Ca2(+)-activated K+ currents in smooth muscle

Calcium-activated potassium currents have been described in a wide variety of cell types. This report summarizes some important properties of these currents in smooth muscle and provides examples from our recent single channel recordings from human cystic artery.     

MCI-154 activates the Ca2+-activated K+ channel of vascular smooth muscle cells

The aim of this study was to examine the effects of MCI-154, a new positive inotropic agent with vasodilating properties, on the Ca²⁺-activated K⁺ channel (K_Ca channel) of vascular smooth muscle cells. Cultured smooth muscle cells from a porcine coronary artery were studied using the patch-clamp technique. Extracellular application of 100 μM MCI-154 activated the K_Ca channel in intact cell-attached patch configurations. In excised inside-out patch configurations, application of 100μM MCI-154 to the cytosolic side activated the K_Ca channel directly, suggesting that the Ca₂₊ sensitivity of the K_Ca channel itself is modulated. Though extracellular application of 100 μM amrinone, a phosphodiesterase inhibitor, activated the K_Ca channel in the cell-attached patch configurations, application of 100 μm amrinone to the cytosolic side could not activate the K_Ca channel in inside-out patch configurations. These results indicate that different from amrinone, MCI-154 can modulate Ca²⁺ sensitivity of the K_Ca channel in vascular smooth muscle cells.     

ATP inhibits smooth muscle Ca2(+)-activated K+ channels

There has been much recent interest in the roles played by smooth-muscle K+ channels in protecting cells against ischemic and anoxic insults and in therapeutic vaso- and bronchodilation (Buckingham 1990; Longmore & Weston 1990). A K+ channel, which is uniquely sensitive to cytoplasmic ATP (KATP), has been identified as a likely candidate for mediating these important functions (Standen et al. 1989). We now show, by using electrophysiological techniques in three different types of smooth muscle, that a large-conductance voltage and Ca2(+)-sensitive channel, otherwise indistinguishable from the the large-conductance Ca2(+)-activated K+ channel (BK channel), is also sensitive to cytoplasmic ATP and cromakalim. ATP, in a dose-dependent manner, decreased the probability of channel opening (Po) of rabbit aortic, rabbit tracheal and pig coronary artery BK channels with a Ki of 0.2-0.6 mM. Cromakalim, 10 microM, partially reversed the ATP induced inhibition and increased Po. Our observations raise the possibility that the ubiquitous BK channel may play a role during pathophysiological events.     

Activation by divalent cations of a Ca2+-activated K+ channel from skeletal muscle membrane

Several divalent cations were studied as agonists of a Ca2+-activated K+ channel obtained from rat muscle membranes and incorporated into planar lipid bilayers. The effect of these agonists on single-channel currents was tested in the absence and in the presence of Ca2+. Among the divalent cations that activate the channel, Ca2+ is the most effective, followed by Cd2+, Sr2+, Mn2+, Fe2+, and Co2+. Mg2+, Ni2+, Ba2+, Cu2+, Zn2+, Hg2+, and Sn2+ are ineffective. The voltage dependence of channel activation is the same for all the divalent cations. The time-averaged probability of the open state is a sigmoidal function of the divalent cation concentration. The sigmoidal curves are described by a dissociation constant K and a Hill coefficient N. The values of these parameters, measured at 80 mV are: N = 2.1, K = 4 X 10(-7) mMN for Ca2+; N = 3.0, K = 0.02 mMN for Cd2+; N = 1.45, K = 0.63 mMN for Sr2+; N = 1.7, K = 0.94 mMN for Mn2+; N = 1.1, K = 3.0 mMN for Fe2+; and N = 1.1 K = 4.35 mMN for Co2+. In the presence of Ca2+, the divalent cations Cd2+, Co2+, Mn2+, Ni2+, and Mg2+ are able to increase the apparent affinity of the channel for Ca2+ and they increase the Hill coefficient in a concentration-dependent fashion. These divalent cations are only effective when added to the cytoplasmic side of the channel. We suggest that these divalent cations can bind to the channel, unmasking new Ca2+ sites.     

Large-conductance Ca2+-activated K+ channels in freshly dissociated smooth muscle cells

Freshly dissociated cells from the stomach muscularis of the toad Bufo marinus have been employed to carry out a systematic set of electrophysiological studies on the membrane properties of smooth muscle. The existence of Ca2+-activated K+ channels became apparent during the first studies under current clamp. In subsequent studies under voltage clamp, a Ca2+-activated. TEA-sensitive outward current was evident, and it was more than an order of magnitude larger than any other current observed in the cells. The channel responsible, at least in part, for this large outward current has been identified on the basis of single-channel records, and some of its main characteristics have been studied. It is similar in many respects to the large-conductance, Ca2+-activated K+ channel seen in other preparations. This channel has now been found in a considerable diversity of smooth muscle types.     

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)- fold weaker.     

The Ca2+-activated K+ channel and its functional roles in smooth muscle cells of guinea pig taenia coli

Currents through single potassium channels were studied in cell-attached or inside-out patches from collagenase-dispersed smooth muscle cells of the guinea pig taenia coli. Under conditions mimicking the physiological state with [K+]i = 135 mM: [K+]o = 5.4 mM, three distinct types of K+ channel were identified with conductances around 0 mV of 147, 94, and 63 pS. The activities of the 94- and 63-pS channel were observed infrequently. The 147-pS channel was most abundant. It has a reversal potential of approximately -75 mV. It is sensitive to [Ca2+]i and to membrane potential. At -30 mV, the probability of a channel being open is at a minimum. At more positive voltages, the probability follows Boltzman distribution. A 10-fold change in [Ca2+]i causes a 25-mV negative shift of the voltage where half of the channels are open; an 11.3-mV change in membrane potential produces an e-fold increase in the probability of the channel being open when P is low. At voltages between -30 and -50 mV, the open probability increases in an anomalous manner because of a large decrease of the channel closed time without much change in the channel open time. This anomalous activity may play a regulatory role in maintaining the resting potential. The histograms of channel open and closed time fit well, respectively, with single and double exponential distributions. Upon step depolarizations by 100-ms pulses, the 147-pS channel opens with a brief delay. The delay shortens and both the number of open channels and the open time increase with increasing positivity of the potential. The averaged currents during the step depolarizations closely resemble the delayed rectifying outward K+ currents in whole-cell recordings.     

Aminoglycoside blockade of Ca2+-activated K+ channel from rat brain synaptosomal membranes incorporated into planar bilayers

Summary Ca²⁺-activated K⁺ channels from rat brain synaptosomal membranes were incorporated into planar lipid bilayers, and the effects of aminoglycoside antibiotics on the single channel conductance (258±13 pS at 100mm K⁺) were investigated. Aminoglycosides reduced the single channel conductance from the cis (cytoplasmic) side in a dose- and voltage-dependent manner. Voltage dependence of the blockade indicated an interaction between positively charged amino residues of aminoglycoside antibiotics and a binding site located within the electric field of the ion-conducting pathway. The order of blocking potency was consistent with that of the number of amino residues of aminoglycosides (neomycin (6)>dibekacin (5)>ribostamycin (4)=kanamycin (4)), while the electrical distance (z=0.46–0.49) of the binding site kept almost constant for each drug. Thesezs were almost the same with those (0.46–0.51) of alkyldiamine blockers with two amino residues (total net charge of +2) and approximately twice of those (0.25–0.26) of alkylmonoamine blockers (total net charge of +1). Assuming that amino residues of aminoglycosides and alkylamines shared the same binding site located at 25% voltage drop from the cytoplasmic surface of the channel, the site would have to be at least large enough to accommodate one diamino sugar residue of the aminoglycoside in order to simultaneously interact with two positively charged amino groups. Dose- and voltage-dependent blockade of the channel by gallamine, an extremely bulky trivalent organic cation, supported the picture that the channel has a wide mouth on the cytoplasmic side and its pore region, where voltage drop occurs, may also be quite wide and nonselective, suddenly tapering to a constriction where most charged cations block the channel by occluding the K⁺-conducting pathway.     

Ketamine blocks Ca2+-activated K+ channels in rabbit cerebral arterial smooth muscle cells

Although ketamine and Ca2+-activated K+ (KCa) channels have been implicated in the contractile activity regulation of cerebral arteries, no studies have addressed the specific interactions between ketamine and the KCa channels in cerebral arteries. The purpose of this study was to examine the direct effects of ketamine on KCa channel activities using the patch-clamp technique in single-cell preparations of rabbit middle cerebral arterial smooth muscle. We tested the hypothesis that ketamine modulates the KCa channel activity of the cerebral arterial smooth muscle cells of the rabbit. Vascular myocytes were isolated from rabbit middle cerebral arteries using enzymatic dissociation. Single KCa channel activities of smooth muscle cells from rabbit cerebral arteries were recorded using the patch-clamp technique. In the inside-out patches, ketamine in the micromolar range inhibited channel activity with a half-maximal inhibition of the ketamine concentration value of 83.8 +/- 12.9 microM. The Hill coefficient was 1.2 +/- 0.3. The slope conductance of the current-voltage relationship was 320.1 +/- 2.0 pS between 0 and +60 mV in the presence of ketamine and symmetrical 145 mM K+. Ketamine had little effect on either the voltage-dependency or open- and closed-time histograms of KCa channel. The present study clearly demonstrates that ketamine inhibits KCa channel activities in rabbit middle cerebral arterial smooth muscle cells. This inhibition of KCa channels may represent a mechanism for ketamine-induced cerebral vasoconstriction.     

Upregulation of intermediate-conductance Ca2+-activated K+ channel (IKCa1) mediates phenotypic modulation of coronary smooth muscle

A hallmark of smooth muscle cell (SMC) phenotypic modulation in atherosclerosis and restenosis is suppression of SMC differentiation marker genes, proliferation, and migration. Blockade of intermediate-conductance Ca(2+)-activated K(+) channels (IKCa1) has been shown to inhibit restenosis after carotid balloon injury in the rat; however, whether IKCa1 plays a role in SMC phenotypic modulation is unknown. Our objective was to determine the role of IKCa1 channels in regulating coronary SMC phenotypic modulation and migration. In cultured porcine coronary SMCs, platelet-derived growth factor-BB (PDGF-BB) increased TRAM-34 (a specific IKCa1 inhibitor)-sensitive K(+) current 20-fold; increased IKCa1 promoter histone acetylation and c-jun binding; increased IKCa1 mRNA approximately 4-fold; and potently decreased expression of the smooth muscle differentiation marker genes smooth muscle myosin heavy chain (SMMHC), smooth muscle alpha-actin (SMalphaA), and smoothelin-B, as well as myocardin. Importantly, TRAM-34 completely blocked PDGF-BB-induced suppression of SMMHC, SMalphaA, smoothelin-B, and myocardin and inhibited PDGF-BB-stimulated migration by approximately 50%. Similar to TRAM-34, knockdown of endogenous IKCa1 with siRNA also prevented the PDGF-BB-induced increase in IKCa1 and decrease in SMMHC mRNA. In coronary arteries from high fat/high cholesterol-fed swine demonstrating signs of early atherosclerosis, IKCa1 expression was 22-fold higher and SMMHC, smoothelin-B, and myocardin expression significantly reduced in proliferating vs. nonproliferating medial cells. Our findings demonstrate that functional upregulation of IKCa1 is required for PDGF-BB-induced coronary SMC phenotypic modulation and migration and support a similar role for IKCa1 in coronary SMC during early coronary atherosclerosis.     

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.     

Ca2(+)-sensitive K+ channel in aortic smooth muscle of rats

We measured K+ channel activity in inside-out patches of cell membrane from aortic vascular smooth muscle cultured (Passages 1-3) from Wistar, Wistar-Kyoto, and spontaneously hypertensive rats (SHR). With [Ca2+]i between 25 and 100 nm and 150 mm K+ on both sides of the membrane, the conductance of this channel was 55 +/- 7 pS (slope of current-voltage curve through 0 mV) and the current was outwardly rectified. There was no difference in single-channel conductance among the three rat strains. Increasing negative holding voltages or increasing [Ca2+]i, increased the probability of this type channel being open (Npo; P less than 0.01); SHR had a larger NPo (P less than 0.01). Compared with cells from Wistar and Wistar-Kyoto, cells from SHR also had the longest mean open time. The increased NPo and mean open time we observed in this K+ channel of cells from SHR could contribute, at least in part, to the increased membrane K+ permeability, reported previously.     

Large-conductance voltage- and Ca2+-activated K+ channels regulate human detrusor smooth muscle function

The large-conductance voltage- and Ca(2+)-activated K(+) (BK) channel is expressed in many smooth muscle types, but its role in human detrusor smooth muscle (DSM) is unclear. With a multidisciplinary approach spanning channel molecules, single-channel activity, freshly isolated human DSM cells, intact DSM preparations, and the BK channel specific inhibitor iberiotoxin, we elucidated human DSM BK channel function and regulation. Native human DSM tissues were obtained during open surgeries from patients with no preoperative history of overactive bladder. RT-PCR experiments on single human DSM cells showed mRNA expression of BK channel α-, β(1)-, and β(4)-subunits. Western blot and immunocytochemistry confirmed BK channel α, β(1), and β(4) protein expression. Native human BK channel properties were described using the perforated whole cell configuration of the patch-clamp technique. In freshly isolated human DSM cells, BK channel blockade with iberiotoxin inhibited a significant portion of the total voltage step-induced whole cell K(+) current. From single BK channel recordings, human BK channel conductance was calculated to be 136 pS. Voltage-dependent iberiotoxin- and ryanodine-sensitive transient BK currents were identified in human DSM cells. In current-clamp mode, iberiotoxin inhibited the hyperpolarizing membrane potential transients and depolarized the cell resting membrane potential. Isometric DSM tension recordings revealed that BK channels principally control the contractions of isolated human DSM strips. Collectively, our results indicate that BK channels are fundamental regulators of DSM excitability and contractility and may represent new targets for pharmacological or genetic control of urinary bladder function in humans.     

Limited autolysis reduces the Ca2+ requirement of a smooth muscle Ca2+-activated protease

Chicken gizzard smooth muscle contains large amounts of Ca2+-activated protease activity. Approximately 15 mg of purified enzyme can be obtained from 1 kg of fresh muscle. The enzyme consists of two subunits (Mr = 80,000 and 30,000) present in a 1:1 molar ratio. In the presence of CaCl2, the 80,000/30,000-dalton heterodimer (form I) is rapidly converted by limited autolysis to a 76,000/18,000-dalton species (form II). Both the 80,000- and 30,000-dalton subunits are degraded simultaneously. Moreover, the Ca2+ dependence for autolysis (K0.5 = 300 microM) is identical for both subunits. Neither the time course nor the Ca2+ dependence of the autolytic conversion reaction is altered by 10- and 20-fold molar excesses of substrate. Limited autolysis markedly reduces the Ca2+ requirement for substrate degradation. Using N-[ethyl-2-3H]maleimide-labeled 27,000-dalton cardiac myosin light chains as substrate, the Ca2+ requirement of form I was found to be quite high (K0.5 = 150 microM). Under similar conditions, the Ca2+ requirement of form II was 30-fold lower (K0.5 = 5 microM). Limited autolysis did not alter the specific activity of the enzyme. Our results demonstrate that smooth muscle contains an abundant amount of Ca2+-activated protease. Moreover, autolysis of this enzyme may play an important regulatory role by converting the native form to a species that is fully active at physiological levels of intracellular calcium ion.     

Big-conductance Ca2+-activated K+ channels in physiological and pathophysiological urinary bladder smooth muscle cells

Contraction and relaxation of urinary bladder smooth muscle cells (UBSMCs) represent the important physiological functions of the bladder. Contractile responses in UBSMCs are regulated by a number of ion channels including big-conductance Ca²⁺- activated K⁺ (BK) channels. Great progress has been made in studies of BK channels in UBSMCs. The intent of this review is to summarize recent exciting findings with respect to the functional interactions of BK channels with muscarinic receptors, ryanodine receptors (RyRs) and inositol triphosphate receptors (IP₃Rs) as well as their functional importance under normal and pathophysiological conditions. BK channels are highly expressed in UBSMCs. Activation of muscarinic M₃ receptors inhibits the BK channel activity, facilitates opening of voltage-dependent Ca²⁺ (Ca_V) channels, and thereby enhances excitability and contractility of UBSMCs. Signaling molecules and regulatory mechanisms involving RyRs and IP₃Rs have a significant effect on functions of BK channels and thereby regulate cellular responses in UBSMCs under normal and pathophysiological conditions including overactive bladders. Moreover, BK channels may represent a novel target for the treatment of bladder dysfunctions.     

Multi-ion conduction and selectivity in the high-conductance Ca++-activated K+ channel from skeletal muscle.

Open-channel ion permeation properties were investigated for Ca++-activated K+ (CaK) channels in solutions of K+ and its analogues T1+, Rb+, and NH4+. Single CaK channels were inserted into planar lipid bilayers composed of neutral phospholipids, and open-channel current-voltage (I-V) relations were measured in symmetrical and asymmetrical solutions of each of these individual ions. For all concentrations studied, the zero-voltage conductance falls in the sequence K+ greater than T1+ greater than NH4+ greater than Rb+. The shape of the I-V curve in symmetrical solutions of a single permeant ion is non-ohmic and is species-dependent. The I-V shape is sublinear for K+ and T1+ and superlinear for Rb+ and NH4+. As judged by reversal potentials under bi-ionic conditions with K+ on one side of the bilayer and the test cation on the other, the permeability sequence is T1+ greater than K+ greater than Rb+ greater than NH4+ at 300 mM, which differs from the conductance sequence. Symmetrical mixtures of K+ or NH4+ with Rb+ show a striking anomalous mole fraction behavior, i.e., a minimum in single-channel conductance when the composition of a two-ion mixture is varied at constant total ion concentration. This result is incompatible with present models that consider the CaK channel a single-ion pore. In total, the results show that the CaK channel finely discriminates among K+-like ions, exhibiting different energy profiles among these species, and that several such ions can reside simultaneously within the conduction pathway.