Potassium channels of adult locust (Schistocerca gregaria) muscle

Two types of K⁺ channels have been identified in patches of plasma membrane of metathoracic extensor tibiae muscle fibres of adult locust, Schistocerca gregaria. One channel had a maximum conductance of 170 pS, fast open-closed kinetics, and a linear current/ voltage relationship. In inside-out patches it was activated by ‘‘internally applied’’ Ca²⁺, but at unexpectedly low levels (between 10⁻¹⁰ and 10⁻⁹M). The other channel had a maximum conductance of 35 pS, slower open-closed kinetics, and was not activated by Ca²⁺. In cell-attached patches, its channel conductance measured in symmetrical salines was about three times greater for hyperpolarisations than for depolarisations. This inward rectification was proved to be due to block by intracellular Mg²⁺. For both channels, open probability (P _o) and mean open time increased during depolarisations and decreased during hyperpolarisations, resulting in outward rectifications in terms of net current (I _n , product of the single-channel current and P _o). For both channels, the K⁺ conductance was 10 times greater than that for Na⁺. Internally applied tetraethylammonium or tetramethylammonium ions blocked both channels. Received: 12 June 1995/Received after revision and accepted: 30 January 1996     

Ketamine blocks voltage-gated K+ channels and causes membrane depolarization in rat mesenteric artery myocytes

Clinical doses of ketamine typically increase blood pressure, heart rate, and cardiac output. However, the precise mechanism by which ketamine produces these cardiovascular effects remains unclear. The voltage-gated K⁺ (K_V) channel is the major regulator of resting membrane potential (E _m) and vascular tone in many arteries. Therefore, we sought to evaluate the effects of ketamine on K_V currents using the standard whole-cell patch clamp recordings in single myocytes, enzymatically dispersed from rat mesenteric arteries. Ketamine [(±)-racemic mixture] inhibited K_V currents reversibly and concentration dependently with a K _d of 566.7 ± 32.3 μM and Hill coefficient of 0.75 ± 0.03. The inhibition of K_V currents by ketamine was voltage independent, and the time courses of channel activation and inactivation were little affected. The effects of ketamine on steady-state activation and inactivation curves were also minimal. Use-dependent inhibition was not observed either. S(+)-ketamine inhibited K_V currents with similar potency and efficacy as the racemic mixture. The average resting E _m in rat mesenteric artery myocytes was −44.1 ± 4.2 mV, and both racemic and S(+)-ketamine induced depolarization of E _m (15.8 ± 3.6 and 24.3 ± 5.0 mV at 100 μM, respectively). We conclude that ketamine induces E _m depolarization in vascular myocytes by blocking K_V channels in a state-independent manner, which may contribute to the increased vascular tone and blood pressure produced by this drug under a clinical setting.     

The Plasmodium falciparum-induced anion channel of human erythrocytes is an ATP-release pathway

Infection with the malaria parasite Plasmodium falciparum induces osmolyte and anion channels in the host erythrocyte membrane involving ATP release and autocrine purinergic signaling. P. falciparum-parasitized but not unstimulated uninfected erythrocytes released ATP in a 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB; 7 μM)-sensitive and serum album (SA; 0.5% w/v)-stimulated manner. Since Plasmodium infection of human erythrocytes induces SA-dependent outwardly (OR) and SA-independent inwardly rectifying (IR) anion conductances, we tested whether the infection-induced OR channels directly generate an ATP release pathway. P. falciparum-parasitized erythrocytes were recorded in whole-cell mode with either Cl⁻ or ATP as the only anion in the bath or pipette. In parasitized cells with predominant OR activity, replacement of bath NaCl by Na–ATP (NMDG–Cl pipette solution) shifted the current reversal potential (V _rev) from −2 ± 1 to +51 ± 3 mV (n = 15). In cells with predominant IR activity, in contrast, the same maneuver induced a shift of V _rev to significantly larger (p ≤ 0.05, two-tailed t test) values (from −3 ± 1 to +66 ± 8 mV; n = 5) and an almost complete inhibition of outward current. The anion channel blocker NPPB reversibly decreased the ATP-generated OR currents from 1.1 ± 0.1 nS to 0.2 ± 0.05 nS and further shifted V _rev to +87 ± 7 mV (n = 12). The NPPB-sensitive fraction of the OR reversed at +48 ± 4 mV suggesting a relative permeability of P _ATP/P _Cl ≈ 0.01. Together, these data raise the possibility that the OR might be the electrophysiological correlate of an erythrocyte ATP release pathway. Canan Akkaya and Ekaterina Shumilina contributed equally to this work and, thus, share first authorship.     

Contribution of BK channels to action potential repolarisation at minimal cytosolic Ca2+ concentration in chromaffin cells

BK channels modulate cell firing in excitable cells in a voltage-dependent manner regulated by fluctuations in free cytosolic Ca²⁺ during action potentials. Indeed, Ca²⁺-independent BK channel activity has ordinarily been considered not relevant for the physiological behaviour of excitable cells. We employed the patch-clamp technique and selective BK channel blockers to record K⁺ currents from bovine chromaffin cells at minimal intracellular (about 10 nM) and extracellular (free Ca²⁺) Ca²⁺ concentrations. Despite their low open probability under these conditions (V₅₀ of +146.8 mV), BK channels were responsible for more than 25% of the total K⁺ efflux during the first millisecond of a step depolarisation to +20 mV. Moreover, BK channels activated about 30% faster (τ = 0.55 ms) than the rest of available K⁺ channels. The other main source of fast voltage-dependent K⁺ efflux at such a low Ca²⁺ was a transient K⁺ (I_A-type) current activating with V ₅₀ = −14.2 mV. We also studied the activation of BK currents in response to action potential waveforms and their contribution to shaping action potentials both in the presence and the absence of extracellular Ca²⁺. Our results show that BK channels activate during action potentials and accelerate cell repolarisation even at minimal Ca²⁺ concentration, and suggest that they could do so also in the presence of extracellular Ca²⁺, before Ca²⁺ entering the cell facilitates their activity.     

Kinetic parameters of the ionic currents in myelinated axons: Characterization of temperature effects in a hibernator and a nonhibernator

Na⁺ and K⁺ currents were measured by the patch-clamp method in the paranodal region of single sciatic nerve fibres of rats and of warm-adapted and cold-adapted golden hamsters. Kinetic parameters and temperature dependence of the Na⁺ currents were determined. The time constant for activation (about 0.2 ms for rats and hamsters) as well as the time constant for inactivation (about 1.6 ms for rats and hamsters) at 15 °C and at −35 mV compared well with single fibre voltage-clamp data from the rat. Differences amongst the three groups of animals were not significant. The temperature coefficient, Q ₁₀, for the activation and the inactivation time constant as well as for the time-to-peak of the Na⁺ current ranged between 2.3 and 3.1. No data have previously been published on the temperature dependence of the delayed-rectifier K channels of mammalian nerve fibres. Most of the K⁺ current was carried by intermediate (K_I) and fast (K_F) K channels. Dendrotoxin block indicated that ≈55% of the K⁺ current was due to K_I channels, with no significant difference amongst the three groups of animals tested. The Arrhenius plot of the time constant of K⁺ current activation, τ_n, yielded a mean Q ₁₀ of 3.3 at −40 mV (4.0 at + 60 mV). No significant differences of the channel kinetics between rats, warm-adapted hamsters and cold-adapted hamsters were detected. We observed, however, a significant decrease of the Na channel density in the paranodal region of cold-adapted hamsters. Received: 10 June 1995/Received after revision: 3 November 1995/Accepted: 17 November 1995     

Adenine nucleotides and intracellular Ca2+ regulate a voltage-dependent and glucose-sensitive potassium channel in neurosecretory cells

Effects of membrane potential, intracellular Ca²⁺ and adenine nucleotides on glucose-sensitive channels from X organ (XO) neurons of the crayfish were studied in excised inside-out patches. Glucose- sensitive channels were selective to K⁺ ions; the unitary conductance was 112 pS in symmetrical K⁺, and the K⁺ permeability (P _K) was 1.3 × 10⁻¹³ cm ⋅s⁻¹. An inward rectification was observed when intracellular K⁺ was reduced. Using a quasi-physiological K⁺ gradient, a non-linear K⁺ current/voltage relationship was found showing an outward rectification and a slope conductance of 51 pS. The open-state probability (P _o) increased with membrane depolarization as a result of an enhancement of the mean open time and a shortening of the longer period of closures. In quasi-physio- logical K⁺ concentrations, the channel was activated from a threshold of about −60 mV, and the activation midpoint was −2 mV. P _o decreased noticeably at 50 μM internal adenosine 5′-triphosphate (ATP), and single-channel activity was totally abolished at 1 mM ATP. Hill analysis shows that this inhibition was the result of simultaneous binding of two ATP molecules to the channel, and the half-blocking concentration of ATP was 174 μM. Internal application of 5′-adenylylimidodiphosphate (AMP-PNP) as well as glibenclamide also decreased P _o. By contrast, the application of internal ADP (0.1 to 2 mM) activated this channel. An optimal range of internal free Ca²⁺ ions (0.1 to 10 μM) was required for the activation of this channel. The glucose--sensitive K⁺ channel of XO neurons could be considered as a subtype of ATP-sensitive K⁺ channel, contributing substantially to macroscopic outward current. Received: 13 November 1995/Received after revision and accepted: 13 December 1995     

Distal colonic Na+ absorption inhibited by luminal P2Y2 receptors

Luminal P2 receptors are ubiquitously expressed in transporting epithelia. In steroid-sensitive epithelia (e.g., lung, distal nephron) epithelial Na⁺ channel (ENaC)-mediated Na⁺ absorption is inhibited via luminal P2 receptors. In distal mouse colon, we have identified that both, a luminal P2Y₂ and a luminal P2Y₄ receptor, stimulate K⁺ secretion. In this study, we investigate the effect of luminal adenosine triphosphate/uridine triphosphate (ATP/UTP) on electrogenic Na⁺ absorption in distal colonic mucosa of mice treated on a low Na⁺ diet for more than 2 weeks. Transepithelial electrical parameters were recorded in an Ussing chamber. Baseline parameters: transepithelial voltage (V _te): −13.7 ± 1.9 mV (lumen negative), transepithelial resistance (R _te): 24.1 ± 1.8 Ω cm², equivalent short circuit current (I _sc): −563.9 ± 63.8 μA/cm² (n = 21). Amiloride completely inhibited I _sc to −0.5 ± 8.5 μA/cm². Luminal ATP induced a slowly on-setting and persistent inhibition of the amiloride-sensitive I _sc by 160.7 ± 29.7 μA/cm² (n = 12, NMRI mice). Luminal ATP and UTP were almost equipotent with IC₅₀ values of 10 μM and 3 μM respectively. In P2Y₂ knock-out (KO) mice, the effect of luminal UTP on amiloride-sensitve Na⁺ absorption was absent. In contrast, in P2Y₄ KO mice the inhibitory effect of luminal UTP on Na⁺ absorption remained present. Semiquantitative polymerase chain reaction did not indicate regulation of the P2Y receptors under low Na⁺ diet, but it revealed a pronounced axial expression of both receptors with highest abundance in surface epithelia. Thus, luminal P2Y₂ and P2Y₄ receptors and ENaC channels co-localize in surface epithelium. Intriguingly, only the stimulation of the P2Y₂ receptor mediates inhibition of electrogenic Na⁺ absorption.     

Ionic selectivity of the mechano-electrical transduction channels in the hair cells of the frog sacculus

In the isolated saccular macula of Ratio, esculenta extracellular hair cell receptor currents evoked by mechanical stimulation of the otolithic membrane were recorded under transepithelial voltage clamp conditions. The ionic selectivity of the mechano-electrical transduction channels of the hair cells was determined by examining the effects of different concentrations of Ca²⁺ and K⁺ in the apical solution on the transepithelial voltage at which the extracellular receptor current was zero (V_rev). Changing the concentration of Ca²⁺ from 0.26 mM to 0.026 and to 2.6 mM at a constant K⁺ concentration caused changes in V_rev of – 15 + 7 mV (mean±SD; n= 9) and 20±6mV (n= 13), respectively. The relative ionic permeabilities of the transduction channels were estimated from a modified Goldman, Hodgkin and Katz equation, assuming that 80% of the transepithelial resistance is located in the apical membranes of the hair cells. The permeability of the transduction channels for Ca²⁺ was found to be two orders of magnitude larger than that for K⁺. The measured effects on V_rev of changing the concentration of K⁺ at constant ionic strength and at different constant Ca²⁺ concentrations were well predicted by the same equation. These results indicate that the transduction channels of the frog saccular hair cells are highly selective to Ca²⁺.     

Pannexin 1 forms an anion-selective channel

Pannexin 1 (Panx1) is expressed in various mammalian tissues including the brain and immune cells. Here, we present evidence that Panx1 when expressed in mammalian cells, forms anion-selective channels, with a rank order of permeabilities: NO₃⁻ > I⁻ > Br⁻ > Cl⁻ > F⁻ ≫ aspartate⁻ ≈ glutamate⁻ ≈ gluconate⁻. Single-channel Panx1-mediated currents have a unitary conductance around 68 pS. Our results show that Panx1 assembles into a membrane anion channel with a relatively low single-channel conductance.     

K+ channel modulation in arterial smooth muscle

Potassium channels play an essential role in the membrane potential of arterial smooth muscle, and also in regulating contractile tone. Four types of K⁺ channel have been described in vascular smooth muscle: Voltage-activated K⁺ channels (K_V) are encoded by the Kv gene family, Ca²⁺-activated K⁺ channels (BK_Ca) are encoded by the slogene, inward rectifiers (K_IR) by Kir2.0, and ATP-sensitive K⁺ channels (K_ATP) by Kir6.0 and sulphonylurea receptor genes. In smooth muscle, the channel subunit genes reported to be expressed are: Kv1.0, Kv1.2, Kv1.4–1.6, Kv2.1, Kv9.3, Kvβ1–β4, slo α and β, Kir2.1, Kir6.2, and SUR1 and SUR2. Arterial K⁺ channels are modulated by physiological vasodilators, which increase K⁺ channel activity, and vasoconstrictors, which decrease it. Several vasodilators acting at receptors linked to cAMP-dependent protein kinase activate K_ATP channels. These include adenosine, calcitonin gene-related peptide, and β-adrenoceptor agonists. β-adrenoceptors can also activate BK_Ca and K_V channels. Several vasoconstrictors that activate protein kinase C inhibit K_ATP channels, and inhibition of BK_Ca and K_V channels through PKC has also been described. Activators of cGMP-dependent protein kinase, in particular NO, activate BK_Ca channels, and possibly K_ATP channels. Hypoxia leads to activation of K_ATP channels, and activation of BK_Ca channels has also been reported. Hypoxic pulmonary vasoconstriction involves inhibition of K_V channels. Vasodilation to increased external K⁺ involves K_IR channels. Endothelium-derived hyperpolarizing factor activates K⁺ channels that are not yet clearly defined. Such K⁺ channel modulations, through their effects on membrane potential and contractile tone, make important contributions to the regulation of blood flow.     

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A

We identified voltage-activated K⁺ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca²⁺-activated K⁺ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 ± 1.7 pS channels (K_DR1), 90.6 ± 5.4 pS channels (K_DR2) and 149 ± 4 pS intermediate-conductance Ca²⁺-activated K⁺ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from K_DR1 channels inactivated with a time constant of 1.7 ± 0.1 s at +60 mV test potential, while K_DR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca²⁺] (10⁻⁶M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). K_DR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1–10 mM) but not to Ca²⁺. K_DR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of K_DR1 channels 3.4-fold and of K_DR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (−60 mV) only openings of K_DR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K⁺ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells. Received: 20 June 1995/Received after revision: 25 January 1996/Accepted: 7 February 1996     

Effect of acidosis on Ca2+-activated K+ channels in cultured porcine coronary artery smooth muscle cells

 Although acidosis induces vasodilation, the vascular responses mediated by large-conductance Ca²⁺-activated K⁺ (K_Ca) channels have not been investigated in coronary artery smooth muscle cells. We therefore investigated the response of porcine coronary arteries and smooth muscle cells to acidosis, as well as the role of K_Ca channels in the regulation of muscular tone. Acidosis (pH 7.3–6.8), produced by adding HCl to the extravascular solution, elicited concentration-dependent relaxation of precontracted, endothelium-denuded arterial rings. Glibenclamide (20 μM) significantly inhibited the vasodilatory response to acidosis (pH 7.3-6.8). Charybdotoxin (100 nM) was effective only at pH 6.9–6.8. When we exposed porcine coronary artery smooth muscle cells to a low-pH solution, K_Ca channel activity in cell-attached patches increased. However, pretreatment of these cells with 10 or 30 μM O, O′-bis(2-aminophenyl)ethyleneglycol-N,N,N′,N′-tetraacetic acid tetrakis(acetoxymethyl)ester (BAPTA-AM), a Ca²⁺ chelator for which the cell membrane is permeable, abolished the H⁺-mediated activation of K_Ca channels in cell-attached patches. Under these circumstances H⁺ actually inhibited K_Ca channel activity. When inside-out patches were exposed to a [Ca²⁺] of 10^–6 M [adjusted with ethyleneglycolbis(β-aminoethylester)-N,N,N′,N′-tetraacetic acid (EGTA) at pH 7.3], K_Ca channels were activated by H⁺ concentration dependently. However, when these patches were exposed to a [Ca²⁺] of 10^–6 M adjusted with BAPTA at pH 7.3, H⁺ inhibited K_Ca channel activity. Extracellular acidosis had no significant direct effect on K_Ca channels, suggesting that extracellular H⁺ exerts its effects after transport into the cell, and that K_Ca channels are regulated by intracellular H⁺ and by cytosolic free Ca²⁺ modulated by acute acidosis. These results indicate that the modulation of K_Ca channel kinetics by acidosis plays an important role in the determination of membrane potential and, hence, coronary arterial tone. Received: 20 January 1998 / Received after revision: 9 April 1998 / Accepted: 22 April 1998     

A stretch-activated K+ channel in the basolateral membrane ofXenopus kidney proximal tubule cells

The present study examined whether a basolateral potassium ion (K⁺) channel is activated by membrane-stretching in the cell-attached patch. A K⁺ channel of conductance of 27.5 pS was most commonly observed in the basolateral membrane ofXenopus kidney proximal tubule cells. Channel activity increased with hyperpolarizing membrane potentials [at more positive pipette potentials (V _p)]. Open probability (P _o) was 0.03, 0.13, and 0.21 atV _p values of 0, 40, and 80 mV, respectively. Barium (0.1 mM) in the pipette reducedP _o by 79% at aV _p of 40 mV. Application of negative hydraulic pressure (−16 to −32 cm H₂O) to the pipette markedly activated outward currents (fromP _o=0.01 to 0.75) at aV _p of −80 mV, but not inward currents at aV _p of 80 mV. The size of the activated outward currents (from cell to pipette) did not change by replacing chloride with gluconate in the pipette. These results indicate that a stretch-activated K⁺ channel exists in the basolateral membrane of proximal tubule cells. It may play an important role as a K⁺ exit pathway when the cell membrane is stretched (for example, by cell swelling).     

The cAMP-regulated and 293B-inhibited K+ conductance of rat colonic crypt base cells

We have shown previously that secretagogues acting via the second messenger adenosine 3′,5′-cyclic monophosphate (cAMP) activate, besides their marked effect on the luminal Cl⁻ conductance, a K⁺ conductance in the basolateral membrane of colonic crypt cells. This conductance is blocked by the chromanol 293B. This K⁺ conductance is examined here in more detail in cell-attached (c.a.) and cell-excised (c.e.) patch- clamp studies. Addition of forskolin (5 μmol/l) to the bath led to the activation of very small-conductance (probably < 3 pS) K⁺ channels in c.a. patches (n = 54). These channels were reversibly inhibited by the addition of 0.1 mmol/l of 293B to the bath (n = 21). Noise analysis revealed that these channels had fast kinetics and produced a Lorentzian noise component with a corner frequency ( f _c) of 308 ± 10 Hz (n = 30). The current/voltage curves of this noise indicated that the underlying ion channels were K⁺ selective. 293B reduced the power density of the noise (S _o) to 46 ± 8.7% of its control value and shifted f _c from 291 ± 26 to 468 ± 54 Hz (n = 8). In c.e. patches from cells previously stimulated by forskolin, the same type of current persisted in 3 out of 18 experiments when the bath solution was a cytosolic-type solution without adenosine 5′-triphosphate (ATP) (CYT). In 15 experiments the addition of ATP (1 mmol/l) to CYT solution was necessary to induce or augment channel activity. In six experiments excision was performed into CYT + ATP solution and channel activity persisted. 293B exerted a reversible inhibitory effect. The channel activity was reduced by 5 mmol/l Ba²⁺ and was completely absent when K⁺ in the bath was replaced by Na⁺. These data suggest that forskolin activates a K⁺ channel of very small conductance which can be inhibited directly and reversibly by 293B. Received: 1 October 1995/Received after revision: 28 December 1995/Accepted: 28 December 1995     

Whole-cell conductive properties of rat pancreatic acini

Acetylcholine-controlled exocrine secretion by pancreatic acini has been explained by two hypotheses. One suggests that NaCl secretion occurs by secondary active secretion as has been originally described for the rectal gland of Squalus acanthias. The other is based on a “push-pull” model whereby Cl⁻ is extruded luminally and sequentially taken up basolaterally. In the former model Cl⁻ uptake is coupled to Na⁺ and basolateral K⁺ conductances play a crucial role, in the latter model, Na⁺ uptake supposedly occurs via basolateral non-selective cation channels. The present whole-cell patch-clamp studies were designed to further explore the conductive properties of rat pancreatic acini. Pilot studies in approximately 300 cells revealed that viable cells usually had a membrane voltage (V _m) more hyperpolarized than −30 mV. In all further studies V _m had to meet this criterion. Under control conditions V _m was −49 ± 1 mV (n = 149). The fractional K⁺ conductance (f _K) was 0.13 ± 0.1 (n = 49). Carbachol (CCH, 0.5 μmol/l) depolarized to −19 ± 1.1 mV (n = 63) and increased the membrane conductance (G _m) by a factor of 2–3. In the seeming absence of Na⁺ [replacement by N-methyl-D-glucamine (NMDG⁺)] V _m hyperpolarized slowly to −59 ± 2 mV (n = 90) and CCH still induced depolarizations to −24 ± 2 mV (n = 34). The hyperpolarization induced by NMDG⁺ was accompanied by a fall in cytosolic pH by 0.4 units, and a very slow and slight increase in cytosolic Ca²⁺. f _K increased to 0.34. The effect of NMDG⁺ on V _m was mimicked by the acidifying agents propionate and acetate (10 mmol/l) added to the bath. The present study suggests that f _K makes a substantial contribution to G _m under control conditions. The NMDG⁺ experiments indicate that the non- selective cation conductance contributes little to V _m in the presence of CCH. Hence the present data in rat pancreatic acinar cells do not support the push-pull model. Received: 8 November 1995/Received after revision: 18 December 1995/Accepted: 3 January 1996     

Effect of repetitive stimulation on cell volume and its relationship to membrane potential in amphibian skeletal muscle

The effect of electrical stimulation on cell volume, V _c, and its relationship to membrane potential, E _m, was investigated in Rana temporaria striated muscle. Confocal microscope xz-plane scanning and histology of plastic sections independently demonstrated significant and reversible increases in V _c of 19.8±0.62% (n=3) and 27.1±8.62% (n=3), respectively, after a standard stimulation protocol. Microelectrode measurements demonstrated an accompanying membrane potential change, ΔE _m, of +23.6±0.98 mV (n=3). The extent to which this ΔE _m might contribute to the observed changes in V _c was explored in quiescent muscle exposed to variations in extracellular potassium concentration, [K⁺]_e. E _m and V _c varied linearly with log [K⁺]_e and [K⁺]_e, respectively, in the range 2.5–15 mM (R ²=0.99 and 0.96), and these results were used to reconstruct an approximately linear relationship between V _c and E _m (ΔV _c=0.85E _m+68.53; R ²=0.99) and hence derive the ΔV _c expected from the ΔE _m during stimulation. This demonstrated that both the time course and magnitude of the increase and recovery of V _c observed in active muscles could be reproduced by the corresponding [K⁺]_e-induced depolarisation in quiescent muscles, suggesting that the depolarisation associated with membrane activity makes a substantial contribution to the cell swelling during exercise. Furthermore, conditions of Cl⁻ deprivation abolished the relationship between E _m and V _c, supporting a mechanism in which the depolarisation of E _m drives a passive redistribution of Cl⁻ and hence cellular entry of Cl⁻ and K⁺ and an accompanying, osmotically driven, increase in V _c.     

Role of calcium-dependent K+ channels in the regulation of arterial and venous tone by nitric oxide in pigs

Effects of inhibition of calcium-dependent potassium channels (K⁺ _Ca channels) on the regulation of arterial and venous tone by nitric oxide (NO) were studied in anaesthetized pigs following vagotomy and blockade of autonomic ganglia. Selective inhibition of K⁺ _Ca channels by charybdotoxin (CTX, 2 μg/kg iv) or iberiotoxin (IbTX, 1 μg/kg) significantly augmented mean total peripheral resistance (TPR) to levels 30–60% above control. Venous and pulmonary vascular tone were assessed by changes in effective compliances of the venous (EVC) and pulmonary (EPC) vascular beds as calculated from changes in central venous and diastolic pulmonary arterial blood pressure during haemorrhagia (−5 ml/kg) and hypervolaemia (+5 ml/kg). Blockade of K⁺ _Ca channels significantly decreased both EVC (−20 to −30%) and EPC (−30 to −50%). Both CTX and IbTX significantly diminished the vasodilation caused by the NO-donor S-nitroso-N-acetylpenicillamine (SNAP) both during control conditions and following experimental vasoconstriction induced by systemic inhibition of NO-synthesis by NG-nitro-L-arginine methyl ester (L-NAME) or infusion of vasoconstrictor agonists. Dilator effects of the adenosine 3′,5′-cyclic monophosphate (cAMP)-dependent agonist adenosine were only slightly reduced. However, blockade of K⁺ _Ca channels did not increase vasoconstriction induced by L-NAME significantly. These results suggest that activation of vascular K⁺ _Ca channels is an important mechanism by which NO attenuates the constrictor tone of resistance and capacitance vessels in vivo. + _Ca channel blockade during vasoconstriction by agonists The effects of CTX on the haemodynamic responses to infusions of AII, AVP, NA and ET-1, at doses producing similar increases in MAP of about 50 mmHg, are listed in Table 1. The increase in TPR caused by NA and ET-1 was significantly smaller after CTX, whereas the responses to AII and AVP were similar both before and after CTX. To characterize this effect of K⁺ _Ca channel blockade further, we constructed dose-response curves for AII and NA with and without pretreatment with IbTX. The results for TPR are shown in Fig. 5. The constrictor responses to the two lower doses of NA were significantly reduced by IbTX. Received: 12 February 1996 / Accepted: 31 March 1996     

Localization of the ergtoxin-1 receptors on the voltage sensing domain of hERG K+ channel by AFM recognition imaging

The inhibition of the human ether-à-go-go-related (hERG) K⁺ channels is the major cause of long QT syndromes inducing fatal cardiac arrhythmias. Ergtoxin 1 (ErgTx1) belongs to scorpion-toxins, which are K⁺ channel-blockers, and binds to hERG channel with 1:1 stoichiometry and high affinity (K _d ∼ 10 nM). Nevertheless, patch-clamp recordings recently demonstrated that ErgTx1 does not establish complete blockade of hERG currents, even at high ErgTx1 concentrations. Such phenomenon is supposed to be consistent with highly dynamic conformational changes of the outer pore domain of hERG. In this study, simultaneous topography and recognition imaging (TREC) on hERG HEK 293 cells was used to visualize binding sites on the extracellular part of hERG channel (on S1–S2 region) for Anti-K_v11.1 (hERG-extracellular-antibody). The recognition maps of hERG channels contained recognition spots, haphazardly distributed and organized in clusters. Recognition images after the addition of ErgTx1 at high concentrations (∼1 μM) revealed subsequent partial disappearance of clusters, indicating that ErgTx1 was bound to the S1–S2 region. These results were supported by AFM force spectroscopy data, showing for the first time that voltage sensing domain (S1–S4) of hERG K⁺ channel might be one of the multiple binding sites of ErgTx1.     

Temporal profile of potassium channel dysfunction in cerebrovascular smooth muscle after experimental subarachnoid haemorrhage

The pathogenesis of cerebral vasospasm after subarachnoid haemorrhage (SAH) involves sustained contraction of arterial smooth muscle cells that is maximal 6–8 days after SAH. We reported that function of voltage-gated K⁺ (K_V) channels was significantly decreased during vasospasm 7 days after SAH in dogs. Since arterial constriction is regulated by membrane potential that in turn is determined predominately by K⁺ conductance, the compromised K⁺ channel dysfunction may cause vasospasm. Additional support for this hypothesis would be demonstration that K⁺ channel dysfunction is temporally coincident with vasospasm. To test this hypothesis, SAH was created using the double haemorrhage model in dogs and smooth muscle cells from the basilar artery, which develops vasospasm, were isolated 4 days (early vasospasm), 7 days (during vasospasm) and 21 days (after vasospasm) after SAH and studied using patch-clamp electrophysiology. We investigated the two main K⁺ channels (K_V and large-conductance voltage/Ca²⁺-activated (K_Ca) channels). Electrophysiologic function of K_Ca channels was preserved at all times after SAH. In contrast, function of K_V channels was significantly decreased at all times after SAH. The decrease in cell size and degree of K_V channel dysfunction was maximal 7 days after SAH. The results suggest that K_V channel dysfunction either only partially contributes to vasospasm after SAH or that compensatory mechanisms develop that lead to resolution of vasospasm before K_V channels recover their function.     

Modification of cloned brain Na+ channels by batrachotoxin

The effects of batrachotoxin (BTX) on cloned α-subunit Na⁺ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na⁺ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at −100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11×10⁶ mol⁻¹·s⁻¹ at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The K _D value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (K _D=14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between −20 mV to +50 mV. Below −30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH₃ cells. Neutral local anesthetic benzocaine, however, drastically enhanced the steady-state inactivation of BTX-modified NaIIA channels, with its maximal effect around −60 mV. We conclude that BTX can bind and modify the NaIIA α-subunit. However, a specific subtype of α-subunits and/or an unidentified modulating process may be required for the optimal steady-state inactivation of BTX-modified Na⁺ channels.