Symmetry and asymmetry of permeation through toxin-modified Na+ channels.

Single Na+ channels from rat skeletal muscle were inserted into planar lipid bilayers in the presence of either 200 nM batrachotoxin (BTX) or 50 microM veratridine (VT). These toxins, in addition to their ability to shift inactivation of voltage-gated Na+ channels, may be used as probes of ion conduction in these channels. Channels modified by either of the toxins have qualitatively similar selectivity for the alkali cations (Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+). Biionic reversal potentials, for example, were concentration independent for all ions studied. Na+/K+ and Na+/Rb+ reversal potentials, however, were dependent on the orientation of the ionic species with respect to the intra- or extracellular face of the channel, whereas Na+/Li+ biionic reversal potentials were not orientation dependent. A simple, four-barrier, three-well, single-ion occupancy model was used to generate current-voltage relationships similar to those observed in symmetrical solutions of Na, K, or Li ions. The barrier profiles for Na and Li ions were symmetric, whereas that for K ions was asymmetric. This suggests the barrier to ion permeation for K ions may be different than that for Na and Li ions. With this model, these hypothetical energy barrier profiles could predict the orientation-dependent reversal potentials observed for Na+/K+ and Na+/Rb+. The energy barrier profiles, however, were not capable of describing biionic Na/Li ion permeation. Together these results support the hypothesis that Na ions have a different rate determining step for ion permeation than that of K and Rb ions.     

Batrachotoxin-modified sodium channels from squid optic nerve in planar bilayers. Ion conduction and gating properties

Squid optic nerve sodium channels were characterized in planar bilayers in the presence of batrachotoxin (BTX). The channel exhibits a conductance of 20 pS in symmetrical 200 mM NaCl and behaves as a sodium electrode. The single-channel conductance saturates with increasing the concentration of sodium and the channel conductance vs. sodium concentration relation is well described by a simple rectangular hyperbola. The apparent dissociation constant of the channel for sodium is 11 mM and the maximal conductance is 23 pS. The selectivity determined from reversal potentials obtained in mixed ionic conditions is Na+ approximately Li+ greater than K+ greater than Rb+ greater than Cs+. Calcium blocks the channel in a voltage-dependent manner. Analysis of single-channel membranes showed that the probability of being open (Po) vs. voltage relation is sigmoidal with a value of 0.5 between -90 and -100 mV. The fitting of Po requires at least two closed and one open state. The apparent gating charge required to move through the whole transmembrane voltage during the closed-open transition is four to five electronic charges per channel. Distribution of open and closed times are well described by single exponentials in most of the voltage range tested and mean open and mean closed times are voltage dependent. The number of charges associated with channel closing is 1.6 electronic charges per channel. Tetrodotoxin blocked the BTX-modified channel being the blockade favored by negative voltages. The apparent dissociation constant at zero potential is 16 nM. We concluded that sodium channels from the squid optic nerve are similar to other BTX-modified channels reconstituted in bilayers and to the BTX-modified sodium channel detected in the squid giant axon.     

Modeling ion permeation through batrachotoxin-modified Na+ channels from rat skeletal muscle with a multi-ion pore.

The mechanism of ion permeation through Na+ channels that have been modified by batrachotoxin (BTX) and inserted into planar bilayers has been generally described by models based on single-ion occupancy, with or without an influence of negative surface charge, depending on the tissue source. For native Na+ channels there is evidence suggestive of a multi-ion conduction mechanism. To explore the question of ion occupancy, we have reexamined permeation of Na+, Li+, and K+ through BTX-modified Na+ channels from rat skeletal muscle. Single-channel current-voltage (I-V) behavior was studied in neutral lipid bilayers in the presence of symmetrical Na+ concentrations ranging from 0.5 to 3,000 mM. The dependence of unitary current on the mole fraction of Na+ was also examined in symmetrical mixtures of Na(+)-Li+ and Na(+)-K+ at a constant total ionic strength of 206 and 2,006 mM. The dependence of unitary conductance on symmetrical Na+ concentration does not exhibit Michaelis-Menten behavior characteristic of single-ion occupancy but can be simulated by an Eyring-type model with three barriers and two sites (3B2S) that includes double occupancy and ion-ion repulsion. Best-fit energy barrier profiles for Na+, Li+, and K+ were obtained by nonlinear curve fitting of I-V data using the 3B2S model. The Na(+)-Li+ and Na(+)-K+ mole-fraction experiments do not exhibit an anomalous mole-fraction effect. However, the 3B2S model is able to account for the biphasic dependence of unitary conductance on symmetrical [Na+] that is suggestive of multiple occupancy and the monotonic dependence of unitary current on the mole fraction of Na+ that is compatible with single or multiple occupancy. The best-fit 3B2S barrier profiles also successfully predict bi-ionic reversal potentials for Na(+)-Li+ and Na(+)-K+ in both orientations across the channel. Our experimental and modeling results reconcile the dual personality of ion permeation through Na+ channels, which can display features of single or multiple occupancy under various conditions. To a first approximation, the 3B2S model developed for this channel does not require corrections for vestibule surface charge. However, if negative surface charges of the protein do influence conduction, the conductance behavior in the limit of low [Na+] does not correspond to a Gouy-Chapman model of planar surface charge.     

Batrachotoxin-resistant Na+ channels derived from point mutations in transmembrane segment D4-S6.

Local anesthetics (LAs) block voltage-gated Na+ channels in excitable cells, whereas batrachotoxin (BTX) keeps these channels open persistently. Previous work delimited the LA receptor within the D4-S6 segment of the Na+ channel alpha-subunit, whereas the putative BTX receptor was found within the D1-S6. We mutated residues at D4-S6 critical for LA binding to determine whether such mutations modulate the BTX phenotype in rat skeletal muscle Na+ channels (mu1/rSkm1). We show that mu1-F1579K and mu1-N1584K channels become completely resistant to 5 microM BTX. In contrast, mu1-Y1586K channels remain BTX-sensitive; their fast and slow inactivation is eliminated by BTX after repetitive depolarization. Furthermore, we demonstrate that cocaine elicits a profound time-dependent block after channel activation, consistent with preferential LA binding to BTX-modified open channels. We propose that channel opening promotes better exposure of receptor sites for binding with BTX and LAs, possibly by widening the bordering area around D1-S6, D4-S6, and the pore region. The BTX receptor is probably located at the interface of D1-S6 and D4-S6 segments adjacent to the LA receptor. These two S6 segments may appose too closely to bind BTX and LAs simultaneously when the channel is in its resting closed state.     

Ion selectivity of porcine skeletal muscle Ca2+ release channels is unaffected by the Arg615 to Cys615 mutation.

The Arg615 to Cys615 mutation of the sarcoplasmic reticulum (SR) Ca2+ release channel of malignant hyperthermia susceptible (MHS) pigs results in a decreased sensitivity of the channel to inhibitory Ca2+ concentrations. To investigate whether this mutation also affects the ion selectivity filter of the channel, the monovalent cation conductances and ion permeability ratios of single Ca2+ release channels incorporated into planar lipid bilayers were compared. Monovalent cation conductances in symmetrical solutions were: Li+, 183 pS +/- 3 (n = 21); Na+, 474 pS +/- 6 (n = 29); K+, 771 pS +/- 7 (n = 29); Rb+, 502 pS +/- 10 (n = 22); and Cs+, 527 pS +/- 5 (n = 16). The single-channel conductances of MHS and normal Ca2+ release channel were not significantly different for any of the monovalent cations tested. Permeability ratios measured under biionic conditions had the permeability sequence Ca2+ >> Li+ > Na+ > K+ > or Rb+ > Cs+, with no significant difference noted between MHS and normal channels. This systematic examination of the conduction properties of the pig skeletal muscle Ca2+ release channel indicated a higher Ca2+ selectivity (PCa2+:Pk+ approximately 15.5) than the sixfold Ca2+ selectivity previously reported for rabbit skeletal (Smith et al., 1988) or sheep cardiac muscle (Tinker et al., 1992) Ca2+ release channels. These results also indicate that although Ca2+ regulation of Ca2+ release channel activity is altered, the Arg615 to Cys615 mutation of the porcine Ca2+ release channel does not affect the conductance or ion selectivity properties of the channel.     

Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells

Single channel and whole cell recordings were used to study ion permeation through Ca channels in isolated ventricular heart cells of guinea pigs. We evaluated the permeability to various divalent and monovalent cations in two ways, by measuring either unitary current amplitude or reversal potential (Erev). According to whole cell measurements of Erev, the relative permeability sequence is Ca2+ greater than Sr2+ greater than Ba2+ for divalent ions; Mg2+ is not measurably permeant. Monovalent ions follow the sequence Li+ greater than Na+ greater than K+ greater than Cs+, and are much less permeant than the divalents. These whole cell measurements were supported by single channel recordings, which showed clear outward currents through single Ca channels at strong depolarizations, similar values of Erev, and similar inflections in the current-voltage relation near Erev. Information from Erev measurements stands in contrast to estimates of open channel flux or single channel conductance, which give the sequence Na+ (85 pS) greater than Li+ (45 pS) greater than Ba2+ (20 pS) greater than Ca2+ (9 pS) near 0 mV with 110-150 mM charge carrier. Thus, ions with a higher permeability, judged by Erev, have lower ion transfer rates. In another comparison, whole cell Na currents through Ca channels are halved by less than 2 microM [Ca]o, but greater than 10 mM [Ca]o is required to produce half-maximal unitary Ca current. All of these observations seem consistent with a recent hypothesis for the mechanism of Ca channel permeation, which proposes that: ions pass through the pore in single file, interacting with multiple binding sites along the way; selectivity is largely determined by ion affinity to the binding sites rather than by exclusion by a selectivity filter; occupancy by only one Ca ion is sufficient to block the pore's high conductance for monovalent ions like Na+; rapid permeation by Ca ions depends upon double occupancy, which only becomes significant at millimolar [Ca]o, because of electrostatic repulsion or some other interaction between ions; and once double occupancy occurs, the ion-ion interaction helps promote a quick exit of Ca ions from the pore into the cell.     

Ionic selectivity of the sodium channel of frog skeletal muscle

The ionic selectivity of the Na channel to a variety of metal and organic cations is studied in frog semitendinosus muscle. Na channel currents are measured under voltage clamp conditions in fibers bathed in solutions with all Na+ replaced by a test ion. Permeability ratios are calculated from measured reversal potentials using the Goldman-Hodgkin-Katz equation. The permeability sequence was Na+ approximately Li+ approximately hydroxylammonium greater than hydrazinium greater than ammonium greater than guanidinium greater than K+ greater than aminoguanidinium in the ratios 1:0.96:0.94:0.31:0.11:0.093:0.048:0.031. No inward currents were observed for Ca++, methylammonium, methylguanidinium, tetraethylammonium, and tetramethylammonium. The results are consistent with the Hille model of the Na channel selectivity filter of the node of Ranvier and suggest that the selectivity filter of the two channels is the same.     

KcsA: it's a potassium channel

Ion conduction and selectivity properties of KcsA, a bacterial ion channel of known structure, were studied in a planar lipid bilayer system at the single-channel level. Selectivity sequences for permeant ions were determined by symmetrical solution conductance (K(+) > Rb(+), NH(4)(+), Tl(+) > Cs(+), Na(+), Li(+)) and by reversal potentials under bi-ionic or mixed-ion conditions (Tl(+) > K(+) > Rb(+) > NH(4)(+) > Na(+), Li(+)). Determination of reversal potentials with submillivolt accuracy shows that K(+) is over 150-fold more permeant than Na(+). Variation of conductance with concentration under symmetrical salt conditions is complex, with at least two ion-binding processes revealing themselves: a high affinity process below 20 mM and a low affinity process over the range 100-1,000 mM. These properties are analogous to those seen in many eukaryotic K(+) channels, and they establish KcsA as a faithful structural model for ion permeation in eukaryotic K(+) channels.     

A study of properties of batrachotoxin modified sodium channels

A further analysis of the effects of the steroidal alkaloid batrachotoxin (BTX) on sodium channels in frog node of Ranvier has been carried out under voltage-clamp conditions. The main properties of modified channels as compared with those of normal ones are as follows: The rate of channel closing is drastically decreased, whereas that of opening is changed slightly if at all; The steady-state voltage dependence of channel activation is shifted towards more negative potentials by 60-70 mV; Currents through modified channels do not show a decay during maintained depolarization as it is typical for normal channels. However modified channels retain the ability to partial inactivation as shown by experiments with depolarizing prepulses; Sodium against potassium selectivity beyond--20 mV suggesting either nonhomogeneity of the modified channels as for their kinetic and selectivity properties or potential-dependence of ionic selectivity for each channel; The selectivity sequence determined from peak current reversal potential measurements is as follows: H: Na :NH4:K = 528:1:0.47: :0.19; The effective pK value of proton block is decreased by about 0.4; 7) The sensitivity of the channels to tetrodotoxin (TTX) block is practically unchanged.     

A mechanosensitive K+ channel in heart cells. Activation by arachidonic acid

Mechanosensitive ion channels have been described in many types of cells. These channels are believed to transduce pressure signals into intracellular biochemical and physiological events. In this study, the patch-clamp technique was used to identify and characterize a mechanosensitive ion channel in rat atrial cells. In cell-attached patches, negative pressure in the pipette activated an ion channel in a pressure-dependent manner. The pressure to induce half-maximal activation was 12 +/- 3 mmHg at +40 mV, and nearly full activation was observed at approximately 20 mmHg. The probability of opening was voltage dependent, with greater channel activity at depolarized potentials. The mechanosensitive channel was identical to the K+ channel previously shown to be activated by arachidonic acid and other lipophilic compounds, as judged by the outwardly rectifying current-voltage relation, single channel amplitude, mean open time (1.4 +/- 0.3 ms), bursty openings, K+ selectivity, insensitivity to any known organic inhibitors of ion channels, and pH sensitivity. In symmetrical 140 mM KCl, the slope conductance was 94 +/- 11 pS at +60 mV and 64 +/- 8 pS at -60 mV. Anions and cations such as Cl-, glutamate, Na+, Cs+, Li+, Ca2+, and Ba2+ were not permeant. Extracellular Ba2+ (1 mM) blocked the inward K+ current completely. GdCl3 (100 microM) or CaCl2 (100 microM) did not alter the K+ channel activity or amplitude. Lowering of intracellular pH increased the pressure sensitivity of the channel. The K+ channel could be activated in the presence of 5 mM intracellular [ATP] or 10 microM glybenclamide in inside-out patches. In the absence of ATP, when the ATP-sensitive K+ channel was active, the mechanosensitive channel could further be activated by pressure, suggesting that they were two separate channels. The ATP-sensitive K+ channel was not mechanosensitive. Pressure activated the K+ channel in the presence of albumin, a fatty acid binding protein, suggesting that pressure and arachidonic acid activate the K+ channel via separate pathways.     

A discrete type of potassium channel conductance in snail neurons

The results of further investigations on a single potential dependent K+ channel are described. It was shown that ionic selectivity of the channel for monovalent ions is too high: Li+, Na+, and Cs+ are practically impermeant ions. Permeability of the channel for Rb+ is approximately 10 times less, and for Tl+ it is 2 times more than permeability for K+. Besides, it was found that open K+ channel has 16 multiple conductance levels.     

A Cationic Channel in the Guard Cell Tonoplast of Allium cepa

Stomatal movements depend on an osmoregulation process in which swelling or shrinking of the guard cells opens or closes the stomatal pore. Ions and water fluxes are an essential aspect of guard cell osmoregulation. Thus far, studies of these fluxes have focused on the guard cell plasma membrane. Guard cells, however, are a multi-compartment system that includes a prominent vacuole, which has a primary role in turgor regulation. This study reports on a detailed characterization of an ion channel at the guard cell tonoplast of Allium cepa (onion). We used patch-clamp methodology with isolated tonoplast patches to study conduction and gating at the single channel level. A voltage-dependent outward-rectifying cationic channel (210 picosiemens) was the dominant conductance. In symmetrical solutions the channel displayed an ohmic behavior in its current-voltage relationship. It also showed a very large rectification in the open probability. The channel was predominantly cationic and its sequence of ionic selectivity was weak (Na+ > K+ > Rb+ > Cs+). The channel conductance was not affected by intravacuolar pH. Analysis of membrane patches with multiple channels showed that the probability of a channel to open was independent of the opening of the other channels present in the patch and that there was a conservation of the open probability for different channels. Ensemble records generated using a pulse protocol showed slow activation and deactivation kinetics. A first-latency analysis of single-channel records in response to protocols with different prepulse duration indicated that this channel has more than one closed state.     

Selectivity and gating of the type L potassium channel in mouse lymphocytes

Type l voltage-gated K+ channels in murine lymphocytes were studied under voltage clamp in cell-attached patches and in the whole-cell configuration. The kinetics of activation of whole-cell currents during depolarizing pulses could be fit by a single exponential after an initial delay. Deactivation upon repolarization of both macroscopic and microscopic currents was mono-exponential, except in Rb-Ringer or Cs-Ringer solution in which tail currents often displayed "hooks," wherein the current first increased or remained constant before decaying. In some cells type l currents were contaminated by a small component due to type n K+ channels, which deactivate approximately 10 times slower than type l channels. Both macroscopic and single channel currents could be dissected either kinetically or pharmacologically into these two K+ channel types. The ionic selectivity and conductance of type l channels were studied by varying the internal and external permeant ion. With 160 mM K+ in the cell, the relative permeability calculated from the reversal potential with the Goldman-Hodgkin-Katz equation was K+ (identical to 1.0) greater than Rb+ (0.76) greater than NH4+ = Cs+ (0.12) much greater than Na+ (less than 0.004). Measured 30 mV negative to the reversal potential, the relative conductance sequence was quite different: NH4+ (1.5) greater than K+ (identical to 1.0) greater than Rb+ (0.5) greater than Cs+ (0.06) much greater than Na+, Li+, TMA+ (unmeasurable). Single channel current rectification resembled that of the whole-cell instantaneous I-V relation. Anomalous mole-fraction dependence of the relative permeability PNH4/PK was observed in NH4(+)-K+ mixtures, indicating that the type l K+ channel is a multi-ion pore. Compared with other K+ channels, lymphocyte type l K+ channels are most similar to "g12" channels in myelinated nerve.     

Demonstration of Na+-selective channels in the luminal-membrane vesicles isolated from pars recta of rabbit proximal tubule

Characteristics of 22Na+ fluxes through Na+ channels in luminal-membrane vesicles isolated from either pars recta or pars convoluta of rabbit proximal tubule were studied. In NaCl-loaded vesicles from pars recta, transient accumulation of 22Na+ is observed, which is inhibited by amiloride. The isotope accumulation is driven by an electrical diffusion potential as shown in experiments using either these membrane vesicles loaded with different anions, or an outwardly directed K+ gradient with a K+ ionophore valinomycin. The vesicles containing the channel show a cation selectivity with the order Li+ greater than Na+ greater than K+. The amiloride-sensitive 22Na+ flux is dependent on intravesicular Ca2+. In NaCl-loaded vesicles from pars convoluta, no overshoot for 22Na+ uptake is observed. Furthermore, addition of amiloride to the incubation medium did not influence the uptake of 22Na+ in these vesicle preparations. It is concluded that Na+ channels are only present in pars recta of rabbit proximal tubule.     

Site-specific mutations in a minimal voltage-dependent K+ channel alter ion selectivity and open-channel block

MinK is a small membrane protein of 130 amino acids with a single potential membrane-spanning alpha-helical domain. Its expression in Xenopus oocytes induces voltage-dependent, K(+)-selective channels. Using site-directed mutagenesis of a synthetic gene, we have identified residues in the hydrophobic region of minK that influence both ion selectivity and open-channel block. Single amino acid changes increase the channel's relative permeability for NH4+ and Cs+ without affecting its ability to exclude Na+ and Li+. Blockade by two common K+ channel pore blockers, tetraethylammonium and Cs+, was also modified. These results suggest that an ion selectivity region and binding sites for the pore blockers within the conduction pathway have been modified. We conclude that the gene encoding minK is a structural gene for a K+ channel protein.     

Interactions in cation permeation through the gramicidin channel. Cs, Rb, K, Na, Li, Tl, H, and effects of anion binding.

As a prototype for binding and interaction in biological Na and K channels, the single channel conductances for Li, Na, K, Rb, Cs, H, and Tl and the membrane potentials for Tl-K mixtures are characterized for gramicidin A over wider concentration rangers than previously and analyzed using an "equilibrium domain" model that assumes a central rate-determining barrier. Peculiarities in the conductance-concentration relationship for TlF, TlNO3, and TlAc suggest that anions bind to Tl-loaded channels, and the theory is extended to allow for this. For concreteness, the selectivity of cation permeation is characterized in terms of individual binding and rate constants of this model, with the conclusions that the strongest site binds Cs greater than Rb greater than K greater than Na greater than Li, while the next strongest binds Na greater than K greater than Li greater than Rb greater than Cs. However, because Schagina, Grinfeldt, and Lev''s recent finding of single filing (personal communication) indicates that the channel sites in gramicidin cannot be at equilibrium with the solution, and work in progress with Hägglund and Enos (Biophys. J. 21:26a. [Abstr.]) indicates that the simplest model adequate to account for the observed concentration-dependences of flux-ratio, conductance, I--V characteristic, and permeability has three barriers and four sites, some implications of additional rate-determining barriers at the mouth of the channel are discussed. The results are summarized using phenomenological "experimental" parameters that provide a model-independent way to represent that data concisely and which can be interpreted physically in terms of any desired model.     

Monovalent ion current through single calcium channels of skeletal muscle transverse tubules.

Akali monovalents, Li, Na, K, Cs, and organic monovalents of molecular cross section less than 20 A2, ammonium, methylammonium, hydrazinium, guanidinium, are shown to have a measurable conductance through Ca channels of muscle transverse tubules reconstituted into planar bilayers. For the alkali series, single channel conductances follow the sequence Cs approximately equal to K greater than Na greater than Li with a conductance ratio [g(Cs)/g(Li)] = 1.7. For permeability ratios, the sequence is Li greater than Na greater than K approximately equal to Cs with [P(Li)/P(Cs)] = 1.5. Monovalent current is only unmasked when Ba ions are not present. In mixtures of Cs and Ba, single channel current reverses close to the Ba equilibrium potential and more than 100 mV away from the Cs equilibrium potential. A cutoff in conduction is found for organic cations larger than trimethylammonium; this suggests an apparent pore aperture of about 5 X 5 A. Even in such a large pore, the fact that the alkali cation permeability sequence and conductance sequence are inverted rules out molecular sieving as the mechanism of selection among monovalents.     

Selectivity characteristics of cation channels in Nitella flexilis plasmalemma

Ionic selectivity of Nitella flexilis plasmalemma cation channels is studied by voltage-clamp method with consecutive replacing of cations in the bathing medium. The selectivity sequence received by measuring the ionic current reversal potentials, psi alpha is: Ba++ approximately equal to Sr++ approximately equal to Ca++ greater than Mg++ greater than Cs+ approximately equal to K+ greater than Na+ greater than Li+. An analysis of results based on the three-barrier channel model suggests that when ions of the same valency are compared, the channel selectivity is determined by specific interactions between the ion and the nearest water molecules, which is possible both in a narrow and wide pore. On the other hand, when monovalent and divalent ions are compared the effects of ions binding in the channel or near the membrane surface prevail, thus causing the channel preference for divalent cations.     

Single-channel recordings from two types of amiloride-sensitive epithelial Na+ channels

We report here the first evidence in intact epithelial cells of unit conductance events from amiloride-sensitive Na+ channels. The events were observed when patch-clamp recordings were made from the apical surface of cultured epithelial kidney cells (A6). Two types of channels were observed: one with a high selectivity to Na+ and one with relatively low selectivity. The characteristics of the low-selectivity channel are as follows: single-channel conductance ranged between 7 and 10 pS (mean = 8.4 +/- 1.3), the current-voltage (I-V) relationship displayed little if any nonlinearity over a range of +/- 80 mV (with respect to the patch pipette) and the channel Na+/K+ selectivity was approximately 3-4:1. Amiloride, a cationic blocker of the channel, reduced channel mean open time and increased channel mean closed times as the voltage of the cell interior was made more negative. Amiloride induced channel flickering at increased negative potentials (intracellular potential with respect to the patch) but did not alter the single-channel conductance or the I-V relationship from that observed in control patches. The characteristics of the high-selectivity channel are: a single-channel conductance of 1-3 pS (mean = 2.8 +/- 1.2), the current-voltage relationship is markedly nonlinear with a Na+/K+ selectivity greater than 20:1. The mean open and closed times for the two types of channels are quite different, the high-selectivity channel being open only about 10% of the time while the low-selectivity channel is open about 30% of the time.     

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.