Role of a Ca2+-activated K+ current in the maintenance of resting membrane potential of isolated, human, saphenous vein smooth muscle cells

 Calcium-activated potassium currents were studied in dissociated smooth muscle cells from human saphenous vein (HSV) using the patch-clamp technique in the whole-cell configuration. The average measured resting membrane potential (V _m) was –41±2 mV (n=39), when the cells were dialysed with an intracellular pipette solution (IPS) containing 0.1 mM ethyleneglycol-bis(β-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA) (IPS-0.1 mM EGTA). When the EGTA concentration was increased to 10 mM (IPS-10 mM EGTA) V _m became significantly less negative: –13±2 mV (n=23, P<0.05). These results suggest that 10 mM EGTA reduces a calcium-dependent current involved in the maintenance of V _m. Depolarizing voltage steps up to +60 mV from holding potentials of –60 mV resulted in large (1–10 nA) time- and voltage-dependent outward currents. The amplitudes of total whole-cell current densities measured at voltages above –20 mV were significantly greater in the cells dialysed with IPS-0.1 mM EGTA than in those dialysed with IPS-10 mM EGTA. In the cells dialysed with IPS-0.1 mM EGTA, 0.1 mM tetraethylammonium chloride (TEA) and 50 nM iberiotoxin (IBTX), which selectively block large conductance Ca²⁺-activated potassium channels (BK_Ca), diminished the total current recorded at +60 mV by 45±14% (P<0.05, n=5) and 50±6% (n=8, P<0.05), respectively. These blockers at the same concentrations did not affect the total current in cells dialysed with IPS-10 mM EGTA. When tested on intact HSV rings, both 0.1 mM TEA and 50 nM IBTX elicited vessel contraction. We conclude that BK_Ca channels present in HSV smooth muscle cells contribute to the maintenance of the V _m and sustain a significant portion of the total voltage-activated, outward current. Finally, BK_Ca channels appear to play a significant role in the regulation of HSV smooth muscle contractile activity. Received: 3 April 1998 / Received after revision: 23 September 1998 / Accepted: 13 October 1998     

Tedisamil inhibits the delayed rectifier K+ current in single smooth muscle cells of the guinea-pig portal vein

Tedisamil is a new bradycardic agent with an inhibitory action on K⁺ channels in cardiac muscle, and secondary beneficial effects in experimentally induced cardiac ischemia. In whole-cell clamp studies in enzymatically dispersed, single smooth muscle cells from the guinea-pig portal vein, tedisamil inhibited the delayed rectifier K⁺ current (determined as the charge transferred through the cell membrane), the mean concentration for half-maximal inhibition being 2.9 M. In contrast to controls in the absence of drugs or in the presence of the classical K⁺ channel blockers barium, tetraethylammonium or 4-aminopyridine, the time course of the delayed rectifier K⁺ current in the presence of tedisamil could no longer be fitted by a single exponential, and signs of an accelerated inactivation by tedisamil were obtained. The slow onset of the response to tedisamil applied to the outside of the vascular myocytes, and the finding that tedisamil applied directly to the cytosol via the pipette was highly effective, suggest an intracellular site of action.     

Membrane rectification in single smooth muscle cells from the rat tail artery

Membrane rectification to depolarization was studied by voltage recording with patch electrodes in freshly isolated cells from the rat tail artery. Injection of depolarizing currents elicited electrotonic potentials that developed with a single-exponential time course (time constant of 94.8 ms). When the cell was depolarized beyond –30 mV, delayed rectification was observed. A second type of rectification, characterized by oscillations, was observed when the cell was depolarized positive to + 30 mV. The threshold of this rectification and the oscillations were sensitive to changes in intracellular Ca²⁺. Delayed rectification was more sensitive to 4-aminopyridine but more resistant to tetraethylammonium and charybdotoxin than the Ca²⁺-sensitive rectification. A 4-aminopyridine-sensitive outward current (I _K,dr) with a threshold of around –30 mV and a second Ca²⁺-sensitive outward current (I _K,Ca) activated at around + 30 mV were observed from whole-cell voltage clamp recordings. I _K,Ca was blocked by tetraethylammonium and charybdotoxin. An 11-pS and a 122-pS channel, having characteristics similar to I _K,dr and I _K,Ca respectively, were identified from single-channel recordings. These observations showed how membrane depolarization of vascular smooth-muscle cells was regulated by these two populations of K⁺ channels under various conditions.     

ATP-Dependent inhibition of Ca2+-activated K+ channels in vascular smooth muscle cells by neuropeptide Y

Neuropeptide Y(NPY) inhibits Ca²⁺-activated K⁺ channels reversibly in vascular smooth muscle cells from the rat tail artery. NPY (200 M) had no effect in the absence of intracellular adenosine 5triphosphate (ATP) and when the metabolic poison cyanide-M-chlorophenyl hydrozone (10 M) was included in the intracellular pipette solution. NPY was also not effective when ATP was substituted by the non-hydrolysable ATP analogue adenosine 5-[, -methylene]-triphosphate (AMP-PCP). NPY inhibited Ca²⁺-activated K⁺ channel activity when ATP was replaced by adenosine 5-O-(3-thiotriphosphate) (ATP [-S]) and the inhibition was not readily reversed upon washing. Protein kinase inhibitor (1 M), a specific inhibitor of adenosine 3, 5-cyclic monophosphatedependent protein kinase, had no significant effect on the inhibitory action of NPY. The effect of NPY on single-channel activity was inhibited by the tyrosine kinase inhibitor genistein (10 M) but not by daidzein, an inactive analogue of genistein. These observations suggest that the inhibition by NPY of Ca²⁺-activated K⁺ channels is mediated by ATP-dependent phosphorylation. The inhibitory effect of NPY was antagonized by the tyrosine kinase inhibitor genistein.     

Ca2+-activated K+ channels in airway smooth muscle are inhibited by cytoplasmic adenosine triphosphate

Large-conductance Ca²⁺-activated K⁺ channels were studied in membranes of cultured rabbit airway smooth muscle cells, using the patch-clamp technique. In cell-attached recordings, channel openings were rare and occurred only at very positive potentials. Bradykinin (10 M), an agonist which releases Ca²⁺ from the sarcoplasmic reticulum, transiently increased channel activity. The metabolic blocker 2,4-dinitrophenol (20 M), which lowers cellular adenosine triphosphate (ATP) levels, induced a sustained increase of channel activity in cell-attached patches. In excised patches, these channels had a slope conductance of 155 pS at 0 mV, were activated by depolarization and by increasing the Ca²⁺ concentration at the cytoplasmic side above 10^–7 mol/l. ATP, applied to the cytoplasmic side of the patches, dose-dependently decreased the channel's open-state probability. An inhibition constant (K _i) of 0.2 mmol/l was found for the ATP-induced inhibition. ATP reduced the Ca²⁺ sensitivity of the channel, shifting the Ca²⁺ activation curve to the right and additionally reducing its steepness. Our results demonstrate that cytoplasmic ATP inhibits a large-conductance Ca²⁺-activated K⁺ channel in airway smooth muscle. This ATP modulation of Ca²⁺-activated K⁺ channels might serve as an important mechanism linking energy status and the contractile state of the cells.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Single K+ channels in membrane evaginations of smooth muscle cells

Attempts have been made to apply the patch-clamp technique to enzymatically dispersed smooth muscle cells of frog and toad stomach. The rate of successful gigaseal formation has been extremely low, but better results can be obtained when patches are taken from membrane evaginations which develop on single cells after mechanical agitation and incubation in Ca²⁺-containing solutions at 25° C. Also ball-shaped single cells formed by the confluence of membrane evaginations were found to be equally useful for patch-clamp studies. Giga-seal formation was obtained in more than 80% of all attempts. Electron micrographs indicate that the myofilaments in membrane evaginations an in ball-shaped cells are separated from the cell membrane. Channel activity in membrane patches of such myoballs or evaginations is similar to the channel activity as found in intact cells. Two types of K⁺ channels (100 and 200 pS) have been observed that can be blocked by tetraethylammonium. Channels with the conductance of 200 pS are activated by intracellular Ca²⁺. The formation of evaginations has also been observed in other cells and may help to apply the patch-clamp technique to cells contaminated with surface coats.     

Cromakalim and lemakalim activate Ca2+-dependent K+ channels in canine colon

The effects of cromakalim (BRL 34915) and its (–) optical isomer, lemakalim (BRL 38227) on the activity of 265-pS Ca²⁺-activated K⁺ channels (BK channels) were examined in cell-attached and inside-out patches from canine colonic myocytes. In cell-attached patches lemakalim increased the open probability (P _o) of BK channels. Mean NP _o, where N is the number of channels per patch, at + 50 mV increased from 0.08 to 0.26 (20 M lemakalim). In inside-out patches, cromakalim and lemakalim increased channel NP _o rapidly and reversibly. This increase in NP _o was due to a shift in half-maximal activation. Glyburide (20 M) prevented the increase in NP _o caused by lemakalim in cell-attached patches and reversed the increase in NP _o in inside-out patches. Under conditions where Ca²⁺-activated K⁺ channels were maximally activated, lemakalim failed to increase current or induce a second type of K⁺ channel activity. When tetraethylammonium (200 M) was added to the pipette solution to block the BK channel half maximally, lemakalim also failed to induce a second type of channel. Adenosine triphosphate (1 or 2 mM) applied to the inner surface of inside-out patches had no effect on P _o of BK channels. Finally, the effects of lemakalim on ensemble average currents, constructed from multiple openings of BK channels in cell-attached patches was found to successfully mimic the effects of the drug on whole-cell membrane currents. We conclude that cromakalim and lemakalim activate BK channels in canine colonic cells. Whether this action participates in the membrane hyperpolarization and the decrease in frequency and duration of slow waves produced by these compounds in intact colonic muscles remains to be investigated.     

NADH and NAD modulates Ca2+-activated K+ channels in small pulmonary arterial smooth muscle cells of the rabbit

We have investigated the effect of NADH and NAD on the gating of large conductance Ca²⁺-activated K(K_Ca) channels in arterial smooth muscle cells isolated from small pulmonary artery(outer diameter <300μm) and ear artery, using the patch clamp technique. In the inside-out configuration, intracellularly applied 2 mM NADH inhibited the activity of K_Ca, channels in pulmonary arterial smooth muscle cells, while it had no significant effect on ear arterial smooth muscle cells. On the other hand, 2 mM NAD increased the opening of K_Ca, channels in pulmonary arterial smooth muscle cells. The effects of another intracellular redox couple, glutathione(GSH) and glutathione disulfide(GSSG) were also dependent on their redox potentials. GSH(5 mM) inhibited K_Ca. channels activity, while GSSG(5 mM) increased the activity of pulmonary arterial smooth muscle cells. It could be concluded that the modulation of K_Ca channels by intracellular redox state contributes, at least in part, to the hypoxic suppression of outward current in pulmonary arterial smooth muscle cells.     

Caffeine induces periodic oscillations of Ca2+-activated K+ current in pulmonary arterial smooth muscle cells

The periodic oscillations of outward currents were studied in smooth muscle cells of the rabbit pulmonary artery. The combined stimuli of superfusion with 1 mM caffeine and depolarization of the membrane potential to 0 mV evoked periodic oscillations of outward currents with fairly uniform amplitudes and intervals. The oscillating outward currents induced by caffeine were dependent on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and had a reversal potential near to the equilibrium potential for K⁺. So the oscillating outward currents are carried by K⁺ through Ca²⁺-dependent K⁺ channels (I _K(Ca)), and may reflect the oscillations of [Ca²⁺]_i. The oscillating outward currents were abolished, or their frequency reduced, by lowering external [Ca²⁺], Ca²⁺ channel blockers, or by 1 M ryanodine, indicating that: (1) there is a continuous influx of Ca²⁺ through the plasma membrane at a holding potential of 0 mV; (2) the periodic transient increases of [Ca²⁺]_i are ascribed to the rhythmic release of Ca²⁺ from ryanodinesensitive intracellular store by the mechanism of Ca²⁺-induced Ca²⁺ release (CICR). On the basis of the above results, we simulated the oscillation of [Ca²⁺]_i induced by caffeine, which is known to lower the threshold of CICR. The patterns of peak amplitude histograms of spontaneous transient outward currents (STOC) in the oscillating cells were different from those in non-oscillating cells. The amplitudes of STOC in the latter were more variable than those in the former. The oscillating outward currents were modulated by 1 M forskolin and 1 M sodium nitroprusside, but STOC were little affected. The above differences between STOC and oscillating outward currents suggest that the two currents are activated by the Ca²⁺ originating from different intracellular Ca²⁺ stores which are functionally heterogeneous.     

Stretch-inactivated cationic channels in single smooth muscle cells

Stretch-inactivated channels (SICs) were identified in single smooth muscle cells freshly dissociated from the stomach of the toad, Bufo marinus. In both cell-attached and excised inside-out patches, negative pressure applied to the extracellular surface of the membrane patch suppressed the activity of SICs. These channels were permeable to cations and were not significantly permeable to Cl^–. The current-voltage relationship showed outward rectification in cell-attached patches with high NaCl in the pipette solution (2 mM MgCl₂), and the slope conductance at negative potentials was approximately 8 pS under these conditions. When divalent cations were eliminated from the pipette solution, the slope conductance at negative potentials increased to 30 pS. No significant voltage dependence of SIC gating could be observed between –100 mV and 60 mV.     

Voltage-dependent K+ channels in the sarcolemma of mouse skeletal muscle

The voltage-dependent K⁺ channels of the mammalian sarcolemma were studied with the patch-clamp technique in intact, enzymatically dissociated fibres from the toe muscle of the mouse. With a physiological solution (containing 2.5 mM K⁺) in the pipette, depolarizing pulses imposed on a cell-attached membrane patch activated K⁺ channels with a conductance of about 17 pS. No channel activity was observed when the pipette solution contained 2mM tetraethylammonium (TEA), or 2 mM 4-aminopyridine (4-AP). Whole cell recordings from these very small muscle fibres showed the well-known delayed rectifier K⁺ outward current with a threshold of about -40mV. The whole-cell current was completely blocked by 2 mM TEA in the bath, suggesting that the TEA-sensitive channels in the patch were also delayed rectifier channels. The inactivation properties of the channels were studied in the cell-attached mode. Averaged single-channel traces showed at least two types of channels discernible by their inactivation time course at a test potential of 60 mV. The fast type inactivated with a time constant of about 150ms, the slow type with a time constant of about 400 ms. A little channel activity always remained during pulses lasting several minutes, indicating either the presence of a very slowly inactivating third type of K⁺ channel, or the tendency of the fast inactivating channels to re-open at constant voltage. No difference was seen in the single-channel amplitudes of the different types of K⁺ channels. The well characterized adenosine-5-triphosphate-(ATP)-sensitive and Ca²⁺-dependent K⁺ channels, although present, were not active under the conditions used. The results suggest that in mouse skeletal muscle the delayed rectifier channels to not only carry the outward current during excitation but are also responsible for the resting K⁺ conductance.     

Tetrandrine blocks a slow,large-conductance,Ca2+-activated potassium channel besides inhibiting a non-inactivating Ca2+ current in isolated nerve terminals of the rat neurohypophysis

The effects of tetrandrine, a bis-benzyl-isoquinoline alkaloid, on voltage-gated Ca²⁺ currents (I _Ca) and on Ca²⁺-activated K⁺ current (I _K(Ca)) and channels in isolated nerve terminals of the rat neurohypophysis were investigated using patch-clamp techniques. The non-inactivating component of I _Ca was inhibited by external tetrandrine in a voltage- and dose-dependent manner, with an IC₅₀=10.1 M. I _K(Ca) was elicited by depolarizations when approximately 10 M Ca²⁺ was present on the cytoplasmic side. Only externally applied tetrandrine, at 1 M, decreased the amplitude of I _K(Ca), whereas the fast inward Na⁺ current and transient outward K⁺ current were not affected. Tetrandrine, applied to the extracellular side of outside-out patches excised from the nerve terminals, induced frequent and short closures of single type II, maxi-Ca²⁺-activated K⁺channels. Tetrandrine decreased the channel-open probability, within bursts, with an IC₅₀=0.21 M. Kinetic analysis of the channel activity showed that the open-time constant decreased linearly with increasing tetrandrine concentrations (0.01–3 M), giving an association rate constant of 8.8×10⁸ M^–1s^–1, whereas the arithmetic mean closed time did not change, giving a dissociation rate constant of 136.6s^–1. These results show that tetrandrine is a high-affinity blocker of the type II, maxi-Ca²⁺-activated K⁺ channel of the rat neurohypophysial terminals.     

Biophysical properties of Ca2+- and Mg-ATP-activated K+ channels in pulmonary arterial smooth muscle cells isolated from the rat

A novel class of Ca²⁺-activated K⁺ channel, also activated by Mg-ATP, exists in the main pulmonary artery of the rat. In view of the sensitivity of these K_Ca,ATP channels to such charged intermediates it is possible that they may be involved in regulating cellular responses to hypoxia. However, their electrophysiological profile is at present unknown. We have therefore characterised the sensitivity of K_Ca,ATP channels to voltage, intracellular Ca²⁺ ([Ca²⁺]_i) and Mg-ATP. They have a conductance of 245 pS in symmetrical K⁺ and are approximately 20 times more selective for K⁺ ions than Na⁺ ions, with a K⁺ permeability (P _K) of 4.6×10^–13cm s^–1. Ca²⁺ ions applied to the intracellular membrane surface of K_Ca,ATP channels causes a marked enhancement of their activity. This activation is probably the result of simultaneous binding of at least two Ca²⁺ ions, determined using Hill analysis, to the channel or some closely associated protein. This results in a shift of the voltage activation threshold to more hyperpolarized membrane potentials. The activation of K_Ca,ATP channels by Mg-ATP has an EC₅₀ of approximately 50 M. Although the EC₅₀ is unaffected by [Ca²⁺]_i, channel activation by Mg-ATP is enhanced by increasing [Ca²⁺]_i. One possible interpretation of these data is that Mg-ATP increases the sensitivity of K_Ca,ATP channels to Ca²⁺. It is therefore possible that under hypoxic conditions, where lower levels of Mg-ATP may be encountered, the sensitivity of K_Ca,ATP channels to Ca²⁺ and therefore voltage is reduced. This would tend to induce a depolarising influence, which would favour the influx of Ca²⁺ through voltage-activated Ca²⁺ channels, ultimately leading to increased vascular tone.     

Hypotonic shock evokes opening of Ca2+-activated K channels in opossum kidney cells

Using the patch clamp technique we show that exposure of opossum kidney cells to hypotonic shock evokes an outward rectifying potassium current. The corresponding single channel slope conductance approaches 15 pS at depolarizing voltages. The K current also becomes activated after addition of the ionophore A23187 to an isotonic bath medium containing Ca²⁺. We therefore conclude that the K selective channels are modulated by an elevation of cytoplasmic Ca²⁺. Evidence is presented that release of Ca²⁺ from internal stores is involved.     

Comparison of Ca2+-dependent K+ channels in the membrane of smooth muscle cells isolated from adult and foetal human aorta

Ca²⁺-activated K⁺ ionic currents in the membrane of cultured smooth muscle cells isolated from foetal and adult human aorta were studied using whole cell and single-channel patch-clamp techniques. Whole cell currents in adult smooth muscle cells were 3–8 times larger than in foetal cells of similar sizes. The elementary conductance and ionic selectivity of single Ca²⁺-activated K⁺ were identical for both types of cells. Channel openings occurred in burst, the duration of which was 3–5-fold longer in adult than in foetal cells. The voltage dependency of the channel activating mechanism and the dependency of the mean open time on the Ca²⁺ concentration on the inner side of the membrane were similar for both types of cells. These results suggest that the main reason for the increase in potassium conductance during development is an alteration in the open time of the Ca²⁺-activated K⁺ channels.     

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     

Neomycin inhibits K+-induced force and Ca2+ channel current in rat arterial smooth muscle

 The techniques of small vessel isometric myography and patch clamp were used to investigate the action of neomycin on K⁺-induced isometric force and voltage-gated Ca²⁺ channel currents in rat arterial smooth muscle. Neomycin and the dihydropyridine (DHP) Ca²⁺ channel antagonist (–)202–791 concentration-dependently and reversibly inhibited 40 mM K⁺-induced isometric force in rings of rat mesenteric and basilar arteries (IC₅₀ values of 70 μM and 1.2 nM, respectively, n = 10 and 4). Elevation of [Ca²⁺]_o by a factor of 2 significantly reduced the IC₅₀ values for inhibition of K⁺-induced force for both neomycin and (–)202–791 (192 μM and 3.7 nM, respectively, n = 6 and 4), but did not affect the Hill coefficient of the concentration/effect relationships. In patch-clamp experiments using freshly isolated basilar arterial myocytes, the voltage-gated inward current carried by Ba²⁺ was reversibly and concentration-dependently inhibited by neomycin (IC₅₀ 32 μM, n = 3). The concentration/effect curve for inhibition of the inward Ba²⁺ current by neomycin was significantly shifted to the right when [Ba²⁺]_o was raised from 1.8 mM to 10 mM (IC₅₀ 144 μM, n = 8). Our findings suggest that neomycin relaxes high-K⁺-induced force in rat isolated mesenteric and basilar arteries largely by inhibition of voltage-dependent and DHP-sensitive Ca²⁺ channels. Received: 1 August 1996 / Received after revision and accepted: 11 September 1996     

Effects of extracellular K+ and Ca2+ on membrane potential,contraction and86Rb+ efflux in guinea-pig mesotubarium

The effects of varying extracellular concentrations of K⁺ and Ca²⁺ [K⁺]_o and [Ca²⁺]_o on force development and membrane potential were investigated in the guinea-pig mesotubarium. At [K⁺]_o up to 40 mM, spontaneous action potentials were present, while higher [K⁺]_o gave sustained contractures at a stable membrane potential (−24 to −12 mV for [K⁺]_o from 60 to 120 mM). Tension decreased successively with increasing [K⁺]_o from 30 to 120 mM. The relaxing potency of the dihydropyridine Ca²⁺ antagonist, felodipine, increased as the membrane was depolarized with increasing [K⁺]_o and action potentials ceased. These results are compatible with the existence of Ca²⁺ channels showing voltage-dependent affinity with dihydrophyridines. Increasing [Ca²⁺]_o from 2.5 to 10 mM caused membrane hyperpolarization by about 11 mV and was accompanied by a lower frequency of spontaneous contractions and a longer duration of the relaxation between contractions.⁸⁶Rb⁺ efflux measurements in 60 mM K⁺ in the absence and presence of felodipine revealed a Ca²⁺-dependent component of the voltage-activated efflux. In normal solution (5.9 mM K⁺), efflux in the presence of felodipine was similar to the minimal value during normal spontaneous activity. The results indicate regulation of the permeability of K⁺ channels by the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and suggest participation of such channels in the generation of the regularly occurring bursts of action potentials characteristic of spontaneous activity in the mesotubarium.     

Inhibitory effects of dihydropyridines on macroscopic K+ currents and on the large-conductance Ca2+-activated K+ channel in cultured cerebellar granule cells

In cultured cerebellar granule cells, we examined the effects of dihydropyridines (DHPs) on K⁺ currents, using the whole-cell recording configuration of the patch-clamp technique and on Ca²⁺-activated K⁺ channels (maxi K⁺ channels) using outside-out patches. We found that micromolar concentrations of nicardipine, nifedipine, (+) and (–) BAY K 8644, nitrendipine, nisoldipine and (–) nimodipine block 10–60% of macroscopic K⁺ currents. The most potent of these DHPs was nicardipine and the least potent, (–) BAY K 8644. (+) Nimodipine had no effect on this current. The inhibitory effects of nifedipine and nicardipine were not additive with those of 1 mM tetraethylammonium (TEA). Outside-out recordings of maxi K⁺ channels showed a main conductance of 200 pS (in 77% of the patches) and two subconductance states (in 23% of the patches). Neither nifedipine nor nicardipine affected the main conductance, but decreased the values of the subconductance levels. In 10% of these patches, nicardipine induced a flickering activity of the channel. These findings show that both Ca²⁺ and K⁺ channels have DHP-sensitive sites, suggesting similarity in electrostatic binding properties of these channels. Furthermore, cerebellar granule cells may express different subtypes of maxi K⁺ channels having different sensitivities to DHPs. These drugs may provide new tools for the molecular study of K⁺ channels.