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.     

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     

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.     

Ca2+ dependence of small Ca2+-activated K+ channels in cultured N1E-115 mouse neuroblastoma cells

Single-channel properties of Ca²⁺-activated K⁺ channels have been investigated in excised membrane patches of N1E-115 mouse neuroblastoma cells under asymmetric K⁺ concentrations at 0 mV. The SK channels are blocked by 3 nM external apamin, are unaffected by 20 mM external tetraethylammonium (TEA) and have a single-channel conductance of 5.4 pS. The half-maximum open probability and opening frequency of SK channels are observed at 1 M internal Ca²⁺. Concentration/effect curves of these parameters are very steep with exponential slope factors between 7 and 13. Opentime distributions demonstrate the existence of at least two open states. The mean short open time increases with [Ca²⁺]_i, whereas the mean long open time is independent of [Ca²⁺]_i. At low [Ca²⁺]_i the short-lived open state predominates. At saturating [Ca²⁺]_i the number of longlived openings is more enhanced than the number of short-lived openings and both open states occur equally frequently. The opening frequency as well as the open times of SK channels are independent of the membrane potential in the range of –16 to +40 mV. The results indicate that activation of K⁺ current through SK channels is mainly determined by the Ca²⁺-dependent single-channel opening frequency. BK channels in N1E-115 cells are insensitive to 100 nM external apamin, are sensitive to external TEA in the millimolar range and have a single-channel conductance of 98 pS. Half-maximum open probability and opening frequency of the BK channel are observed at 7.5–21 M internal Ca²⁺. The slope factors of concentration/effect curves range between 1.7 and 2.9. As the BK channel open time is markedly enhanced at raised [Ca²⁺]_i, the Ca²⁺ dependence of the current through BK channels is determined by the single-channel opening frequency as well as the open time. SK as well as BK channels appear to be clustered and interact in a negative cooperative manner in multiple channel patches. The differences in Ca²⁺ dependence suggest that BK channels are activated by a local high [Ca²⁺]_i associated with Ca²⁺ influx, whereas SK channels may be activated by Ca²⁺ released from internal stores as well.     

Miniature Ca2+ channels in excised plasma-membrane patches: activation by IP3

 In the present work, we characterized the receptor properties and the conductive features of the inositol (1,4,5)-trisphosphate (IP₃)-activated Ca²⁺ channels present in excised plasma-membrane patches obtained from mouse macrophages and A431 cells. We found that the receptor properties of the channels tested were similar to those of the IP₃ receptor (IP₃R) expressed in the endoplasmic reticulum (ER) membrane. These properties include activation by IP₃, inhibition by heparin, time-dependent inactivation by high IP₃ concentrations, activation by guanosine 5′o-thiotriphosphate and regulation by arachidonic acid. On the other hand, in terms of conductive properties, the channel closely resembles Ca²⁺-release-activated Ca²⁺ channels (I _crac). These conductive properties include extremely low conductance (≈1 pS), very high selectivity for Ca²⁺ over K⁺ (P _Ca/P _K>1000), inactivation by high intracellular Ca²⁺ concentration and, importantly, strong inward rectification. Notably, the same channel was activated by: (1) agonists in the cell-attached mode of channel recording, and (2) cytosolic IP₃ after patch excision. Although the possibility cannot be completely excluded that a novel type of IP₃R is expressed exclusively in the plasma membrane, in their entirety our findings suggest that the plasma membrane of mouse macrophages and A431 cells contains I _crac-like Ca²⁺ channels coupled to an IP₃-responsive protein which displays properties similar to those of the IP₃R expressed in the ER membrane. Received: 16 June 1998 / Received after revision: / 24 August 1998 / Accepted: 1 September 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     

Changes of the biophysical properties of calcium-activated potassium channels of rat skeletal muscle fibres during aging

 In the present work, we have investigated the effects of the aging process on Ca²⁺-activated K⁺ channels (K_Ca2+) of rat skeletal muscle fibres. K_Ca2+ channels of adult (5–7 months old) and aged (24–26 months old) rats were surveyed by the patch-clamp technique. In aged rats, K_Ca2+ channels were routinely detected on the surface membrane of the fibres in both cell-attached and inside-out configurations. Conversely, in adult rat fibres, K_Ca2+ channels were rarely detected. In the cell-attached configuration, the open probability of the aged rat K_Ca2+ channel, measured in the range of potentials from –60 mV to +20 mV, was about 1.5–2 times higher than that of the adult one. The number of functional channels was abnormally increased by aging. An average of three channels per patch/area was counted in the inside-out patches of aged rat fibres, whereas no more than one open channel per patch/area was detected in the adult rat fibres. The frequency of finding channels in the patches also increased with aging, i.e. 11.5% and 30.1% in the adult and in the aged rat fibres, respectively. However, no significant change in the single-channel conductance has been observed with aging: it was 227 pS and 231 pS for adult and aged rat channels, respectively. In detached patches, both the adult and aged rat channels showed a similar voltage dependence of open probability and a similar sensitivity to Ca²⁺ ions. The aging process did not alter the response of the single channel to charybdotoxin, or its modulation by nucleotides, MgATP and adenosine 5’-O-(3-thiotriphosphate) (ATP[γ-S]). On the other hand, charybdotoxin reduced the abnormally high resting macroscopic K⁺ conductance of the aged rat fibres, recorded using the two-intracellular-microelectrode technique. These findings indicate that, in skeletal muscle, the activity of K_Ca2+ channels increases with advancing age. Received: 10 April 1997 / Received after revision and accepted: 4 June 1997     

Calcium channels in smooth muscle cells from cerebral precapillary arterioles activate at more negative potentials than those from basilar artery

We compared Ca²⁺ channels in cell-attached patches of smooth muscle cells from cerebral precapillary arterioles and basilar artery of guinea pig. Patches were studied without Ca²⁺ channel activators in the pipette solution. In both preparations, a 23 pS channel (40 mM Ba²⁺) sensitive to block by nifedipine was identified. In arteriolar but not in basilar artery patches, channel activity was recorded without apparent inactivation at potentials of –40 to –20 mV. Values for the number of channels in a patch x probability of channel opening (n·P_o) at various potentials were fit to a Boltzmann function. For the arteriolar patches (n=5) and for patches from basilar artery (n=5), the midpoint potentials for the voltage dependence of n·P_o were –9.3 mV and +8.9 mV, and maximum values of n·P_o at positive potentials were 1.23 and 0.33. At potentials 0 mV, the average for the maximum number of superimposed openings in basilar artery patches was 1.7 (n=17) and in arteriolar patches was 6.5 (n=6). For both preparations, histograms of channel open times at –10 mV required two time constants, 0.48 and 3.95 ms, and the shorter open state accounted for 88% of openings. Our data indicate that Ca²⁺ channel activity is likely to be more prominent near resting membrane potentials in arteriolar cells than in basilar artery cells.     

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     

Ca2+ channels,ryanodine receptors and Ca2+-activated K+ channels: a functional unit for regulating arterial tone

Local calcium transients (‘Ca²⁺ sparks’) are thought to be elementary Ca²⁺ signals in heart, skeletal and smooth muscle cells. Ca²⁺ sparks result from the opening of a single, or the coordinated opening of many, tightly clustered ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). In arterial smooth muscle, Ca²⁺ sparks appear to be involved in opposing the tonic contraction of the blood vessel. Intravascular pressure causes a graded membrane potential depolarization to approximately ?40 mV, an elevation of arterial wall [Ca²⁺]_i and contraction (‘myogenic tone’) of arteries. Ca²⁺ sparks activate calcium-sensitive K⁺ (K_Ca) channels in the sarcolemmal membrane to cause membrane hyperpolarization, which opposes the pressure induced depolarization. Thus, inhibition of Ca²⁺ sparks by ryanodine, or of K_Ca channels by iberiotoxin, leads to membrane depolarization, activation of L -type voltage-gated Ca²⁺ channels, and vasoconstriction. Conversely, activation of Ca²⁺ sparks can lead to vasodilation through activation of K_Ca channels. Our recent work is aimed at studying the properties and roles of Ca²⁺ sparks in the regulation of arterial smooth muscle function. The modulation of Ca²⁺ spark frequency and amplitude by membrane potential, cyclic nucleotides and protein kinase C will be explored. The role of local Ca²⁺ entry through voltage-dependent Ca²⁺ channels in the regulation of Ca²⁺ spark properties will also be examined. Finally, using functional evidence from cardiac myocytes, and histological evidence from smooth muscle, we shall explore whether Ca²⁺ channels, RyR channels, and K_Ca channels function as a coupled unit, through Ca²⁺ and voltage, to regulate arterial smooth muscle membrane potential and vascular tone.     

Effects of caffeine on intracellular calcium,calcium current and calcium-dependent potassium current in anterior pituitary GH3 cells

Caffeine elicits physiological responses in a variety of cell types by triggering the mobilization of Ca²⁺ from intracellular organelles. Here we investigate the effects of caffeine on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and ionic currents in anterior pituitary cells (GH₃) cells. Caffeine has a biphasic effect on Ca²⁺-activated K⁺ current [I _K(Ca)]: it induces a transient increase superimposed upon a sustained inhibition. While the transient increase coincides with a rise in [Ca²⁺]_i, the sustained inhibition of I _K(Ca) is correlated with a sustained inhibition of the L-type Ca²⁺ current. The L-type Ca²⁺ current is also inhibited by other agents that mobilize intracellular Ca²⁺, including thyrotropin releasing hormone (TRH) and ryanodine, but in a matter distinct from caffeine. Unlike the caffeine effect, the TRH-induced inhibition washes-out under whole-cell patch-clamp conditions and is eliminated by intracellular Ca²⁺ chelators. Likewise, the ryanodine-induced inhibition desensitizes while the caffeine-induced inhibition does not. Simultaneous [Ca²⁺]_i and Ca²⁺ current measurements show that caffeine can inhibit Ca²⁺ current without changing [Ca²⁺]_i. Single-channel recordings show that caffeine reduces mean open time without affecting single-channel conductance of L-type channels. Hence the effects of caffeine on ion channels in GH₃ cells are attributable both to mobilization of intracellular Ca²⁺ and to a direct effect on the gating of L-type Ca²⁺ channels.     

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.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

[Ca2+]i elevation evoked by Ca2+ readdition to the medium after agonist-induced Ca2+ release can involve both IP 3-, and ryanodine-sensitive Ca2+ release

 Sustained Ca²⁺ elevation (”Ca²⁺ response”), caused by subsequent readdition of Ca²⁺ to the medium after application of adenosine 5’-triphosphate (ATP, 15 μM) in a Ca²⁺-free medium, was studied using single bovine aortic endothelial (BAE) cells. In cells in which the resting intracellular Ca²⁺ concentration ([Ca²⁺]_i) was between about 50 and 110 nM, a massive Ca²⁺ response occurred and consisted of phasic and sustained components, whereas cells with a resting [Ca²⁺]_i of over 110 nM displayed small plateau-like Ca²⁺ responses. An increase of internal store depletion resulted in loss of the phasic component. When the store was partly depleted, the dependence of the Ca²⁺ response amplitude on resting [Ca²⁺]_i was biphasic over the range of 50 to 110 nM. The greatest degree of store depletion was associated with small monophasic Ca²⁺ responses, the amplitudes of which were almost constant and in the same range as resting [Ca²⁺]_i. Ni²⁺, known to partly block Ca²⁺ entry, caused no change in the half-decay time of [Ca²⁺]_i down to the level of the sustained phase [57 ± 4 s in control and 54 ± 3 s (n = 13) in the presence of 10 mM Ni²⁺] when added at the peak of the phasic component of the Ca²⁺ response. However, it lowered the sustained phase of the Ca²⁺ response by 42%. When applied at the start of the readdition of Ca²⁺, Ni²⁺ blocked the phasic component of the Ca²⁺ response, there being a threefold decrease in the initial rate of [Ca²⁺]_i rise. In cells with a resting [Ca²⁺]_i of 75–80 nM, pre-treatment with ryanodine (10 μM) did not affect the peak amplitude of the Ca²⁺ response, but it did increase the level of the sustained component. In some cells, ryanodine caused an oscillatory Ca²⁺ response. In conclusion, partial depletion of the inositol 1,4,5-trisphosphate-(IP ₃-) sensitive store by a submaximal concentration of agonist (in Ca²⁺-free medium) was followed, on readdition of Ca²⁺, by Ca²⁺ entry, which appeared to trigger IP ₃-sensitive Ca²⁺ release (IICR) which, in turn, initiated Ca²⁺-sensitive Ca²⁺ release (CICR), thus resulting in a massive elevation of [Ca²⁺]_i. Received: 3 July 1996 / Received after revision and accepted: 9 September 1996     

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.     

Voltage-sensitive calcium channels in a human small-cell lung cancer cell line

Pancrazio , J.J., Oie , H.K. & Kim , Y.I. 1992. Voltage-sensitive calcium channels in a human small-cell lung cancer cell line. Acta Physiol Scand 144 , 463468. Received 1 October 1 991 , accepted 24 October 1991. ISSN 0001–6772. Departments of Biomedical Engineering and Neurology, University of Virginia Health Sciences Center, and the National Cancer Institute, Navy Medical Oncology Branch, Naval Hospital Bethesda, USA. Utilizing the whole-cell patch-clamp method we assessed the Ca²⁺ current (I_ca) in well-established cell lines from human small-cell carcinoma (SCC) of the lung, NCI-H209 and NCI-H187. The Ca²⁺ current was readily observed in H209 tumour cells (90% of the cells tested), whereas H187 tumour cells only occasionally expressed Ca^z+ channels (26% of the cells tested). H209 Ca²⁺ current was evoked by potentials greater than -30 mV and exhibited partial inactivation over the duration of a 40 ms command potential. This inward current was unchanged by alteration of the holding potential from - 80 to - 40 mV and the activation phase of the Ca²⁺ current was best fitted by Hodgkin-Huxley m(t)² kinetics. H209 Ca²⁺ current was reduced over 80% by verapamil (100 μM), whereas w-conotoxin (5 μM) appeared to be without effect. In contrast, H209 Ca²⁺ current was rapidly abolished by nifedipine (10 μm), strongly suggesting the presence of L-type Ca²⁺ channels. Voltage-gated Ca²⁺ channels may be important to the secretion of ectopic hormones and the etiology and pathogenesis of Lambert-Eaton syndrome, an autoimmune disorder of the motor nerve terminal in which autoantibodies directed against voltage-gated Ca²⁺ channels are produced.     

Effects of ruthenium red on membrane ionic currents in urinary bladder smooth muscle cells of the guinea-pig

 Three major ionic currents, Ca²⁺-dependent K⁺ current (I _K-Ca), delayed rectifier type K⁺ current (I _kd) and Ca²⁺ current (I _Ca), were activated by depolarization under whole-cell clamp in single smooth muscle cells isolated from guinea-pig urinary bladder. Externally applied ruthenium red (RuR) reduced the amplitude of I _K-Ca and I _Ca at 0 mV (IC₅₀ values were 4.2 and 5.6 μM, respectively), but did not affect I _Kd. Spontaneous transient outward currents (STOCs) and caffeine-induced outward currents (I _caf) at –30 mV were reduced by external 10 μM RuR. When 10 μM RuR was added to the pipette solution, I _K-Ca during depolarization, STOCs and I _caf significantly decreased with time. RuR did not change the unitary current amplitude of the large-conductance Ca²⁺-dependent K⁺ (BK) channels, but reduced the open probability of the channel under excised patch-clamp recording mode. RuR reduced the channel activity more effectively from the cytosolic face than from the other. This inhibition decreased when the cytosolic Ca²⁺ concentration was increased. These results indicate that RuR blocks BK and Ca²⁺ channels in urinary bladder smooth muscle cells. The decrease in I _K-Ca, STOCs and I _caf by RuR is attributable to the direct inhibition of BK channel activity, probably in addition to the inhibition of Ca²⁺ release from storage sites. The direct inhibition of BK channel activity by RuR may be related to the interaction of RuR with the Ca²⁺-binding sites of the channel protein. Received: 15 October 1997 / Received after revision and accepted: 25 November 1997     

Hypoxia increases the activity of Ca2+-sensitive K+ channels in cat cerebral arterial muscle cell membranes

The cellular mechanisms mediating hypoxia-induced dilation of cerebral arteries have remained unknown, but may involve modulation of membrane ionic channels. The present study was designed to determine the effect of reduced partial pressure of O₂, PO ₂, on the predominant K⁺ channel type recorded in cat cerebral arterial muscle cells, and on the diameter of pressurized cat cerebral arteries. A K⁺-selective single-channel current with a unitary slope conductance of 215 pS was recorded from excised inside-out patches of cat cerebral arterial muscle cells using symmetrical KCl (145 mM) solution. The open state probability (NP _o) of this channel displayed a strong voltage dependence, was not affected by varying intracellular ATP concentration [(ATP]_i) between 0 and 100 M, but was significantly increased upon elevation of intracellular free Ca²⁺ concentration ([Ca²⁺]_i). Low concentrations of external tetraethylammonium (0.1–3 mM) produced a concentration-dependent reduction of the unitary current amplitude of this channel. In cell-attached patches, where the resting membrane potential was set to zero with a high KCl solution, reduction of O₂ from 21% to < 2% reversibly increased the NP _o, mean open time, and event frequency of the Ca²⁺-sensitive, high-conductance single-channel K⁺ current recorded at a patch potential of + 20 mV. A similar reduction in PO₂ also produced a transient increase in the activity of the 215-pS K⁺ channel measured in excised inside-out patches bathed in symmetrical 145 mM KCl, an effect which was diminished, or not seen, during a second application of hypoxic superfusion. Hypoxia had no effect on [Ca²⁺]_i or intracellular pH (pH_i) of cat cerebral arterial muscle cells, as measured using Ca²⁺- or pH-sensitive fluorescent probes. Reduced PO₂ caused a significant dilation of pressurized cerebral arterial segments, which was attenuated by pre-treatment with 1 mM tetraethylammonium. These results suggest that reduced PO₂ increases the activity of a high-conductance, Ca²⁺-sensitive K⁺ channel in cat cerebral arterial muscle cells, and that these effects are mediated by cytosolic events independent of changes in [Ca²⁺]_i and pH_i.     

Shear stress induced membrane currents and calcium transients in human vascular endothelial cells

We have measured membrane currents induced by shear stress together with intracellular calcium signals in endothelial cells from human umbilical cord veins. In the presence of extracellular calcium (Ca²⁺]_o), shear stress induced an inward current at a holding potential of 0 mV which is accompanied by a rise in intracellular Ca²⁺ ([Ca²⁺]_i). In the absence of extracellular calcium shear stress was unable to evoke a calcium signal but still induced a membrane current. The voltage dependence of the shear stress induced current was obtained from difference currents evoked by linear voltage ramps before and during application of shear stress. Its reversal potential E_rev shifted from –2.3±0.8 mV (n=4) in a nominally Ca²⁺ free solution to +1.5±1.6 mV at 1.5 mM [Ca²⁺]_o (n=4) and to +21.9±4.4 mV (n=7) at 10 mM [Ca²⁺]_o. From our data we conclude that shear stress opens an ion channel that is 12.5±2.9 (n=7) times more permeable for calcium than for sodium or cesium.     

Monitoring transient Ca dynamics with large-conductance Ca-dependent K channels at active zones in frog saccular hair cells

Neurotransmitter release from the basolateral surface of auditory and vestibular hair cells is mediated by Ca²⁺ influx through voltage-gated Ca²⁺ channels. Co-localization of large-conductance Ca²⁺-activated K⁺ (BK) channels at the active zones of these cells affords them with an optimal location to act as reporters of the Ca²⁺ concentration changes at active zones of transmitter release. In this report we use BK channels in frog (Rana pipiens) hair cells to monitor dynamic changes in intracellular Ca²⁺ concentration during transient influxes of Ca²⁺, showing that BK current magnitude and delay to onset are correlated with the rate and duration of Ca²⁺ entry through Ca²⁺ channels. We also show that BK channels exhibit a much higher Ca²⁺ binding affinity in the open state than in the closed state.