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     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

Ion channels in isolated mouse jejunal crypts

 The patch-clamp technique was used to characterise the ion channels in cells located in the mid region of mouse jejunal crypts. Six different channels were seen. A large outwardly rectified K⁺ channel (BK) (conductance, g at 0 mV = 92 ± 6 pS), which was highly selective for K⁺ [P _K ⁺ (1) > P _Rb ⁺ (0.6) >> P _Cs ⁺ (0.09) ≈ P _Na ⁺ (0.07) > P _Li ⁺ (0.04)], had a low, voltage-independent open probability (P _o) in the on-cell (O/C) configuration and appeared in 66% of the patches. In inside-out (I/O) patches, this channel had a linear current/voltage (I/V) relationship (g = 132 ± 3 pS), P _o was voltage dependent and it was blocked by cytoplasmic Ba²⁺ (5 mmol/l). An intermediate K⁺ channel (IK) which was present in 49% of O/C patches, had a linear I/V (g = 38 ± 3 pS), ran-down in O/C patches, and was not seen in I/O patches. A number of smaller channels (SC) with conductances ranging from 5 to 20 pS were seen in 16% of O/C patches. Also present in the basolateral membrane were a Cl^– channel (ICOR) and a nonselective cation channel (NSCC). These channels were only seen in I/O patches. ICOR had an outwardly rectified conductance (g at 0 mV = 36 ± 2 pS), its P _o was independent of voltage and unaffected by variations in cytoplasmic Ca²⁺ (100 nmol/l to 1 mmol/l) or ATP (0–1 mmol/l). The NSCC had a linear conductance (20 ± 1 pS), its P _o increased with depolarisation and elevation of cytoplasmic [Ca²⁺] (≥ 10 μmol/l), but was reduced by cytoplasmic ATP. None of the basolateral channels described here were activated by cAMP-dependent secretagogues, although a Cl^– conductance was activated. This cAMP-dependent Cl^– conductance was distinct from the basolateral Cl^– channel and thus is most likely located in the apical membrane. Received: 25 June 1997 / Accepted: 14 October 1997     

Identification of a voltage- and calcium-dependent non-selective cation channel in cultured adult and fetal human nasal epithelial cells

The apical membranes of cultured human nasal epithelial cells from adults and fetuses were investigated with the patch-clamp technique. Amiloride-insensitive, calcium- and voltage-dependent, non-selective cation channels were found in 4% of the cell-attached, and 18% of the inside-out and outside-out patches (n=412). Multiple functional channels were present in more than 90% of these patches, with a mean of 3.9 channels per patch (n=55). The current-voltage relationship can be described by the Goldman equations and the single channel conductance was 20.1±0.3 pS (n=29) in adult and 20.7±0.4 pS (n=44) in fetal cells in symmetrical 150mM NaCl solutions. The channels were highly selective for cations: P_Na/P_Cl was 30 in adult and 45 in fetal experiments. They were equally permeable for K⁺ and Na⁺, somewhat less for Cs⁺, and impermeable for choline⁺ and tetraethylammonium⁺. The open probability was voltage dependent: it increased approximately 2-fold with 30mV depolarization in the potential range from −60mV to +60mV. The channels were activated by Ca²⁺ concentrations of about 10⁻⁴M at the cytoplasmic side, but were insensitive to extracellular Ca²⁺ and amiloride (10⁻⁴M). The non-selective cation channels found in apical membranes of cultured fetal nasal epithelial cells were not different from the adult ones.     

Small (SKCa) Ca2+-activated K+ channels in cultured rat hippocampal pyramidal neurones

 Small (SK_Ca) Ca²⁺-activated K⁺ channels were identified in membrane patches excised from cultured CA1-CA3 pyramidal neurones of the neonatal rat hippocampus. When recorded in low-K⁺ extracellular solution ([K⁺]_o=2.5 mM), SK_Ca channels had a low conductance (@3 pS at 0 mV), were activated by ≥175 nM Ca²⁺ (P _o=0.54 at 500 nM Ca²⁺) and there were two open-time components (2.1 and @70 ms) to their activity. These properties of single SK_Ca channels are similar to those of slow after-hyperpolarization channels (sAHP) previously inferred from fluctuation analysis of the sAHP current. It is concluded that the SK_Ca channel reported here may be the channel that generates the sAHP in hippocampal pyramidal neurones. Received: 9 July 1998 / Received after revision: 5 October 1998 / Accepted: 7 October 1998     

Ryanoids change the permeability of potassium channels of locust (Schistocerca gregaria) muscle

The plasma membrane of locust skeletal muscle contains two types of K⁺ channel; a maxi, Ca²⁺-activated 170 pS channel (BK channel) and an inward rectifier of 35 pS conductance (IK channel). The effects of ryanodine, 9, 21-didehydroryanodine and 9, 21-didehydroryanodol on these channels have been investigated. In the concentration domain 10⁻⁹ M to 10⁻⁵ M, ryanodine irreversibly induced a dose-dependent reduction of the reversal potential (V _rev) of the channel currents, measured under physiologically normal K⁺ and Na⁺ gradients, i.e. from ≈60 mV in the absence of ryanodine to ≈15 mV for 10⁻⁵ M ryanodine. The alteration of K⁺ channel selectivity was Ca²⁺ independent. 9, 21-Didehydroryanodine and 9, 21-didehydroryanodol reduced V _rev, but only to ≈35 mV during application of 10⁻⁵ M of these compounds. 9, 21-Didehydroryanodine also diminished the conductances of the locust K⁺ channels. The three ryanoids reduced V _rev of a Ca²⁺-activated, high-conductance channel in inside-out patches excised from mouse interosseal muscle, although the changes were in each case less pronounced than those for the locust K⁺ channels. Also, the action of 9, 21-didehydroryanodine on mouse K⁺ (BK) channels was restricted to a shift of V _rev. For all three ryanoids, the IC₅₀ coefficients (i.e. the concentration of toxin that gave a 50% reduction in V _rev) for the shifts in V _rev were similar for the locust and mouse muscle K⁺ channels. Received: 12 June 1995/Received after revision and accepted: 30 January 1996     

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     

Characteristics of the delayed rectifier K current compared in myocytes isolated from the atrioventricular node and ventricle of the rabbit heart

The delayed rectifier potassium current (I _K) is known to be important in action potential repolarisation and may contribute to the diastolic pacemaker depolarisation in pacemaker cells from the heart. In this study, using whole-cell patch clamp, we investigated the characteristics of I _K in morphologically normal cells from the atrioventricular node (AVN) and ventricle of the rabbit heart. Cells were held at −40 mV and 5 μM external nifedipine was used to block L-type calcium current (I _Ca,L). Significant I _K was observed with pulses to potentials more positive than −30 mV. The steady-state activation curve in both cell types showed maximal activation at between + 10 and + 20 mV. Half-maximal activation of I _K occurred at −4.9 and −4.1 mV with slope factors of 8.3 and 12.4 mV in ventricular and AVN cells, respectively. Using pulses of increasing duration, significant I _K tails after repolarisation from + 40 mV were observed with pulses of 20 ms and increased with pulses up to 100–120 ms in both cell types. Pulses of longer duration did not activate further I _K and this suggested that only the rapid component of I _K, called I _Kr, was present in either cell type. Moreover, I _K tails after pulses to all potentials were blocked completely by E-4031, a selective blocker of I _Kr. The reversal potential of I _K varied with the concentration of external K. Superfusion of AVN cells with medium containing 4, 15 and 40 mM [K⁺]_o resulted in reversal potentials of −81, −56 and −32 mV, respectively, which are close to values predicted if the I _K channel were highly selective for K. The time constants for deactivation of I _K in ventricle and AVN on return to −40 mV after a 500-ms activating pulse to + 60 mV were 480 ms and 230 ms, respectively. The faster deactivation of I _K in AVN cells was a distinguishing feature and suggests that there may be differences in the I _Kr channel protein between ventricular and AVN cells. Received: 24 July 1995 /Received after revision: 20 October 1995 /Accepted: 23 October 1995     

Regulation of ion channels in rat hepatocytes

 Patch-clamp studies have been performed to elucidate single ion channels in rat hepatocytes. In rat hepatocytes two types of ion channel have been identified: an inwardly rectifying K⁺ channel with a mean inward conductance of 55 ± 6.5 pS (n = 20) and a mean outward conductance of 25 ± 3.2 pS (n = 20) in the inside-out configuration with 145 mmol/l KCl on either side of the patch as well as an outwardly rectifying Cl^– channel with a mean outward conductance of 30 ± 4.5 pS (n = 8) and a mean inward conductance of 10 ± 2.3 pS (n = 6) in the inside-out configuration with symmetrical 145 mmol/l KCl. The open probability of these channels is virtually insensitive to Ca²⁺ activity on the intracellular side. Accordingly, the Ca²⁺ ionophore ionomycin had no effect on cell membrane potential. Dibutyryl-cAMP (db-cAMP) hyperpolarizes the cell membrane and increases the activity of the 55-pS inwardly rectifying K⁺ channel by reducing the duration of closure between bursts. Forskolin similarly hyperpolarizes the cell membrane. The inwardly rectifying K⁺ channel is inhibited by progesterone, while the outwardly rectifying Cl^– channel is insensitive to progesterone. Received: 21 May 1997 / Received after revision: 7 August 1997 / Accepted: 19 August 1997     

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     

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     

Exogenous lysophosphatidylcholine increases non-selective cation current in guinea-pig ventricular myocytes

Whole cell, patch-clamp studies were performed to examine the effect of lysophosphatidylcholine (LPC) on the membrane current in guinea-pig ventricular myocytes. The addition of 10 μM LPC to the external solution induced a membrane current which had a reversal potential of 0 mV. When Na⁺, the main cation in the external solution, was replaced by either K⁺, N-methyl-D-glucamine (NMG) or 90 mM Ca²⁺, LPC induced a current with the reversal potential near 0 mV, indicating that the current passed through a Ca²⁺-permeable non-selective cation channel. The order of the cationic permeability calculated from the reversal potential of the current was Cs⁺ > K⁺ > NMG > Na⁺ > Ca²⁺. Cl⁻ did not pass through the LPC-induced channel. The LPC-induced current was not blocked by Gd³⁺ in the external solution, nor by the absence of Ca²⁺ in the pipette solution. In conclusion, LPC induces a Ca²⁺-permeable non-selective cation channel in guinea-pig ventricular myocytes. Received: 11 September 1995/Received after revision: 3 January 1996/Accepted: 12 February 1996     

Differential distribution of two Ca2+-dependent and -independent K+ channels throughout receptive and basolateral membranes of bullfrog taste cells

We could identify two types of K⁺ channels, of 80 and 40 pS conductance, respectively, in the bullfrog taste cell membrane using excised and cell-attached configurations of the patch-clamp technique. The taste cell membrane could be divided into four membrane parts — receptive area, apical process, cell body and proximal process. The 80-pS K⁺ channels were dependent on voltage and Ca²⁺ and were located exclusively on the receptive membrane and the apical process membrane. The 40-pS K⁺ channels were independent of voltage and Ca²⁺. The open probability of 40-pS K⁺ channels was decreased by the simultaneous presence of cyclic adenosine monophosphate (cAMP) and adenosine triphosphate (ATP), and the suppressive effect was antagonized by protein kinase inhibitor (PKI). Although 40-pS K⁺ channels were found in a high density on the receptive and apical process membranes, the channels also were present in the other two parts of the taste cell membrane. These results suggest that the two different types of K⁺ channel in the bullfrog taste cells may play different roles in gustatory transduction.     

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.     

A Na+-activated K+ current (I K,Na) is present in guinea-pig but not rat ventricular myocytes

 The effects of removing extracellular Ca²⁺ and Mg²⁺ on the membrane potential, membrane current and intracellular Na⁺ activity (a ⁱ _Na) were investigated in guinea-pig and rat ventricular myocytes. Membrane potential was recorded with a patch pipette and whole-cell membrane currents using a single-electrode voltage clamp. Both guinea-pig and rat cells depolarize when the bathing Ca²⁺ and Mg²⁺ are removed and the steady-state a ⁱ _Na increases rapidly from a resting value of 6.4± 0.6 mM to 33±3.8 mM in guinea-pig (n=9) and from 8.9±0.8 mM to 29.3±3.0 mM (n=5) in rat ventricular myocytes. Guinea-pig myocytes partially repolarized when, in addition to removal of the bathing Ca²⁺ and Mg²⁺, K⁺ was also removed, however rat cells remained depolarized. A large diltiazem-sensitive inward current was recorded in guinea-pig and rat myocytes, voltage-clamped at –20 mV, when the bathing divalent cations were removed. When the bathing K⁺ was removed after Ca²⁺ and Mg²⁺ depletion, a large outward K⁺ current developed in guinea-pig, but not in rat myocytes. This current had a reversal potential of –80±0.7 mV and was not inhibited by high Mg²⁺ or glybenclamide indicating that it is not due to activation of non-selective cation or adenosine triphosphate (ATP)-sensitive K channels. The current was not activated when Li⁺ replaced the bathing Na⁺ and was blocked by R-56865, suggesting that it was due to the activation of K_Na channels. Received: 15 October 1998 / Received after revision: 22 January 1999 / Accepted: 5 February 1999     

Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. In excised inside-out patches exposed to symmetrical 150 mM KCl, two channel types with conductances in the ranges of 250–300 pS and 9–14 pS were routinely observed. The activity of the large conductance channel is enhanced by internal Ca²⁺ and by depolarization of the patch membrane. This channel contributes to the repolarization of Ca²⁺ action potentials but has a Ca²⁺ sensitivity at –50 mV that is too low for it to contribute to the resting membrane conductance. The small conductance channel is activated by much lower concentrations of Ca²⁺ at –50 mV, ad its open probability is not strongly voltage sensitive. In cell-attached patches from voltage-clamped cells, the small conductance channels were found to be active during slowly decaying Ca²⁺-activated K⁺ tails currents and during Ca²⁺-activated K⁺ currents stimulated by thyrotropin-releasing hormone induced elevations of cytosolic calcium. In cell-attached patches on unclamped cells, the small conductance channels were also active at negative membrane potentials when the frequency of spontaneously firing action potentials was high or during the slow afterhyperpolarization following single spontaneous action potentials of slightly prolonged duration. The small conductance channel may thus contribute to the regulation of membrane excitability.     

Membrane potential modulation of ionomycin-stimulated Ca<Superscript>2+</Superscript> entry via Ca<Superscript>2+</Superscript>/H<Superscript>+</Superscript> exchange and SOC in rat submandibular acinar cells

Ionomycin (IM) at 5 μM mediates the Ca²⁺/H⁺ exchange, while IM at 1 μM activates the store-operated Ca²⁺ entry channels (SOCs). In this study, the effects of depolarization on both pathways were examined in rat submandibular acinar cells by increasing extracellular K⁺ concentration ([K⁺]_o). IM (5 μM, the Ca²⁺/H⁺ exchange) increased the intracellular Ca²⁺ concentration ([Ca²⁺]_i) to an extremely high value at 151 mM [K⁺]_o. However, with increasing [K⁺]_o, the rates of Ca²⁺ entry decreased in a linear relationship. The reversal potential (E _rev) for the Ca²⁺/H⁺ exchange was +93 mV, suggesting that IM (5 μM) exchanges 1 Ca²⁺ for 1 H⁺. Thus, depolarization decreases the Ca²⁺ influx via the Ca²⁺/H⁺ exchange because of its electrogenicity (1 Ca²⁺ for 1 H⁺). On the other hand, IM (1 μM, the SOCs) abolished an increase in [Ca²⁺]_i at 151 mM [K⁺]_o. With increasing [K⁺]_o, the rate of Ca²⁺ entry immediately decreased linearly. The E _rev for the SOC was +3.7 mV, suggesting that the SOCs are nonselective cation channels and less selective for Ca²⁺ over Na⁺ (P _Ca/P _Na = 8.2). Moreover, an increase in extracellular Ca²⁺ concentration (20 mM) enhanced the Ca²⁺ entry via the SOCs at 151 mM [K⁺]_o, suggesting depolarization does not inhibit the SOCs and decreases the driving force for the Ca²⁺ entry. This suggests that membrane potential changes induced by a secretory stimulation finely regulate the [Ca²⁺]_i via the SOCs in rat submandibular acinar cells. In conclusion, IM increases [Ca²⁺]_i via two pathways depending on its concentration, the exchange of 1 Ca²⁺ for 1 H⁺ at 5 μM and the SOCs at 1 μM.     

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     

Block of BK (maxi K) channels of rat pituitary melanotrophs by Na+ and other alkali metal ions

The block of large-conductance calcium-activated potassium (BK) channels by internal and external alkali metal ions was studied in adult rat melanotrophs. Internal but not external 20 mM Na⁺ produced a strongly voltage-dependent, flickery block that was well-fitted to the Woodhull model by using a value of 140 mM for the dissociation rate constant at 0 mV [K _d(0)] and an equivalent valence (zδ) of 0.9. At a concentration of 20 mM external K⁺, Cs⁺ and Rb⁺, but not Li⁺, caused a rightward shift of the voltage dependence of the intracellular Na⁺ (Na⁺ _i ) block. This effect of K⁺, Cs⁺ and Rb⁺ was modelled by an equilibrium knock-out mechanism in which the block-relieving ion binds to a site located within the voltage field and consequently increases the off-rate of Na⁺. Internal Li⁺ caused little or no block whereas internal Cs⁺ caused a voltage-dependent block [K _d(0) ≈150 mM]. Flickery channel block observed in cell-attached patches was consistent with a cytoplasmic Na⁺ activity between 1 and 10 mM. Received: 22 January 1996 /Accepted: 26 March 1996