Ca(2+)-activated K+ channels in human leukemic T cells

Using the patch-clamp technique, we have identified two types of Ca(2+)-activated K+ (K(Ca)) channels in the human leukemic T cell line. Jurkat. Substances that elevate the intracellular Ca2+ concentration ([Ca2+]i), such as ionomycin or the mitogenic lectin phytohemagglutinin (PHA), as well as whole-cell dialysis with pipette solutions containing elevated [Ca2+]i, activate a voltage-independent K+ conductance. Unlike the voltage-gated (type n) K+ channels in these cells, the majority of K(Ca) channels are insensitive to block by charybdotoxin (CTX) or 4-aminopyridine (4-AP), but are highly sensitive to block by apamin (Kd less than 1 nM). Channel activity is strongly dependent on [Ca2+]i, suggesting that multiple Ca2+ binding sites may be involved in channel opening. The Ca2+ concentration at which half of the channels are activated is 400 nM. These channels show little voltage dependence over a potential range of -100 to 0 mV and have a unitary conductance of 4-7 pS in symmetrical 170 mM K+. In the presence of 10 nM apamin, a less prevalent type of K(Ca) channel with a unitary conductance of 40-60 pS can be observed. These larger-conductance channels are sensitive to block by CTX. Pharmacological blockade of K(Ca) channels and voltage-gated type n channels inhibits oscillatory Ca2+ signaling triggered by PHA. These results suggest that K(Ca) channels play a supporting role during T cell activation by sustaining dynamic patterns of Ca2+ signaling.     

Potassium channel types in arterial chemoreceptor cells and their selective modulation by oxygen.

Single K+ channel currents were recorded in excised membrane patches from dispersed chemoreceptor cells of the rabbit carotid body under conditions that abolish current flow through Na+ and Ca2+ channels. We have found three classes of voltage-gated K+ channels that differ in their single-channel conductance (gamma), dependence on internal Ca2+ (Ca2+i), and sensitivity to changes in O2 tension (PO2). Ca(2+)-activated K+ channels (KCa channels) with gamma approximately 210 pS in symmetrical K+ solutions were observed when [Ca2+]i was greater than 0.1 microM. Small conductance channels with gamma = 16 pS were not affected by [Ca2+]i and they exhibited slow activation and inactivation time courses. In these two channel types open probability (P(open)) was unaffected when exposed to normoxic (PO2 = 140 mmHg) or hypoxic (PO2 approximately 5-10 mmHg) external solutions. A third channel type (referred to as KO2 channel), having an intermediate gamma(approximately 40 pS), was the most frequently recorded. KO2 channels are steeply voltage dependent and not affected by [Ca2+]i, they inactivate almost completely in less than 500 ms, and their P(open) reversibly decreases upon exposure to low PO2. The effect of low PO2 is voltage dependent, being more pronounced at moderately depolarized voltages. At 0 mV, for example, P(open) diminishes to approximately 40% of the control value. The time course of ensemble current averages of KO2 channels is remarkably similar to that of the O2-sensitive K+ current. In addition, ensemble average and macroscopic K+ currents are affected similarly by low PO2. These observations strongly suggest that KO2 channels are the main contributors to the macroscopic K+ current of glomus cells. The reversible inhibition of KO2 channel activity by low PO2 does not desensitize and is not related to the presence of F-, ATP, and GTP-gamma-S at the internal face of the membrane. These results indicate that KO2 channels confer upon glomus cells their unique chemoreceptor properties and that the O2-K+ channel interaction occurs either directly or through an O2 sensor intrinsic to the plasma membrane closely associated with the channel molecule.     

Charybdotoxin and noxiustoxin, two homologous peptide inhibitors of the K+ (Ca2+) channel

We show that noxiustoxin (NTX), like charybdotoxin (CTX) described by others, affects Ca2+-activated K+ channels of skeletal muscle (K+(Ca2+) channels). Chemical characterization of CTX shows that it is similar to NTX. Although the amino-terminal amino acid of CTX is not readily available, the molecule was partially sequenced after CNBr cleavage. A decapeptide corresponding to the C-terminal region of NTX shows 60% homology to that of CTX, maintaining the cysteine residues at the same positions. While CTX blocks the K+ (Ca2+) channels with a Kd of 1-3 nM, for NTX it is approx. 450 nM. Both peptides can interact simultaneously with the same channel. NTX and CTX promise to be good tools for channel isolation.     

Characterization of ion channels in human preadipocytes

Ion channels participate in regulation of cell proliferation. However, though preadipocyte (the progenitor of fat cell) is a type of highly proliferating cells, ion channel expression and their role in proliferation is not understood in human preadipocytes. The present study was designed to characterize ion channels using whole-cell patch clamp technique, RT-PCR, and Western blotting. It was found that a 4-aminopyridine- (4-AP) sensitive transient outward K(+) current (I(to)) was present in a small population of (32.0%) cells, and an outward "noisy" big conductance Ca(2+)-activated K(+) current (I(KCa)) was present in most (92.7%) preadipocytes. The noisy current was inhibited by the big conductance I(KCa) channel blocker paxilline (1 microM), and enhanced by the Ca(2+) ionophore A23187 (5 microM) and the big conductance I(KCa) channel activator NS1619 (10 microM). RT-PCR and Western blot revealed the molecular identities (i.e., KCa1.1 and Kv4.2) of the functional ionic currents I(KCa) and I(to). Blockade of I(KCa) or I(to) with paxilline or 4-AP reduced preadipocyte proliferation, and similar results were obtained with specific siRNAs targeting to KCa1.1 and Kv4.2. Flow cytometric analysis showed ion channel blockade or knockdown of KCa1.1 or Kv4.2 with specific siRNA increased the cell number of G0/G1 phase. The present study demonstrates for the first time that two types of functional ion channel currents, I(to) and big conductance I(KCa), are present in human preadipocytes and that these two types of ion channels participate in regulating proliferation of human preadipocytes.     

Interaction of charybdotoxin with permeant ions inside the pore of a K+ channel.

Charybdotoxin (CTX) blocks high conductance Ca(2+)-activated K+ channels by binding to a receptor site in the externally facing "mouth." Toxin bound to the channel can be destabilized from its site by K+ entering the channel from the opposite, internal, solution. By analyzing point mutants of CTX expressed in E. coli, assayed with single Ca(2+)-activated K+ channels reconstituted into planar lipid bilayers, we show that a single positively charged residue of the peptide, Lys-27, wholly mediates this interaction of K+ with CTX. If position 27 carries a positively charged residue, internal K+ accelerates the dissociation rate of CTX in a voltage-dependent manner; however, if a neutral Asn or Gln is substituted at this position, the dissociation rate is completely insensitive to either internal K+ or applied voltage. Position 27 is unique in this respect; charge-neutral substitutions made at other positions fail to eliminate the K+ destabilization phenomenon. The results argue that CTX bound to the channel positions Lys-27 physically close to a K(+)-specific binding site on the external end of the conduction pathway and that a K+ ion occupying this site destabilizes CTX via direct electrostatic repulsion with the epsilon-amino group of Lys-27.     

Charybdotoxin blocks voltage-gated K+ channels in human and murine T lymphocytes

A variety of scorpion venoms and purified toxins were tested for effects on ion channels in human T lymphocytes, a human T leukemia cell line (Jurkat), and murine thymocytes, using the whole-cell patch-clamp method. Nanomolar concentrations of charbdotoxin (CTX), a purified peptide component of Leiurus quinquestriatus venom known to block Ca2+-activated K+ channels from muscle, blocked "type n" voltage-gated K+ channels in human T lymphoid cells. The Na+ channels occasionally expressed in these cells were unaffected by the toxin. From the time course of development and removal of K+ channel block we determined the rates of CTX binding and unbinding. CTX blocks K+ channels in Jurkat cells with a Kd value between 0.5 and 1.5 nM. Of the three types of voltage-gated K+ channels present in murine thymocytes, types n and n' are blocked by CTX at nanomolar concentrations. The third variety of K+ channels, "type l," is unaffected by CTX. Noxiustoxin (NTX), a purified toxin from Centruroides noxius known to block Ca2+-activated K+ channels, also blocked type n K+ channels with a high degree of potency (Kd = 0.2 nM). In addition, several types of crude scorpion venoms from the genera Androctonus, Buthus, Centruroides, and Pandinus blocked type n channels. We conclude that CTX and NTX are not specific for Ca2+ activated K+ channels and that purified scorpion toxins will provide useful probes of voltage-gated K+ channels in T lymphocytes. The existence of high-affinity sites for scorpion toxin binding may help to classify structurally related K+ channels and provide a useful tool for their biochemical purification.     

Ca2+-activated K+ channel in rat pancreatic islet B cells: permeation, gating and blockade by cations

Activation of Ca2+-dependent K+ conductance has long been postulated to contribute to the cyclical pauses in glucose-induced electrical activity of pancreatic islet B cells. Here we have examined the gating, permeation and blockade by cations of a large-conductance, Ca2+-activated K+ channel in these cells. This channel shares many features with BK (or maxi-K+) Ca2+-activated K+ channels in other cells. (1) Its 'permeability' selectivity sequence is PT1+: PK+: PRb+: PNH4+: PNa+, Li+, Cs+ = 1.3:1.0:0.5:0.17: less than 0.05. Permeant, as well as impermeant, cations reduce channel conductance. (2) Its conductance saturates at 325-350 pS with bath KCl greater than 400 mM (144 mM KCl pipette). (3) It shows asymmetric blockade by tetraethylammonium ion (TEA) and Na+. (4) It is sensitive to Ca2+i over the range 5 nM-100 microM; over the range 50-200 nM, channel activity varies as [Ca2+ free]1-2. (5) It is sensitive to internal pH over the range 6.85-7.35, but the decrease in channel activity seen with reduced pHi may be partially compensated by the increase in free Ca2+ concentration which occurs on acidification of buffered Ca2+/EGTA solutions.     

Potassium channel activity recorded from the apical membrane of freshly isolated epithelial cells in rat caudal epididymis.

K+ channels were recorded in excised, inside-out patches from the apical membrane of the freshly isolated tubule of the caudal portion of the rat epididymis. With asymmetric K+ concentrations in bath and pipette (140 mM K+in/6 mM K+out), the channels had a slope conductance of 54.2 pS at 0 mV. The relative permeability of K+ over Na+ was about 171 to 1. The channels were activated by intracellular Ca2+ and by membrane depolarization. These channels belong to a class defined as "intermediate-conductance Ca2+-activated K+ channel. " External tetraethylammonium ions (TEA+) caused a flickery block of the channel with reduction in single-channel current amplitude measured at a range of holding membrane potentials (-40 to 60 mV). Activity of the K+ channels was inhibited by intracellular ATP (KD =1.188 mM). The channel activity was detected only occasionally in patches from the apical membrane (about 1 in 17 patches containing active channels). The presence of the intermediate-conductance Ca2+-activated K+ channels indicates that they could provide a route for K+ secretion in a Ca2+-dependent process responsible for a high luminal K+ concentration found in the epididymal duct of the rat.     

Transformation of renal tubule epithelial cells by simian virus-40 is associated with emergence of Ca(2+)-insensitive K+ channels and altered mitogenic sensitivity to K+ channel blockers.

We compared the pattern of K+ channels and the mitogenic sensitivity to K+ channel blocking agents in primary cultures of rabbit proximal tubule cells (PC.RC) (Ronco et al., 1990) and two derived SV-40-transformed cell lines exhibiting specific functions of proximal (RC.SV1) and more distal (RC.SV2) tubule cells (Vandewalle et al., 1989). First, K+ channel equipment surveyed by the patch-clamp technique was modified after SV-40 transformation in both cell lines; although a high conductance Ca(2+)-activated K+ channel [K+200 (Ca2+)] remained the most frequently recorded K+ channel, the transformed state was characterized by emergence of three Ca(2+)-insensitive K+ channels (150, 50, and 30 pS), virtually absent from primary culture, contrasting with reduced frequency of two Ca(2+)-sensitive K+ channels (80 and 40 pS). Second, quinine (Q), tetraethylammonium ion (TEA) and charybdotoxin (CTX), at concentrations not affecting cell viability, all decreased 3H-TdR incorporation and cell growth in PC.RC cultures, but only TEA had similar effects in transformed cells. The latter were further characterized by paradoxical effects of Q that induced a marked increase in thymidine incorporation. Q also exerted contrasting effects on channel activity: it inhibited the [K+200 (Ca2+)] when the channel was highly active, with a Ki (0.2 mM) similar to that measured for 3H-TdR incorporation in PC.RC cells (0.3 mM), but increased the mean current through poorly active channels. TEA blocked all K+ channels with conductance greater than or equal to 50 pS, including the [K+200 (Ca2+)], in a range of concentrations that substantially affected cell proliferation. The unique effect of TEA on SV-40-transformed cells might be related to broad inhibition of K+ channels.     

KCa channel insensitivity to Ca2+ sparks underlies fractional uncoupling in newborn cerebral artery smooth muscle cells

In smooth muscle cells, localized intracellular Ca2+ transients, termed "Ca2+ sparks," activate several large-conductance Ca2+-activated K+ (KCa) channels, resulting in a transient KCa current. In some smooth muscle cell types, a significant proportion of Ca2+ sparks do not activate KCa channels. The goal of this study was to explore mechanisms that underlie fractional Ca2+ spark-KCa channel coupling. We investigated whether membrane depolarization or ryanodine-sensitive Ca2+ release (RyR) channel activation modulates coupling in newborn (1- to 3-day-old) porcine cerebral artery myocytes. At steady membrane potentials of -40, 0, and +40 mV, mean transient KCa current frequency was approximately 0.18, 0.43, and 0.26 Hz and KCa channel activity [number of KCa channels activated by Ca2+ sparksxopen probability of KCa channels at peak of Ca2+ sparks (NPo)] at the transient KCa current peak was approximately 4, 12, and 24, respectively. Depolarization between -40 and +40 mV increased KCa channel sensitivity to Ca2+ sparks and elevated the percentage of Ca2+ sparks that activated a transient KCa current from 59 to 86%. In a Ca2+-free bath solution or in diltiazem, a voltage-dependent Ca2+ channel blocker, steady membrane depolarization between -40 and +40 mV increased transient KCa current frequency up to approximately 1.6-fold. In contrast, caffeine (10 microM), an RyR channel activator, increased mean transient KCa current frequency but did not alter Ca2+ spark-KCa channel coupling. These data indicate that coupling is increased by mechanisms that elevate KCa channel sensitivity to Ca2+ sparks, but not by RyR channel activation. Overall, KCa channel insensitivity to Ca2+ sparks is a prominent factor underlying fractional Ca2+ spark uncoupling in newborn cerebral artery myocytes.     

Functional modification of a Ca2+-activated K+ channel by trimethyloxonium

Single Ca2+-activated K+ channels from rat skeletal muscle plasma membranes were studied in neutral phospholipid bilayers. Channels were chemically modified by briefly exposing the external side to the carboxyl group modifying reagent trimethyloxonium (TMO). TMO modification, in a "multi-hit" fashion, reduces the single-channel conductance without affecting ion selectivity. Modification also shifts the voltage activation curve toward more depolarized voltages and reduces the affinity of the channel blocker charybdotoxin (CTX). CTX, bound to the channel during the TMO exposure, prevents the TMO-induced reduction of the single-channel conductance. These data suggest that the high-conductance Ca2+-activated K+ channel has carboxyl groups on its external surface. These groups influence ion conduction, gating, and the binding of CTX.     

A developmental model for generating frequency maps in the reptilian and avian cochleas.

Hair cells in the turtle cochlea are frequency-tuned by a mechanism involving the combined activation of voltage-sensitive Ca2+ channels and Ca(2+)-activated K+ (KCa) channels. The main determinants of a hair cell's characteristic frequency (Fo) are the KCa channels' density and kinetics, both of which change systematically with location in the cochlea in conjunction with the observed frequency map. We have developed a model based on the differential expression of two KCa channel subunits, which when accompanied by concurrent changes in other properties (e.g., density of Ca2+ channels and inwardly rectifying K+ channels), will generate sharp tuning at frequencies from 40 to 600 Hz. The kinetic properties of the two subunits were derived from previous single-channel analysis, and it was assumed that the subunits (A and B) combine to form five species of tetrameric channel (A4, A3B, A2B2, AB3, and B4) with intermediate kinetics and overlapping distribution. Expression of KCa and other channels was assumed to be regulated by diffusional gradients in either one or two chemicals. The results are consistent with both current- and voltage-clamp data on turtle hair cells, and they show that five channel species are sufficient to produce smooth changes in both Fo and kinetics of the macroscopic KCa current. Other schemes for varying KCa channel kinetics are examined, including one that allows extension of the model to the chick cochlea to produce hair cells with Fo's from 130 to 4000 Hz. A necessary assumption in all models is a gradient in the values of the parameters identified with the cell's cytoplasmic Ca2+ buffer.     

Charybdotoxin block of single Ca2+-activated K+ channels. Effects of channel gating, voltage, and ionic strength

Charybdotoxin (CTX), a small, basic protein from scorpion venom, strongly inhibits the conduction of K ions through high-conductance, Ca2+-activated K+ channels. The interaction of CTX with Ca2+-activated K+ channels from rat skeletal muscle plasma membranes was studied by inserting single channels into uncharged planar phospholipid bilayers. CTX blocks K+ conduction by binding to the external side of the channel, with an apparent dissociation constant of approximately 10 nM at physiological ionic strength. The dwell-time distributions of both blocked and unblocked states are single-exponential. The toxin association rate varies linearly with the CTX concentration, and the dissociation rate is independent of it. CTX is competent to block both open and closed channels; the association rate is sevenfold faster for the open channel, while the dissociation rate is the same for both channel conformations. Membrane depolarization enhances the CTX dissociation rate e-fold/28 mV; if the channel's open probability is maintained constant as voltage varies, then the toxin association rate is voltage independent. Increasing the external solution ionic strength from 20 to 300 mM (with K+, Na+, or arginine+) reduces the association rate by two orders of magnitude, with little effect on the dissociation rate. We conclude that CTX binding to the Ca2+-activated K+ channel is a bimolecular process, and that the CTX interaction senses both voltage and the channel's conformational state. We further propose that a region of fixed negative charge exists near the channel's CTX-binding site.     

Coassembly of big conductance Ca2+-activated K+ channels and L-type voltage-gated Ca2+ channels in rat brain

Based on electrophysiological studies, Ca(2+)-activated K(+) channels and voltage-gated Ca(2+) channels appear to be located in close proximity in neurons. Such colocalization would ensure selective and rapid activation of K(+) channels by local increases in the cytosolic calcium concentration. The nature of the apparent coupling is not known. In the present study we report a direct coassembly of big conductance Ca(2+)-activated K(+) channels (BK) and L-type voltage-gated Ca(2+) channels in rat brain. Saturation immunoprecipitation studies were performed on membranes labeled for BK channels and precipitated with antibodies against alpha(1C) and alpha(1D) L-type Ca(2+) channels. To confirm the specificity of the interaction, precipitation experiments were carried out also in reverse order. Also, additive precipitation was performed because alpha(1C) and alpha(1D) L-type Ca(2+) channels always refer to separate ion channel complexes. Finally, immunochemical studies showed a distinct but overlapping expression pattern of the two types of ion channels investigated. BK and L-type Ca(2+) channels were colocalized in various compartments throughout the rat brain. Taken together, these results demonstrate a direct coassembly of BK channels and L-type Ca(2+) channels in certain areas of the brain.     

Identification of an intermolecular disulfide bond in barley hemoglobin

The present study was designed to investigate properties of ion channels in undifferentiated rabbit mesenchymal stem cells (MSCs) from bone marrow using whole-cell patch-clamp and RT-PCR techniques. It was found that three types of outward currents were present in rabbit MSCs, including an inward rectifier K(+) current (I(Kir)), a noise-like Ca(2+)-activated K(+) current (I(KCa)) co-present with delayed rectifier K(+) current (IK(DR)). I(Kir) was inhibited by Ba(2+), while I(KCa) was inhibited by paxilline (a blocker of big conductance I(KCa) channels) and clotrimazole (an inhibitor of intermediate conductance I(KCa) channels). IK(DR) exhibited a slow inactivation, "U-shaped" voltage-dependent inactivation, and slow recovery from inactivation, and the current was inhibited by tetraethylammonium or 4-aminopyridine. RT-PCR revealed the molecular identities for the functional ionic currents, including Kir1.1 (possibly responsible for I(Kir)), KCa1.1 and KCa3.1 (possibly responsible for I(KCa)), and Kv1.2, Kv2.1, and Kv2.2 (possibly responsible for IK(DR)). These results demonstrate for the first time that three types of functional ion channel currents (i.e., I(Kir), I(KCa), and IK(DR)) are present in rabbit MSCs from bone marrow.     

Internal blockade of a Ca(2+)-activated K+ channel by Shaker B inactivating "ball" peptide.

Shaker B inactivating peptide ("ball peptide", BP) interacts with Ca(2+)-activated K+ (KCa) channels from the cytoplasmic side only, producing inhibition of channel activity. This effect was reversible and dose and voltage dependent (stronger at depolarized potentials). The inhibition of KCa channels by BP cannot be mimicked by an inactive point mutation of the BP, L7E. BP binds to KCa channels in a bimolecular reaction (dissociation constant of 95 microM at +40 mV). The binding site is probably located in the internal "mouth" or conduction pathway, since both external K+ and internal tetraethylammonium relieve BP-induced inhibition. These results suggest that KCa channels possess a binding site for the BP with some properties similar to the ball receptor found in Shaker B K+ channels.     

Phosphatidylinositol 3-phosphate indirectly activates KCa3.1 via 14 amino acids in the carboxy terminus of KCa3.1

KCa3.1 is an intermediate conductance Ca2+-activated K+ channel that is expressed predominantly in hematopoietic cells, smooth muscle cells, and epithelia where it functions to regulate membrane potential, Ca2+ influx, cell volume, and chloride secretion. We recently found that the KCa3.1 channel also specifically requires phosphatidylinositol-3 phosphate [PI(3)P] for channel activity and is inhibited by myotubularin-related protein 6 (MTMR6), a PI(3)P phosphatase. We now show that PI(3)P indirectly activates KCa3.1. Unlike KCa3.1 channels, the related KCa2.1, KCa2.2, or KCa2.3 channels do not require PI(3)P for activity, suggesting that the KCa3.1 channel has evolved a unique means of regulation that is critical for its biological function. By making chimeric channels between KCa3.1 and KCa2.3, we identified a stretch of 14 amino acids in the carboxy-terminal calmodulin binding domain of KCa3.1 that is sufficient to confer regulation of KCa2.3 by PI(3)P. However, mutation of a single potential phosphorylation site in these 14 amino acids did not affect channel activity. These data together suggest that PI(3)P and these 14 amino acids regulate KCa3.1 channel activity by recruiting an as yet to be defined regulatory subunit that is required for Ca2+ gating of KCa3.1.     

HIV-1 nef expression inhibits the activity of a Ca2+-dependent K+ channel involved in the control of the resting potential in CEM lymphocytes

The HIV-1 Nef protein plays an important role in the development of the pathology associated with AIDS. Despite various studies that have dealt with different aspects of Nef function, the complete mechanism by which it alters the physiology of infected cells remains to be established. Nef can associate with cell membranes, therefore supporting the hypothesis that it might interact with membrane proteins as ionic channels and modify their electrical properties. By using the patch-clamp technique, we found that Nef expression determines a 25-mV depolarization of lymphoblastoid CEM cells. Both charybdotoxin (CTX) and the membrane-permeable Ca2+ chelator BAPTA/AM depolarized the membrane of native cells without modifying that of Nef-transfected cells. These data suggested that the resting potential in native CEM cells is settled by a CTX- and Ca2+-sensitive K+ channel (KCa,CTX), whose activity is absent in Nef-expressing cells. This was confirmed by direct measurements of whole-cell KCa,CTX currents. Single-channel recordings on excised patches showed that a KCa,CTX channel of 35 pS with a half-activation near 400 nM Ca2+ was present in both native and Nef-transfected cells. The measurements of free intracellular Ca2+ were not different in the two cell lines, but Nef-transfected cells displayed an increased Ca2+ content in ionomycin-sensitive stores. Taken together, these results indicate that Nef expression alters the resting membrane potential of the T lymphocyte cell line by inhibiting a KCa,CTX channel, possibly by intervening in the regulation of intracellular Ca2+ homeostasis.     

Low-conductance states of K+ channels in adult mouse skeletal muscle

Single-channel currents were recorded from Ca2+-activated or ATP-sensitive K+ channels in inside-out membrane patches excised from isolated mouse toe muscles. In addition to the closed and fully open configurations, both types of channels may exhibit several intermediate low-conductance states which are clustered near multiples of elementary conductance units. The units are 1/8 or 1/6 of the channel conductance for Ca2+-activated channels and 1/4 or 1/3 for ATP-sensitive channels. Normally, low-conductance states are rare, but they occur more frequently directly after patch excision. An increased probability of low-conductance states of ATP-sensitive K+ channels was also observed in the presence and during washout of the internal channel blocker adenine. The results suggest that Ca2+-activated and ATP-sensitive K+ channels are composed of several membrane pores with strong positive cooperativity among the elementary conductance units.     

Calcium-activated potassium channels in resting and activated human T lymphocytes. Expression levels, calcium dependence, ion selectivity, and pharmacology

Ca(2+)-activated K+[K(Ca)] channels in resting and activated human peripheral blood T lymphocytes were characterized using simultaneous patch-clamp recording and fura-2 monitoring of cytosolic Ca2+ concentration, [Ca2+]i. Whole-cell experiments, using EGTA-buffered pipette solutions to raise [Ca2+]i to 1 microM, revealed a 25-fold increase in the number of conducting K(Ca) channels per cell, from an average of 20 in resting T cells to > 500 channels per cell in T cell blasts after mitogenic activation. The opening of K(Ca) channels in both whole-cell and inside-out patch experiments was highly sensitive to [Ca2+]i (Hill coefficient of 4, with a midpoint of approximately 300 nM). At optimal [Ca2+]i, the open probability of a K(Ca) channel was 0.3-0.5. K(Ca) channels showed little or no voltage dependence from - 100 to 0 mV. Single-channel I-V curves were linear with a unitary conductance of 11 pS in normal Ringer and exhibited modest inward rectification with a unitary conductance of approximately 35 pS in symmetrical 160 mM K+. Permeability ratios, relative to K+, determined from reversal potential measurements were: K+ (1.0) > Rb+ (0.96) > NH4+ (0.17) > Cs+ (0.07). Slope conductance ratios were: NH4+ (1.2) > K+ (1.0) > Rb+ (0.6) > Cs+ (0.10). Extracellular Cs+ or Ba2+ each induced voltage-dependent block of K(Ca) channels, with block increasing at hyperpolarizing potentials in a manner suggesting a site of block 75% across the membrane field from the outside. K(Ca) channels were blocked by tetraethylammonium (TEA) applied externally (Kd = 40 mM), but were unaffected by 10 mM TEA applied inside by pipette perfusion. K(Ca) channels were blocked by charybdotoxin (CTX) with a half-blocking dose of 3-4 nM, but were resistant to block by noxiustoxin (NTX) at 1-100 nM. Unlike K(Ca) channels in Jurkat T cells, the K(Ca) channels of normal resting or activated T cells were not blocked by apamin. We conclude that while K(Ca) and voltage-gated K+ channels in the same cells share similarities in ion permeation, Cs+ and Ba2+ block, and sensitivity to CTX, the underlying proteins differ in structural characteristics that determine channel gating and block by NTX and TEA.