 is known as the voltage sensor in voltage-gated potassium channels, the carboxyl terminus of S3 (S3C) is of particular interest concerning the site for gating modifier toxins like hanatoxin. The thus derived helical secondary structural arrangement for S3C, as well as its surrounding environment, has since been intensively and vigorously debated. Our previous structural analysis based on molecular simulation has provided sufficient information to describe reasonable docking conformation and further experimental designs (Lou et al., 2002. J. Mol. Recognit. 15: 175-179). However, if one only relies on such information, more advanced structure-functional interpretations for the roles S3C may play in the modification of gating behavior upon toxin binding will remain unknown. In order to have better understanding of the molecular details regarding this issue, we have performed the docking simulation with the S3C sequence from the hanatoxin-insensitive K+-channel, shaker, and analyzed the conformational changes resulting from such docking. Compared with other functional data from previous studies with respect to the proximity of the S3-S4 linker region, we suggested a significant movement of drk1 S3C, but not shaker S3C, in the direction presumably towards S4, which was comprehended as a possible factor interfering with S4 translocation during drk1 gating in the presence of toxin. In combination with the discussions for structural roles of the length of the S3-S4 linker, a possible molecular mechanism to illustrate the hanatoxin binding-modified gating is proposed.