Potassium channels in airway smooth muscle: A tale of two channels

Pharmac. Ther. Vol. 58, pp. 1-12, 1993 Printed in Great Britain. All rights reserved

0163-7258/93 $24.00 "f~ 1993 Pergamon Press Ltd

Associate Editor: I. W. RODGER

POTASSIUM CHANNELS IN AIRWAY SMOOTH MUSCLE: A TALE OF TWO CHANNELS MICHAEL I. KOTLIKOFF Department of Animal Biology, School of Veterinary Medicine and Department of Medicine, School of Medicine, University of Pennsylvania, Philadelphia PA 19104-6046, U.S.A. Abstract--Potassium channels are an important determinant of smooth muscle excitability and force generation. Two potassium channels have been fully described in airway smooth muscle: large conductance, calcium-activated potassium channels and voltage-dependent delayed rectifier channels. This article will review the biophysics and pharmacology of these channels and discuss what is currently known with respect to their regulation and physiological significance.

CONTENTS 1. Introduction 2. Overview of Potassium Channels 3. Calcium-Activated Potassium Channels 3.1. Single channel measurements 3.2. Whole-cell measurements 3.3. Agonist regulation of Kca channels 4. Delayed Rectifier Potassium Channels 5. Conclusion References

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!. INTRODUCTION It is the best of times, it is the worst of times. Scientific information about potassium channels at the cellular and molecular levels has increased markedly in the past decade. At the same time, many of these advances have not been integrated into physiological information that might provide useful insights into cell and organ physiology, as well as practical targets for drug development. with respect to airway smooth muscle, the principal characters in the drama have been introduced. Details of the behavior of these players and their specific roles in the processes of excitation--contraction and relaxation, however, are still poorly understood. This review will attempt to summarize information about two potassium channel proteins - - large-conductance calcium-activated (Kca) and delayed rectifier (KDR) channels - - in airway smooth muscle cells and to describe the relevant genetic information that has emerged for these channels from other tissues. I will not attempt to review information about ATP-sensitive potassium channels, since detailed electrophysiologic recordings of these channels in airway smooth muscle cells have not yet been reported.

2. OVERVIEW OF POTASSIUM CHANNELS The importance of potassium conductance for the maintenance of normal electrical behavior in smooth muscle has long been known (Casteels, 1981). Potassium channels appear to play important roles in setting resting membrane potential and limiting electrical responses to excitatory stimuli.

Abbreviations--4-AP, 4-aminopyridine; BK, large conductance Kc~; Kc,, calcium-activated potassium; KDR, delayed-rectifier potassium; TEA, tetraethylammonium.

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Additionally, various agents that relax smooth muscle hyperpolarize the tissue by activating potassium channels (Allen et al., 1985, 1986; Honda et al., 1986; Weston and Abbott, 1987). Airway smooth muscle generally behaves as an electrically quiescent tissue that depolarizes during activation in a graded fashion, without prominent spike depolarizations (Kirkpatrick, 1975; Kroeger and Stephens, 1975; Coburn, 1977). In the presence of substances known to block potassium channels, however, spontaneous electrical activity and spike depolarizations, as well as increases in tonic force, are initiated. It can thus be inferred that a dominant potassium conductance acts to stabilize electrical and contractile activity of airway smooth muscle and conversely that excitation-contraction coupling events are probably associated with an inhibition of potassium conductance. These indirect data are now bolstered by direct measurements of channel behavior at the whole-cell and single-channel levels, some information about channel regulation and in some cases, precise pharmacological studies using specific channel antagonists to examine the role of specific potassium channels on airway smooth muscle function. Over the past few years, several families of potassium channels have been cloned, their relationship to other voltage-dependent, cation selective channels has been determined and some information about channel tertiary structure has emerged from mutations of specific sequences and the development of chimera proteins. Careful study of the pharmacology of re-expressed mutants has provided the first structure/function information at the molecular level.

3. CALCIUM-ACTIVATED POTASSIUM CHANNELS Ion channels that are selective for potassium ions and activated by cytosolic calcium constitute a family of channel proteins that is widely distributed in biology. Calcium-activated, potassium-selective channels are not only found throughout the phylogenetic ladder, but are distributed in numerous tissues throughout the mammalian body. While these channels are described as 'calcium-activated,' a more precise description would indicate that the open-state probability of the channel is markedly increased at higher concentrations of cytosolic calcium. The mechanism by which calcium ions act upon the channel protein to influence open-state probability is not yet certain. At the same time, these channels belong to the general class of voltage-dependent ion channels, in that depolarization of the cell membrane also increases the open-state probability. While the specific functions of these channels in most cells is not known, the linkage between cytosolic calcium and channel activity provokes the suggestion that they comprise a general mechanism by which excitatory signalling processes leading to rises in cytosolic calcium are limited, or terminated, as a consequence of the hyperpolarization associated with channel opening. A prominent member of the family of Kca channels is the large conductance Kca channel (also termed maxi-K, or BK), which was easily observed at the single-channel level in early patch-clamp experiments (for review see Blatz and Magleby, 1987). This channel is now recognized as a ubiquitous channel found in high copy number in smooth muscle and other tissues. The discovery of potent and selective peptide inhibitors of BK channels (Miller et al., 1985; Galvez et al., 1990) has proven quite valuable in selectively inhibiting this channel, as well as providing a potential means of isolating the channel protein. 3.1. SINGLECHANNELMEASUREMENTS The first measurements of potassium channels in airway smooth muscle cells were made by McCann and Welsh in canine tracheal myocytes (McCann and Welsh, 1986). They reported a large conductance, calcium-sensitive potassium channel in on-cell and inside-out patches. This channel has now been measured by several laboratories and there is substantial agreement about its single-channel characteristics, pharmacology and distribution (McCann and Welsh, 1986; Kume et al., 1990; Kume and Kotlikoff, 1991; Muraki et al., 1990; Saunders and Farley, 199 !; Green et al., i 991; Stockbridge et al., 1991; Boyle et al., 1992). Additionally, the channel has been isolated from canine and bovine trachealis membranes and reconstituted in planar lipid bilayers (Savaria et al., 1992). Under conditions of symmetrical potassium, this channel has a conductance of over 220 pS and open-state probability is markedly increased as the cytosolic calcium concentration is

Potassium channels in airway smooth muscle

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increased. Channel activity is also markedly dependent on cytoplasmic pH, with higher concentrations of hydrogen ions decreasing open-state probability at any given voltage or calcium concentration (Kume et al., 1990). Several smaller calcium-activated potassium conductances have been reported in swine tracheal myocytes (Saunders and Farley, 1991); however, well-described subconductance states for the large conductance Kca channel (Stockbridge et al., 1991) and the block of these smaller currents in outside-out patches from airway myocytes by charybdotoxin (H. Kume and M. I. Kotlikoff, unpublished data), indicates caution with respect to the interpretation that smaller, calcium-sensitive unitary currents in single-channel records are unique channels. A large conductance (268 pS) calcium-independent potassium channel with somewhat different pharmacology than the large conductance Kca channel has also been reported in inside-out patches from bovine myocytes (Green et al., 1991); however, the charybdotoxin sensitivity of these channels could not be determined because they were not present in outside-out patches. In our experience, charybdotoxin or iberiotoxin blocks all large unitary currents in outside-out patches from airway smooth muscle cells (Kume and Kotlikoff, 1991; Kume et al., 1992; Boyle et al., 1992). Pharmacologic studies at the single channel level have provided good evidence of selective block as well as information about the molecular interaction between the blocking compound and the channel protein. Kca channels in airway smooth muscle cells are sensitive to charybdotoxin (Boyle et al., 1992; Savaria et al., 1992) and blocked by low concentrations of tetraethylammonium (TEA). A quantitative study of the effect of TEA on smooth muscle Kca channels indicates that the dissociation constant for TEA block is less than 200/~M in arterial myocytes (Langton et al., 1991). We have reported similar sensitivity of the channel in airway cells (Boyle et al., 1992). The sensitivity of Kca channels to TEA has relevance for a number of studies, indicating that application of concentrations of TEA between 10 and 30 mM are required to block outward rectification (Kirkpatrick, 1975) and initiate spontaneous electrical and contractile activity (Kroeger and Stephens, 1975; Imaizumi and Watanabe, 1981). Effects requiring these concentrations of TEA are likely associated with inhibition of other potassium channels (see Section 4). Conversely, Kc~ channels are not blocked by glibenclamide (10-100 #M) (Boyle et al., 1992; Langton et al., 1991), an inhibitor of ATP-sensitive potassium channels, or 4-aminopyridines (1-5 mM) (Muraki et al., 1990; Green et al., 1991; Boyle et al., 1992), inhibitors of several potassium currents in other cell types. While one study indicated that ATP blocks Kc~ channels in airway smooth muscle cells (Groschner et al., 1991), it appears that this finding resulted from the chelation of calcium by ATP (Klockner and Isenberg, 1992). Thus the most selective antagonist of Kc~ channels in smooth muscle appears to be charybdotoxin, which does not appear to block any potassium channels other than large-conductance Kc~ channels in airway smooth muscle cells. However, it should be noted that effects on intrinsic airway nerves have not been evaluated. In vitro pharmacologic studies should therefore be careful to exclude results associated with depolarization of airway nerve terminals and the attendant release of neurotransmitters. 3.2. WHOLE-CELLMEASUREMENTS Several groups have reported the measurement of macroscopic, whole-cell currents in dialyzed airway smooth muscle cells. Dialyzed cell recordings are particularly difficult to interpret with respect to the degree to which they reflect the physiological availability of Kc~ current at a given potential, since the available current is a function of both intracellular calcium concentration and the cytosolic calcium buffering capacity. In traditional whole-cell experiments, these factors are determined by the choice of the particular dialysis conditions, which therefore predetermine the experimental result, the published data on potassium channels in airway smooth muscle cells reflects this complication. As in other smooth muscle cells, under very low calcium buffering conditions, calcium-activated potassium currents are observed in airway smooth muscle cells (Kotlikoff, 1990; Muraki et al., 1990). These currents are activated by step depolarizations and are blocked by charybdotoxin or TEA. Under these conditions, some investigators find a rapidly activating and inactivating current, the kinetics of which presumably mirror the activation of calcium channels and their rapid inactivation (Hisada et al., 1990; Muraki et al., 1990). Other investigators report activation of sustained currents that are calcium sensitive and do not inactivate rapidly (Hisada et al., 1990; Green et al., 1991). These currents probably reflect the voltage-

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dependent (as opposed to calcium-dependent) activation of Kc~ channels under conditions of less rigorous cytosolic calcium buffering. At the other extreme, experimental conditions with aggressive calcium buffering eliminate calcium-dependent currents and have been used to study other voltage-dependent potassium conductances (Kotlikoff, 1990). Whole-cell experiments have generally consistently reported that calcium-activated currents are TEA sensitive and aminopyridine resistant (Kotlikoff, 1990; Muraki et al., 1990), although Hisada et al. (1990) have reported calcium and 4-aminopyridine-sensitive currents in the guinea pig. Since cell dialysis may result in either overbuffering or underbuffering of cytosolic calcium, the most accurate way to determine the degree of current availability under physiological conditions is by using a non-dialyzed whole-cell configuration such as the nystatin permeabilization method (Horn and Marry, 1988), which provides good electrical access and voltage-clamp characteristics. Nystatin forms pores in the membrane patch that are permeant to monovalent ions, but relatively impermeant to cations. Calcium imaging of airway smooth muscle cells loaded with fura 2AM indicates that cytosolic calcium concentration does not change over the course of seal formation and access in experiments using the nystatin method (Murray et al., 1992). Nystatin-permeabilized whole-cell experiments reveal very little calcium-dependent outward current over physiological voltage ranges (Fleischmann et al., 1992). Moreover, in such experiments 4-aminopyridine (1-5 mM) depolarizes smooth muscle cells and markedly increases tone in muscle strips, whereas charybdotoxin (100 riM) does neither. These experiments suggest that the predominate voltage-dependent potassium current in unstimulated cells is the calcium-insensitive, delayed-rectifier current (see Section 4). It should be noted, however, that this argument may not hold for all airway smooth muscle. Small and colleagues point to the effect of charybdotoxin on mechanical and electrical activity in guinea-pig trachealis to suggest that Kca channels determine outward rectification and resting membrane potential (Murray et al., 1991). Since guinea pig trachealis, unlike most airway smooth muscles, is spontaneously active, this may indicate a greater degree of resting activity of Kca channels associated with spontaneous fluctuations of intracellular calcium. Suarez-Kurtz et al. (1991) and Jones et al. (1990), however, have reported no significant contractile effect of charybdotoxin or iberiotoxin on guinea pig tracheal rings. The varying results may relate to the use of indomethacin to eliminate spontaneous tone. Additionally, since these experiments did not block neurotransmitter release, it is difficult to exclude neural effects. Peptidyl Kc~ channel blockers do appear to increase spontaneous tone in tissues such as the bladder, in which delayed rectifier channels are not prominent (Suarez-Kurtz et al., 1991; Klockner and Isenberg, 1985). The available evidence at present suggests that the large calcium-activated currents observed in voltage-clamp experiments reflect non-physiologic calcium-buffering conditions in the dialysis solutions. This evidence includes the lack of availability of calcium-activated potassium currents at physiological potentials in non-dialyzed cells (Fleischmann et al., 1992), including acutely dissociated human cells (B. K. Fleischmann and M. I. Kotlikoff, unpublished data) and a lack of effect of charybdotoxin on resting mechanical activity (Jones et al., 1990; Suarez-Kurtz et al., 1991; Fleischmann et al., 1992). Moreover, an examination of reported data on the open-state probability of Kca channels under physiological conditions of cytosolic calcium, pH and membrane potential suggest that single Kc~ channels are rarely open under these conditions (McCann and Welsh, 1986; Kume et al., 1990; Kume and Kotlikoff, 1991; Green et al., 1991; Saunders and Farley, 1992). 3.3. AGONISTREGULATIONOF Kca CHANNELS The question then arises as to the role of Kca channels in normal physiological processes. Substantial evidence now exists to indicate that Kca channels are important target proteins involved in agonist-induced contraction and relaxation. As described above, Kc~ channels are activated by membrane depolarization and by a rise in cytosolic calcium, two events that accompany excitation-contraction coupling in airway smooth muscle. Since the single channel conductance and the density of these channels in ASM membranes are quite high, an increase in open-state probability might be expected to occur following exposure to contractile agonists. Following exposure of isolated smooth muscle cells to contractile agonists, a brief burst of channel activity occurs, followed by a potent inhibition of channel activity (Benham and BoRon, 1986; Cole and Sanders, 1989; Kotlikoff, 1990; Saunders and Farley, 1992). The burst of outward current appears

Potassium channels in airway smooth muscle to result from the release of intracellular calcium stores and the resulting cytoplasmic calcium transient, since it can be stimulated by calcium ionophores (Kotlikoff, 1990) and by caffeine (Saunders and Farley, 1992). It is likely, however, that the subsequent long-lasting inhibition of channel activity to levels below baseline (Benham and Bolton, 1986; Saunders and Farley, 1992; Cole and Sanders, 1989) is the physiologically more important response, since it would appear to enable the long-lasting tonic depolarization that attends excitation-contraction coupling. We have recently shown that exposure of the extracellular surface of membrane patches to the cholinergic agonist metacholine results in a potent and reversible inhibition of the opening of single Kc, channels (Kume and Kotlikoff, 1991). This receptor-channel coupling is blocked by guanosine 5'-O-(2-thiodiphosphate) and by pretreatment of cells with pertussis toxin. Moreover, inhibitory coupling is observed in inside-out patches following addition of exogenous guanine nucleotides to patches that have been pre-exposed to a muscarinic agonist (Kume et al., 1992). These data indicate that stimulation of muscarinic receptors (probably M2) results in inhibition of Kca channel opening by means of the membrane delimited action of a pertussis toxin-sensitive G protein, reminiscent of the muscarinic receptor/Gi/potassium channel linkage in atrial myocytes (Yatani et al., 1987). Additional mechanisms of receptor/channel coupling are not excluded, however, by these data. In this regard, it is interesting to note that Saunders and Farley report channel inhibition after exposure to caffeine, suggesting that channel inactivation may occur by a calcium-dependent process (Saunders and Farley, 1992). It is likely that inhibition of Kca channels plays an important role in lowering potassium conductance during contraction. Since these channels are quite numerous and the single channel conductance is very high, even modest increases in open-state probability associated with increased cytosolic calcium would be likely to influence resting membrane potential. Experiments that remove this negative feedback to channel opening would be expected to markedly inhibit the response to contractile agonists; such experiments are currently in progress. Augmentation of potassium channel activity by fl-agonists appears to be a consistent feature of their action (Allen et al., 1985; Honda et aL, 1986). The first suggestion that the Kc, channel was responsible for this action was presented by Kume et al. (1989), who demonstrated that B-agonists activate single Kca channels in on-cell recordings. The fact that this action was increased by a phosphatase inhibitor and that cAMP-dependent protein kinase itself activated the channel in inside-out patches, indicated that a traditional phosphorylation-dependent coupling exists between B-receptor stimulation and Kc, channels. An identical regulatory mechanism had previously been demonstrated for Kc, channels in the brain (Ewald et al., 1985). It now appears that this is not the only mechanism by which B-receptors couple to Kc a channels, however. Recent experiments have demonstrated that KCa channels are activated by isoproterenol in outside-out patches under conditions that are extremely unlikely to support phosphorylation (Kume et al., 1992). Additionally, the g subunit of the stimulatory G protein, Gs, directly activates Kc~ channels in inside-out patches and this activation is not affected by the presence of a competitive inhibitor of ATP, or by an inhibitor of cAMP-dependent protein kinase (Fig. 1). It thus appears that a membrane-delimited activation of Kca channels by Gs-linked receptors occurs in airway smooth muscle cells, analogous to the stimulation of adenylyl cyclase (Gilman, 1987) and cardiac calcium channels (Yatani et al., 1988) by ats. The degree to which each of these pathways contributes to the overall hyperpolarizing effect is not yet certain; however, the demonstration that the B-agonist isoproterenol (1 #M) stimulates the channel in outside-out patches (Kume et al., 1992), suggests that this pathway is a physiologically important one. The above description of the regulation of Kc, channels by nauscarinic- and adrenergic-receptor binding is the first demonstration at the single channel level that a mammalian ion channel can be both positively and negatively modulated by G proteins. Stimulation and inhibition of channel activity can be sequentially demonstrated in the same membrane patch (Fig. 2). The dual G protein-dependent regulation of Kc, channels described above bears several important similarities with the regulation of another transmembrane protein, adenylyl cyclase. The activity of both proteins appears to be stimulated by a direct interaction with the 0t subunit of the heterotrimeric G protein complex. In inside-out patches, stimulatory or inhibitory channel modulation can be demonstrated by pre-exposing the external membrane surface to a Gs- or G~-linked receptor agonist, presumably by means of the guanine nucleotide releasing protein-like activity of the

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L_ FIG. 1. The ~ subunit of GS opens Kca channels. Experiments from inside-out patches from porcine tracheal myocytes demonstrate the action of GTP~cS-activated recombinant ~cs proteins on Kca channels. (Top) Dose-dependent action of ~s proteins on channel activity. (Below) Effect on channel activity is not altered by competitive inhibition of endogenous ATP, indicating that the mechanism of activation is independent of phosphorylation. Calibration bar: 10 sec, 5 pA. Reprinted from Kume et al. (1992). receptor agonist which promotes the release of bound G D P from the 0c subunit. Release of G D P from the subgroup of G proteins bound to a specific receptor type allows selective activation of either stimulatory or inhibitory modulatory effects (Kume et al., 1992). These regulatory features are illustrated in Fig. 3. Several proteins have been identified as key regulatory targets involved in mediating fl-adrenergic relaxation. Whereas it is likely that numerous components of the contractile system are modulated

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Time (rain) Fro. 2. Muscarinic inhibition and adrenergic stimulation of K ~ channel activity in the same outside-out patch. Sequential exposure of the same outside-out patch from a ferret tracheal myocyte to methacholine (10 /~M) and isoproterenol (1 /~M) inhibits and stimulates channel activity, respectively. Open-state probability is shown as a function of time before, during and after exposure to the agonists. The intracellular patch surface is exposed to GTP (100 #M) to optimize G protein coupling. Each point represents the average open-state probability for I min. Reprinted from Kume et al. (1992).

Potassium channels in airway smooth muscle

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FIG. 3. Model of inhibitory and stimulatory G protein modulation of Kc~channel activity and of phosphorylation-dependent and -independent stimulatory mechanisms. Muscarinic- and fl-adrenergic regulation of channel activity is schematicallydepicted. Binding of a molecule of acetylcholine(ACH) results in the release of GDP and the binding of GTP at the ~tsubunit. the dotted line depicts the uncertainty as to the molecular action of the subunits on the channel following dissociation of the heterotrimer. The unstimulated fl-receptor is associated with a GDP-bound Gs complex. The solid lines show the phosphorylation independent, presumably direct interaction of ~ts with the channel and the phosphorylation-dependentaction of A kinase on the channel protein. The receptor subtypes shown have not been experimentallyverified.

by fl-adrenergic stimulation, evidence at the tissue level suggests that Kc~ channels represent functionally important target proteins in airway smooth muscle relaxation. Experiments in guinea pig tracheal rings (Jones et al., 1990) and in human bronchial segments (Suarez-Kurtz et al., 1991), indicate that charybdotoxin markedly reduces the ability of isoproterenol and salbutamol to relax precontracted airway smooth muscle strips. The selectivity of charybdotoxin as a Kc~ channel inhibitor in this tissue (Boyle et al., 1992) supports the suggestion that the stimulation of these channels by fl-adrenergic receptors is a key element of their action. Moreover, the toxin had no effect on contractile responses in the absence of the bronchodilators and did not antagonize the relaxant actions of ATP-sensitive potassium channel openers (Jones et al., 1990). Similar results have been reported for the relaxant effect of stimulators of cGMP in bovine tracheal smooth muscle (Jones et al., 1990; Hamaguchi et al., 1992). Interestingly, the results of Jones et al. indicate that the shift was less prominent when relaxation was produced by by-passing the fl-adrenergic receptor and more directly stimulating cAMP-dependent protein kinase using a phosphodiesterase inhibitor or cAMP analog. These results are consistent with the mechanisms of receptor/channel coupling proposed above, in that a substantial component of this coupling may occur independent of rises of cAMP. The decreased potency of cAMP elevating agents as bronchodilators (Jones et al., 1990), as well as the dissociation between relaxation and cAMP concentration observed during equivalent relaxations to forskolin and isoproterenol in airway smooth muscle (Zhou et al., 1992), suggest that .cAMP-independent actions of Gs may be important in mediating smooth muscle relaxation.

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While the large single-channel conductance of BK channels have resulted in numerous descriptions of these currents, fewer studies have focussed on another potentially extremely important group of potassium channels. Delayed rectifier potassium currents that activate with characteristic kinetics and show classical inactivation (Kotlikoff, 1989, 1990; Boyle et al., 1992; Fleischmann et al., 1992) are voltage-gated, calcium-insensitive delayed rectifier channels, which appear to belong to the well-characterized family of channel proteins that have been identified following the cloning of the Shaker locus in Drosophila (Papazian et al., 1987). These channels have been characterized in other tissues by cloning, re-expression and mutation studies and a consensus model of channel structure has emerged (Pongs, 1992; Jan and Jan, 1992). Although KDR channels from smooth muscle have not yet been cloned, the biophysics and pharmacology of the channels have been reported and generally correspond to the observed features of re-expressed Shaker proteins. In airway smooth muscle cells these characteristics have been reported. Voltage-dependent outward currents activate at a threshold positive to - 5 0 mV under physiological conditions (Fleischmann et al., 1992); the currents inactivate in a voltage- and time-dependent fashion (Kotlikoff, 1989, 1990; Boyle et al., 1992). The inactivating characteristics of the macroscopic current were utilized as a biophysical fingerprint for the identification of the

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FIG. 4(B) FIG. 4. Single channel and whole-cell delayed rectifier potassium currents in airway smooth muscle. (A) Single, delayed rectifier currents from an on-cell patch from a canine tracheal myocyte; depolarizing clamp steps from - 7 0 mV to the test potential shown evoked voltage-dependent opening of small, unitary potassium currents. The pipette contained 100 nM charybdotoxin, demonstrating the insensitivity of KoR channels to this toxin. (B) Voltage-dependent inactivation of single channel and whole-cell delayed rectifier currents recorded from porcine tracheal myocytes. Above: On-cell single channel records recorded in the presence of TEA (1 mM) in the pipette. The amplitude of Kca currents (larger openings) is substantially decreased and the characteristic flickery block is apparent; the smaller conductance KOR channels are unaffected. Middle: Amplitude histograms indicate the decrease in KDRopenings as the pre-pulse potential (10 sec conditioning pulses) is more positive. Below: The same protocol in whole cell experiments demonstrates the voltage-dependent inactivation of the inactivating current. Reprinted from Boyle et al. (1992), with permission of the copyright holder, Cambridge University Press, Cambridge.

underlying unitary currents (Boyle et al., 1992), revealing a small unitary conductance of approximately 13 pS that displays similar voltage-dependent and time-dependent inactivation (Fig. 4). The pharmacology at the single channel level was indistinguishable from that observed in whole-cell experiments (Kotlikoff, 1990; Boyle et al., 1992). Delayed rectifier currents are relatively insensitive to TEA (IC50 approximately 50 mM at the whole-cell level; no block up to 1 mM in single channel experiments) and are unaffected by charybdotoxin (200 nM) or glibenclamide (20 /~M). The channel is potently blocked by 4-aminopyridine (5 mM), however and the fact that 4-AP does not block Kca channels (Boyle et al., 1992) makes this compound a useful pharmacological tool to explore the function of delayed rectifier channels in airway smooth muscle. We have recently examined the physiological importance of KDR channels using a combination of whole-cell voltage-clamp, current-clamp and organ bath techniques (Fleischmann et al., 1992). Since the traditional whole-cell patch-clamp method results in the dialysis of the intracellular

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solution and an artificial determination of intraceilular calcium concentration and buffering capacity, experiments were performed using the nystatin whole-cell method, as described above. Under non-dialyzing conditions performed at near physiological temperature, voltage-clamped ferret tracheal myocytes displayed prominent delayed rectifier currents with activation thresholds in the range of - 40 to - 50 mV. This current was almost completely inhibited by 4-AP. Only a small current component was charybdotoxin sensitive. Moreover, in current-clamp experiments, 4-AP depolarized myocytes, whereas charybdotoxin did not and in organ bath measurements of tension, 4-AP increased tension in the presence of atropine and tetrodotoxin, whereas charybdotoxin had minimal effects; the 4-AP-induced tension was completely blocked by nifedipine (1/~ M). As discussed above, these experiments suggest that KOR channels determine the outward rectification and resting membrane potential of ferret tracheal myocytes, whereas BK channels are closed under resting conditions. 5. CONCLUSION Studies to date have clearly identified two major potassium channels and the electrophysiology and pharmacology of these channels is fairly clear. Substantial experimental evidence indicates that large-conductance calcium-activated potassium channels are important hormone targets, whose open probability is modulated by agents that alter smooth muscle tone. The density of these channels in airway smooth muscle cell membranes, as well as the high single channel conductance, provide an enormous reservoir of potassium permeability. Selective Kca channel agonists would therefore constitute potentially potent hyperpolarizing agents. The demonstration that fl-adrenergic agonists act, at least partially, by opening these channels (Kume et al., 1989, 1992; Jones et al., 1990), presents interesting therapeutic possibilities for the use of potential Kca agonists as bronchodilators. This would provide the ability to bypass the fl-adrenergic receptor, stimulating the ultimate target protein directly. While such agents might be expected to be less effective than fl-agonists to the extent that fl-receptors activate other functional target proteins, they would avoid problems related to receptor desensitization and fl-adrenergic side effects. Of course, issues of the specificity of the channel agonist would be vital in the development of selective bronchodilators. Similar strategies might apply for delayed rectifier potassium channels. These channels are open under resting conditions and channel blockade results in depolarization (Fleischmann et al., 1992). Experiments performed under physiologic conditions of calcium buffering indicate that they account for the outwardly rectifying electrical behavior that is characteristic of this tissue. At present, the extent to which these potassium channels are also modulated by agents that alter the contractile state of smooth muscle is unknown. No channel agonists for delayed rectifiers currently exist, but the molecular characterization of this family of channel proteins, as well as the ability to easily re-express the channels, is likely to facilitate the development of such drugs. Activation of these channels would also be likely to hyperpolarize and relax airway smooth muscle, since only a small fraction of the maximum conductance is available at hyperpolarized potentials. The study of the membrane ion channels of airway smooth muscle cells is far from complete and the role of these important membrane proteins in contraction and relaxation processes is only beginning to emerge. The next decade holds the promise of a more thorough understanding of the biophysics and molecular biology of airway potassium channels, as well as the discovery of the role of these channels in contraction, growth and excitability. REFERENCES ALLEN,S. L., BEECH,D. J., FOS~R, R. W., MORGAN,G. P. and SMALL,R. C. 0985) Electrophysiological and other aspects of the relaxant action of isoprenaline in guinea-pig isolated trachealis. Br. J. Pharmac. 86: 843-854. ALLEN,S. L., CORTIJO,J., Fos'rER, R. W., MORGAN,G. P., SMALL,R. C. and WESTON,A. H. (1986) Mechanical and electrical aspects of the relaxant action of aminophylline in guinea-pig isolated trachealis. Br. J. Pharmac. 88: 473--483. BENHAM,C. D. and BOLTON,T. B. (1986) Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit. J. Physiol. 381: 385--406.

Potassium channels in airway smooth muscle

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