Effect of caffeine on K+ efflux in frog skeletal muscle

 The exposure of frog skeletal muscle to caffeine (3–4 mM) generates an increase of the K⁺ (⁴²K⁺) efflux rate coefficient (k _K,o) which exhibits the following characteristics. First it is promoted by the rise in cytosolic Ca²⁺ ([Ca²⁺]_i), because the effect is mimicked by ionomycin (1.25 μM), a Ca²⁺ ionophore. Second, the inhibition of caffeine-induced Ca²⁺ release from the sarcoplasmic reticulum (SR) by 40 μM tetracaine significantly reduced the increase in k _K,o (Δk _K,o). Third, charybdotoxin (23 nM), a blocker of the large-conductance Ca²⁺-dependent K⁺ channels (BK_Ca channels) reduced Δk _K,o by 22%. Fourth, apamin (10 nM), a blocker of the small-conductance Ca²⁺-dependent K⁺ channels (SK_Ca channels), did not affect Δk _K,o. Fifth, tolbutamide (800 μM), an inhibitor of K_ATP channels, reduced Δk _K,o by about 23%. Sixth, Ba²⁺, a blocker of most K⁺ channels, did not preclude the caffeine-induced Δk _K,o. Seventh, omitting Na⁺ from the external medium reduced Δk _K,o by about 40%. Eight, amiloride (5 mM) decreased Δk _K,o by 65%. It is concluded that the caffeine-induced rise of [Ca²⁺]_i increases K⁺ efflux, through the activation of: (1) two channels (BK_Ca and K_ATP) and (2) an external Na⁺-dependent amiloride-sensitive process. Received: 13 March 1998 / Received after revision: 17 June 1998 / Accepted: 14 September 1998     

Ca2+ regulated K+ and non-selective cation channels in the basolateral membrane of rat colonic crypt base cells

 We have previously shown that a new type of K⁺ channel, present in the basolateral membrane of the colonic crypt base (blm), is necessary for cAMP-activated Cl^- secretion. Under basal conditions, and when stimulated by carbachol (CCH) alone, this channel is absent. In the present patch clamp-study we examined the ion channels present in the blm under cell-attached and in cell-excised conditions. In cell-attached recordings with NaCl-type solution in the pipette we measured activity of a K⁺ channel of 16 ± 0.3 pS (n = 168). The activity of this channel was sharply increased by CCH (0.1 mmol/l, n = 26). Reduction of extracellular Ca²⁺ to 0.1 mmol/l (n = 34) led to a reversible reduction of activity of this small channel (SK_Ca). It was also inactivated by forskolin (5 μmol/l, n = 38), whilst the K⁺ channel noise caused by the very small K⁺ channel increased. Activity of non-selective cation channels (NS_cat) was rarely observed immediately prior to the loss of attached basolateral patches and routinely in excised patches. The NS_cat, with a mean conductance of 49 ± 1.0 pS (n = 96), was Ca²⁺ activated and required >10 μmol/l Ca²⁺ (cytosolic side = cs). It was reversibly inhibited by ATP (<1 mmol/l, n = 13) and by 3′,5-dichloro-diphenylamine-2-carboxylate (10–100 μmol/l, n = 5). SK_Ca was also Ca²⁺ dependent in excised inside-out basolateral patches. Its activity stayed almost unaltered down to 1 μmol/l (cs) and then fell sharply to almost zero at 0.1 μmol/l Ca²⁺ (cs, n = 12). SK_Ca was inhibited by Ba²⁺ (n = 31) and was charybdotoxin sensitive (1 nmol/l) in outside-out basolateral patches (n = 3). Measurements of the Ca²⁺ activity ([Ca²⁺]_i) in these cells using fura-2 indicated that forskolin and depolarization, induced by an increase in bath K⁺ concentration to 30 mmol/l, reduced [Ca²⁺]_i markedly (n = 8–10). Hyperpolarization had the opposite effect. The present data indicate that the blm of these cells contains a small-conductance Ca²⁺-sensitive K⁺ channel. This channel is activated promptly by very small increments in [Ca²⁺]_i and is inactivated by a fall in [Ca²⁺]_i induced by forskolin. Received: 15 April 1996 / Received after revision and accepted: 17 June 1996     

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor

 In this study, we have investigated the effect of the vasoconstrictor peptide endothelin-1 (ET-1) on voltage-sensitive Ca²⁺ channels in rat cerebellar granule neurones using the patch-clamp technique. Using amphotericin B perforated-patch recording of whole-cell currents, the Ca²⁺ channel current was inhibited by 28.4±6.4% by 400 nM ET-1, but was unaffected when experiments were repeated using the whole-cell, ruptured-patch configuration. In cell-attached patches, 400 nM ET-1 inhibited unitary L-type Ca²⁺ channel currents (I _Ba) by 85±5%. ET-1 decreased the open probability (NP _o) and the frequency of channel opening and increased the mean closed time of channels. No effects on the mean open time or the time constants for channel opening or closure were observed. L-type Ca²⁺ channel inhibition was dose dependent with an IC₅₀ of 19 nM. The effect of ET-1 was prevented by the combined endothelin-A and -B receptor antagonist PD145065 (10 μM), indicating a receptor-mediated effect. The ET-A receptor antagonist BQ-123 (10 μM) prevented Ca²⁺ channel inhibition by ET-1, while the ET-B receptor agonist sarafotoxin 6c (500 nM) had no effect. The inhibition by ET-1 was not due to a change in the voltage of channel activation. Fura-2 Ca²⁺ imaging showed that no substantial rise in intracellular Ca²⁺ levels occurred during ET-1 application excluding a Ca²⁺-dependent inhibition of the channels. Thus in cultured rat cerebellar granule neurones, ET-1 inhibits L-type Ca²⁺ channels via activation of the ET-A receptor. Inhibition may be mediated by an as yet unidentified cytoplasmic second messenger. Received: 13 March 1998 / Received after revision and accepted: 14 May 1998     

Ca2+-transients induced by K+ channel openers in isolated coronary capillaries

 To study the role of endothelial ATP-sensitive K⁺ channels in the regulation of vascular tone we examined the intracellular calcium concentration ([Ca²⁺]_i) in coronary capillaries consisting only of endothelial cells. Coronary capillary fragments were isolated enzymatically from the guinea-pig heart and [Ca²⁺]_i was determined by microfluorometry of fura-2 loaded cells. Low concentrations of the K⁺ channel opener diazoxide, which caused pronounced glibenclamide-sensitive hyperpolarization in capillaries, induced a rapid, transient rise in [Ca²⁺]_i followed by a sustained elevation of [Ca²⁺]_i (19 of 40 experiments). [Ca²⁺]_i in the endothelial cells increased from 32 ± 7 nM at rest to 66 ± 11 nM at the peak (n = 19). One third of the [Ca²⁺]_i-transients showed irregular oscillations of [Ca²⁺]_i. No significant difference in the [Ca²⁺]_i-response induced by 100 nM or 1 μM diazoxide was found. Similar results were obtained with the K⁺ channel opener rilmakalim. Simultaneous measurements of the membrane potential and [Ca²⁺]_i with fluorometric methods indicated that the hyperpolarization but not the [Ca²⁺]_i-transient could be repeatedly induced in a single capillary by the K⁺ channel openers. Electrophysiological recordings of the membrane potential using the ”perforated patch” method (n = 4), showed that rilmakalim (1 μM) induced hyperpolarization of capillaries towards the K⁺ equilibrium potential, confirming our fluorometric measurements. In conclusion, for the first time, these data indicate that K⁺ channel openers induce [Ca²⁺]_i-transients in microvascular endothelial cells. This raises the possibility that these drugs not only act as synthetic vasoactive factors via hyperpolarizing smooth muscle cells but also via NO release of microvascular endothelial cells. Interestingly, only 100 nM diazoxide was sufficient for a maximal response, suggesting the expression of a new type of K_ATP-channel in coronary capillaries characterised by high sensitivity to diazoxide. Received: 22 August 1997 / Received after revision and accepted: 7 November 1997     

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     

Hypertonic cell shrinkage reduces the K+ conductance of rat colonic crypts

 It has previously been shown in studies of a renal epithelial cell line that nonselective cation (NSC) channels are activated by exposure to hypertonic solution. We have also found such channels in excised patches of colonic crypt cells. They require high Ca²⁺ activities on the cytosolic side and a low ATP concentration for their activation and have not been recorded from cell-attached patches of colonic crypts. We examine here whether this type of channel is activated by hypertonic cell shrinkage. Bath osmolality was increased by addition of 25, 50 or 100 mmol/l mannitol. Cell-attached and whole-cell patch recordings were obtained from rat base and mid-crypt cells. In whole-cell recordings we found that addition of 50 or 100 mmol/l mannitol depolarized these cells significantly from –78±2.0 to –66±3.8 mV (n=22) and from –78±1.3 to –56±2.6 mV (n=61), respectively, and reduced the whole-cell conductance from 20±8.0 to 14±6.6 nS (n=7) and from 20±3.0 to 9.8±1.6 nS (n=19), respectively. In cell-attached patches K⁺ channels with a single-channel conductance of ≈16 pS were found in most recordings. The activity of these channels (N×P _o, N=number, P _o=open channel probability) was reduced from 2.08±0.37 to 0.98±0.23 (n=15) by the addition of 50 mmol/l mannitol and from 1.75±0.26 to 0.77±0.20 (n=12) by 100 mmol/l mannitol. No NSC channel activity was apparent in any of these recordings. Previously we have shown that the 16-pS K⁺ channel is controlled by cytosolic Ca²⁺ ([Ca²⁺]_i). Therefore we measured [Ca²⁺]_i by the fura-2 method and found that hypertonic solution reduced [Ca²⁺]_i significantly (n=16). These data indicate that exposure of rat colonic crypts to hypertonic solutions does not activate NSC channels; [Ca²⁺]_i falls in hypertonic solution leading to a reduction in the value of K⁺ channel N×Po, a reduced whole-cell conductance and depolarization of mid-crypt cells. These processes probably assist volume regulation inasmuch as they reduce KCl losses from the cell. Received: 21 July 1997 / Received after revision: 24 November 1997 / Accepted: 15 December 1997     

Modulation of Ca2+-activated K+ channels of human erythrocytes by endogenous cAMP-dependent protein kinase

 Single Ca²⁺-activated K⁺ channels of human erythrocytes were studied with the patch-clamp technique, to identify the mechanisms of their modulation by phosphorylation. In the cell-attached configuration, the openings of these channels were infrequent, as expected by the low cell Ca²⁺ content. After patch excision, the activity increased to levels determined by the Ca²⁺ concentration (0.5–10 μM) in the bath solution, then the channel activity ran down within a few minutes, to reach values of open probability lower than 0.10. The perfusion of the patch with MgATP increased the channel activity, with delayed and variable effects. Furthermore, the application of a mixture of cAMP (1 mM), MgATP (1 mM) and theophylline (1 mM) to the cytoplasmic side of excised patches led to dramatic enhancement of channel activity, which appeared within 20–120 s and decayed in tens of seconds after wash-out. The activation of the channel by the mixture was reversibly blocked by PKI_5–24, a peptide inhibitor specific to cAMP-dependent protein kinase (PKA). The level of activation promoted by cAMP and ATP was dependent on the Ca²⁺ concentration in the bathing solution. These results provide direct evidence that an endogenous PKA modulates the calcium sensitivity of Ca²⁺-activated K⁺ channels of human erythrocytes. Received: 19 February 1998 / Received after revision: 14 April 1998 / Accepted: 20 April 1998     

Glibenclamide suppresses stretch-activated ANP secretion: involvements of K+ ATP channels and L-type Ca2+ channel modulation

 The mechanism by which glibenclamide regulates mechanically activated atrial natriuretic peptide (ANP) secretion was investigated using isolated perfused rat atria. A reduction in atrial pressure from an experimentally imposed distending pressure stimulated the secretion of ANP and caused concomitant translocation of extracellular fluid (ECF) into the atrial lumen. The activation of ANP secretion and ECF translocation were closely correlated with atrial volume changes and the increase in ANP secretion was a function of the ECF translocation. Glibenclamide (1, 10, 100 μM), an ATP-sensitive K⁺ (K⁺ _ATP) channel blocker, had no effect on the basal secretion of ANP, suppressed the stimulation of stretch-activated ANP secretion in a dose-dependent manner, but not the translocation of the ECF. Glipizide (100 μM) and tolbutamide (100 μM), other K⁺ _ATP channel blockers, had similar effects to those of glibenclamide. Suppression by glibenclamide (100 μM) of the stretch-induced ANP secretion was not observed in atria that had been pretreated with pinacidil (200 μM), an ATP-sensitive K⁺ channel opener: pinacidil alone had no effect on ECF translocation and ANP secretion. Furthermore, blocking Ca²⁺ influx by using the Ca²⁺ channel blocker diltiazem (10 nM), or a Ca²⁺-depleted medium prevented the suppression of stretch-induced ANP secretion by glibenclamide. In other experiments, atrial distension produced a slight membrane depolarization of cardiomyocytes; this was accentuated in the presence of glibenclamide. Furthermore, in single cardiomyocytes, glibenclamide increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in a dose-dependent manner. From these results, we suggest that glibenclamide suppresses atrial release of ANP by blocking K⁺ _ATP channels and increasing Ca²⁺ influx and that the K⁺ _ATP channels are associated with the regulation of the mechanically activated ANP secretion from the atria. Received: 13 May 1996 / Received after revision: 10 February 1997 / Accepted: 5 March 1997     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

Block of cloned BKCa channels (rSlo)expressed in HEK 293 cells by N-methyl d -glucamine

We have investigated the conductance properties of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels formed by stable expression of the rSlo gene in HEK 293 cells. Single-channel recordings were obtained from inside-out patches excised into solution containing 100 μM Ca²⁺ to ensure a relatively high open probability over the range of membrane potentials studied (–120 to +100 mV). The unitary conductance of these channels at +80 mV was 221.6±5.4 pS in symmetrical 140 mM K⁺. Decreasing the K⁺ concentration on either side of the membrane, while maintaining ionic strength by adding N-methyl d-glucamine (NMDG⁺), reduced the unitary conductance. The reduction in conductance was greater when internal K⁺ was lowered by replacement with NMDG⁺. However, if sucrose was used as the internal K⁺ substitute instead of NMDG⁺ the reduction in unitary conductance was similar to that seen on reducing external K⁺. A rate-theory model whereby NMDG⁺ produces a very rapid block of the BK_Ca channel from the inside, but not the outside, is able to describe our results. Received:18 May 1998 / Received after revision: 17 June 1998 / Accepted: 2 July 1998     

Spontaneous Na+ and Ca2+ spike firing of cerebellar Purkinje neurons at high pressure

 The effects of high pressure (up to 10.1 MPa) on the spontaneous firing of Purkinje neurons in guinea-pig cerebellar slices were studied using the macropatch clamp technique. Pressure did not significantly alter the single somatic Na⁺ spike parameters or the frequency of regular Na⁺ spike firing. When Na⁺ currents were blocked by 0.5–1 μM tetrodotoxin (TTX), a pressure of 10.1 MPa slightly reduced the dendritic Ca²⁺ spike amplitude to 90.2±3.1% of its control value, and slowed its kinetics. The effects of pressure on the single Ca²⁺ spike were even less prominent when K⁺ currents were blocked by 5 mM 4-aminopyridine (4-AP). Pressure prolonged the active period of Ca²⁺ spike firing to 152.2±10.4% of the control value. Within the active period pressure increased the inter-spike interval to 164.9±8.7% and suppressed the typical firing of doublets. The latter changes were reversed by a high extracellular potassium concentration ([K⁺]_o) and 1 μM 4-AP, whereas in the presence of 5 mM 4-AP the pattern was insensitive to pressure. A high [Ca²⁺]_o reduced the firing frequency and suppressed doublet firing in a manner reminiscent of the pressure effect, but these changes could not be reversed by 4-AP. A low [Ca²⁺]_o slightly increased the firing of doublets. These results show that the single somatic Na⁺ spike is insensitive and the dendritic Ca²⁺ spike is only mildly sensitive to pressure. However, alterations in Ca²⁺ spike firing pattern suggest that modulation of dendritic K⁺ currents induce depression of dendritic excitability at pressure. Received: 19 May 1998 / Received after revision: 15 July 1998 / Accepted: 3 September 1998     

Na+-dependent regulation of the free Mg2+ concentration in neuropile glial cells and P neurones of the leech Hirudo medicinalis

 To investigate the Mg²⁺ regulation in neuropile glial (NG) cells and pressure (P) neurones of the leech Hirudo medicinalis the intracellular free Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) concentrations, as well as the membrane potential (E _m), were measured using Mg²⁺- and Na⁺-selective microelectrodes. The mean steady-state values of [Mg²⁺]_i were found to be 0.91 mM (mean E _m=–63.6 mV) in NG cells and 0.20 mM (mean E _m=–40.6 mV) in P neurones with a [Na⁺]_i of 6.92 mM (mean E _m=–61.6 mV) and 7.76 mM (mean E _m=–38.5 mV), respectively. When the extracellular Mg²⁺ concentration ([Mg²⁺]_o) was elevated, [Mg²⁺]_i in P neurones increased within 5–20 min whereas in NG cells a [Mg²⁺]_i increase occurred only after long-term exposure (6 h). After [Mg²⁺]_o was reduced back to 1 mM, a reduction of the extracellular Na⁺ concentration ([Na⁺]_o) decreased the inwardly directed Na⁺ gradient and reduced the rate of Mg²⁺ extrusion considerably in both NG cells and P neurones. In P neurones Mg²⁺ extrusion was reduced to 15.4% in Na⁺-free solutions and to 6.0% in the presence of 2 mM amiloride. Mg²⁺ extrusion from NG cells was reduced to 6.2% in Na⁺-free solutions. The results suggest that the major [Mg²⁺]_i-regulating mechanism in both cell types is Na⁺/ Mg²⁺ antiport. In P neurones a second, Na⁺-independent Mg²⁺ extrusion system may exist. Received: 11 August 1998 / Received after revision: 14 October 1998 / Accepted: 15 October 1998     

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.     

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     

Intracellular Na+ modulates large conductance Ca2+-activated K+ currents in human umbilical vein endothelial cells

We studied the effects of Na⁺ influx on large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels in cultured human umbilical vein endothelial cells (HUVECs) by means of patch clamp and SBFI microfluorescence measurements. In current-clamped HUVECs, extracellular Na⁺ replacement by NMDG⁺ or mannitol hyperpolarized cells. In voltage-clamped HUVECs, changing membrane potential from 0 mV to negative potentials increased intracellular Na⁺ concentration ([Na⁺]_i) and vice versa. In addition, extracellular Na⁺ depletion decreased [Na⁺]_i. In voltage-clamped cells, BK_Ca currents were markedly increased by extracellular Na⁺ depletion. In inside-out patches, increasing [Na⁺]_i from 0 to 20 or 40 mM reduced single channel conductance but not open probability (NPo) of BK_Ca channels and decreasing intracellular K⁺ concentration ([K⁺]_i) gradually from 140 to 70 mM reduced both single channel conductance and NPo. Furthermore, increasing [Na⁺]_i gradually from 0 to 70 mM, by replacing K⁺, markedly reduced single channel conductance and NPo. The Na⁺–Ca²⁺ exchange blocker Ni²⁺ or KB-R7943 decreased [Na⁺]_i and increased BK_Ca currents simultaneously, and the Na⁺ ionophore monensin completely inhibited BK_Ca currents. BK_Ca currents were significantly augmented by increasing extracellular K⁺ concentration ([K⁺]_o) from 6 to 12 mM and significantly reduced by decreasing [K⁺]_o from 12 or 6 to 0 mM or applying the Na⁺–K⁺ pump inhibitor ouabain. These results suggest that intracellular Na⁺ inhibit single channel conductance of BK_Ca channels and that intracellular K⁺ increases single channel conductance and NPo. GH Liang and MY Kim contributed equally to this publication and therefore share the first authorship.     

Reversible inhibition of I K,I AHP,I h and I Ca currents by internally applied gluconate in rat hippocampal pyramidal neurones

 Previously, we reported that the spike frequency adaptation and slow afterhyperpolarizations (sAHP) in hippocampal pyramidal neurones are best preserved during whole-cell recording with a methylsulfate (MeSO₄ ^–)- based internal solution, but undergo a fast rundown when gluconate- (Gluc^–)- based internal solution is used. Here we show, with internal perfusion of patch pipettes, the reversibility of the inhibitory effects of Gluc^–on spike frequency adaptation and sAHP, and extend these observations to fast and medium-duration AHPs. Contrary to what might be expected based on Gluc^–binding of Ca²⁺, the sAHP and its underlying current could be temporarily enhanced by adding 1–3 mM of the calcium chelator BAPTA to the internal solution in the presence of Gluc^–. Replacement of internal MeSO₄ ^–with Gluc^–did not affect the membrane resting potential or the amplitude and duration of action potentials, but reversibly increased the cell input resistance and decreased the threshold current for spike generation. Gluc^–reversibly inhibited the hyperpolarization-activated non-selective cationic current (I _h), the depolarization-activated delayed rectifier K⁺ current (I _K), the high-voltage-activated Ca²⁺ current and the Ca²⁺-activated K⁺ current that underlies the sAHP. The combination of these effects of Gluc^–significantly alters the electrophysiological ”fingerprint” of the neurone. Received: 19 April 1996 / Received after revision: 12 July 1996 / Accepted: 3 September 1996     

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     

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.     

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.     

cAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na⁺ channels in the oocyte plasma membrane. Poly(A)⁺ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na⁺ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)⁺ RNA from HS-fed animals [from hereon referred to as HS-poly(A)⁺ RNA] into oocytes induced the expression of amiloride-sensitive Na⁺ conductances. On the other hand, oocytes injected with poly(A)⁺ RNA from LS-fed animals [LS-poly(A)⁺ RNA] expressed a markedly larger amount of amiloride-blockable Na⁺ conductances. LS-poly(A)⁺ RNA-induced conductances were completely inhibitable by amiloride with a K _i of 77 nM, and were also blocked by benzamil with a K _i of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 μM), had no detectable effect on the Na⁺ conductances. Expressed amiloride-sensitive Na⁺ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na⁺ was replaced by K⁺, amiloride (1 μM) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (P _o) between 0.4 and 0.9, and slope conductances of 3.8 pS for Na⁺ and 5.6 pS for Li⁺. The expressed channels showed to be highly selective for Na⁺ over K⁺ with a permeability ratio P _Na/P _K > 20. Amiloride (500 nM) reduced channel P _o to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na⁺ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels. Received: 16 October 1995/Received after revision: 30 November 1995/Accepted: 12 December 1995