Na activation delays and their relation to inactivation in frog skeletal muscle

Summary Delays in the development of activation of Na currents were studied using voltage-clamped frog skeletal muscle fibers. Na currents elicited by a depolarizing voltage step from a hyperpolarized membrane potential were delayed in their activation when compared to Na currents elicited from the resting potential. The magnitude of the delay increased with larger hyperpolarizing potentials and decreased with larger depolarizing test potentials. Delays in activation observed following chloramine-T treatment that partially removes inactivation did not differ from delays observed before treatment. Longer exposures of the muscle fiber to chloramine-T led to a complete loss of inactivation, coincident with an elimination of the hyperpolarization-induced delays in activation. Steady-state slow inactivation was virtually unaffected by prolonged exposures of the fibers to chloramine-T that eliminated fast inactivation. The results show that chloramine-T acts at a number of sites to alter both activation and inactivation. Markov model simulations of the results show that chloramine-T alters fundamental time constants of the system by altering both activation and inactivation rate constants.     

Effects of internal Na and external K concentrations on Na/K coupling of Na,K-pump in frog skeletal muscle

Summary To clarify the dependency of the Na/K coupling of the Na,K-pump on internal Na and external K concentrations in skeletal muscle, the ouabain-induced change in membrane potential, the ouabain-induced change in Na efflux and the membrane resistance were measured at various internal Na and external K concentrations in bullfrog sartorius muscle.Upon raising the internal Na concentration from 6 mmol/kg muscle water to 20 mmol/kg muscle water, the magnitude of the ouabain-induced change in membrane potential increased about eightfold and the magnitude of the ouabain-induced change in Na efflux increased about fivefold while the membrane resistance was not significantly changed. As the external K concentration increased from 1 to 10mm, the magnitude of the ouabain-induced change in membrane potential decreased (1/5.5 fold), while the magnitude of the ouabain-induced change in Na efflux increased (about 1.5-fold). The membrane resistance decreased upon raising the external K concentration from 1 to 10mm (1/2-fold). These observations imply that the values of the Na/K coupling of the Na,K-pump increases upon raising the internal Na concentration and decreases upon raising the external K concentration.     

Na+-sensitive component of 3-O-methylglucose uptake in frog skeletal muscle

Summary A Na⁺-sensitive uptake of 3-O-methylglucose (3-O-MG), a nonmetabolized sugar, was characterized in frog skeletal muscle. A removal of Na⁺ from the bathing solution reduced 3-O-MG uptake, depending on the amount of Na⁺ removed. At a 3-O-MG concentration of 2mm, the Na⁺-sensitive component of uptake in Ringer's solution was estimated to be about 26% of the total uptake. The magnitude of Na⁺-sensitive component sigmoidally increased with an increase of 3-O-MG in bathing solution, whereas in Na⁺-free Ringer's solution the uptake was proportional to the concentration. The half saturation of the Na⁺-sensitive component was at a 3-O-MG concentration of about 13mm, and the Hill coefficient was 1.4 to 1.6. Phlorizin (5mm), a potent inhibitor specific for Na⁺-coupled glucose transport, reduced the uptake in a solution containing Na⁺ to the level in Na⁺-free Ringer's solution. Glucose of concentrations higher than 20mm suppressed 3-O-MG uptake to a level slightly lower than that in Na⁺-free Ringer's solution. These observations indicate that there are Na⁺-coupled sugar transport systems in frog skeletal muscle which are shared by both glucose and 3-O-MG.     

Effects of external K concentration on the electrogenicity of the insulin-stimulated Na,K-pump in frog skeletal muscle

Summary Insulin hyperpolarized the membrane of frog skeletal muscle by stimulating the electrogenic Na,K-pump. At external K concentrations of 1, 2, 5 and 10mm, both the insulin-induced hyperpolarization and the insulin-stimulated ouabain-sensitive Na efflux (an index of Na, K-pump activity) were observed. By increasing the external K concentration, the insulin-stimulated Na efflux increased, but the magnitude of the insulin-induced hyperpolarization decreased; i. e., although the activity of the insulin-stimulated Na,K-pump increased, on the contrary, the magnitude of the hyperpolarization decreased. To clarify the causes of this phenomenon, the specific membrane resistance was measured and found to decrease upon increasing the external K concentration.One of the reasons for the decrease in magnitude of the hyperpolarization is the decrease in the specific membrane resistance. However, the decrease in magnitude of the hyperpolarization with a rise of the external K concentration, which increased the insulin-stimulated Na,K-pump activity, cannot be explained only by the decrease in the specific membrane resistance. It is suggested that the decrease in magnitude of the hyperpolarization is mainly caused by a decrease in the electrogenicity of the insulin-stimulated Na,K-pump upon an increase in the external K concentration. The conclusion of the present study is that the electrogenicity of the insulin-stimulated Na,K-pump in muscles is variable and decreases with increasing the external K concentration.     

Saturation effects and rectifier properties of sodium channels in human skeletal muscle

Sodium outward currents were measured in human myoballs with the whole-cell recording method. The electro-chemical gradient of the sodium ions across the cell membrane was modified over a wide range by variations of the clamped membrane potential and of the internal and external soidum concentration. Up to 50 mV positive to the sodium equilibrium potential, E_Na, the current-voltage relation is linear. At a potential 80 mV positive to E_Na the sodium outward current has a maximum and decreases with a further increase in electrochemical gradient. Investigating the instantaneous current change in experiments in which the membrane potential was changed while the channels were already open we could exclude the possibility that the gates of activation or inactivation are responsible for this effect. Therefore we postulate that the sodium channel has a valve-like mechanism producing a negative slope conductance at highly positive membrane potentials, a current saturation with self-inhibition by the intracellular sodium concentration, and a blockade of the channel on reduction of the extracellular sodium concentration.This work was supported by the Deutsche Forschungsgemeinschaft (Ru 138/15-1, 15-2)     

Inactivation of I(A) channels of frog skeletal muscle is modulated by ATP

Whole cell, voltage clamp experiments were performed in vesicles derived from frog skeletal muscle plasma membranes to characterize the influence of ATP on the kinetic properties of fast inactivating K(+) currents (I(A)). I(A) was recorded in ATP-free solutions. Peak I(A) decayed with a time constant of 27 ms at large depolarizations. Steady state inactivation reached half maximal values at -66 mV. In the presence of ATP, these values were 196 ms and -41 mV, respectively, indicating a major effect of ATP on inactivation. In contrast, activation of I(A) was unaffected by ATP. The protein kinase C (PKC) inhibitors, H7 and staurosporine, greatly prevented the effects of ATP on inactivation. Inactivation remained unchanged by the protein kinase A inhibitor HA1004 or by the catalytic subunit of cAMP protein kinase. We conclude that ATP decreases inactivation of skeletal muscle I(A) and that this effect may be mediated by protein kinase C.     

Direct measurement of intracellular free magnesium in frog skeletal muscle using magnesium-selective microelectrodes

Mg²⁺-selective microelectrodes have been used to measure the intracellular free Mg²⁺ concentration in frog skeletal muscle fibers. Glass capillaries with a tip diameter of less than 0.4 μm were backfilled with the Mg²⁺ sensor, ETH 1117. In the absence of interfering ions, they gave Nernstian responses between 1 and 10 mM free Mg²⁺. In the presence of an ionic environment resembling the myoplasm, the microelectrode response was sub Nernstian (18–24 mV) but still useful. The electrodes were calibrated before and after muscle-fiber impalements. In quiescent fibers from sartorius muscle (Rana pipiens), with resting membrane potentials not less than ?82 mV, the intracellular free Mg²⁺ concentration was 3.8±0.41 (S.E.) mM (n=58) at 22°C. No significant change in the intracellular free Mg²⁺ was observed following extensive (approx. 6 h) incubation in Mg²⁺-free media. Increasing the external concentration of magnesium from 4 to 20 mM (approx. 15 min) produced a slow and small enhancement (1.8 mM) of [Mg²⁺]_i, which was fully reverted when the divalent cation was removed from the bathing solution. No change in ionic magnesium resting concentration was observed when the muscle fibers were treated either with caffeine 3 mM or with Na⁺-free solutions. In depolarized muscle fibers (?23±2.7 mV) treated with 100 mM K⁺, the myoplasmic [Mg²⁺] was 3.7±0.45 (S.E.) mM, n=6, immediately after the spontaneous relaxation of the contracture. Similar determinations in muscle fibers during stimulation at low frequency (5 Hz), and after fatigue development, showed no changes in the concentration of free cytosolic Mg²⁺. These results point out that [Mg²⁺]_i is not modified under these three different experimental conditions.     

Potassium inward rectifier and acetylcholine receptor channels in embryonic Xenopus muscle cells in culture

Embryonic muscle cells of the frog Xenopus laevis were isolated and grown in culture and single-channel recordings of potassium inward rectifier and acetylcholine (ACh) receptor currents were obtained from cell-attached membrane patches. Two classes of inward rectifier channels, which differed in conductance, were apparent. With 140 mM potassium chloride in the electrode, one channel class had a conductance of 28.8 ± 3.4 pS (n = 21), and, much more infrequently, a smaller channel class with a conductance of 8.6 ± 3.6 pS (n = 7) was recorded. Both channel classes had relatively long mean channel open times, which decreased with membrane hyperpolarization. The probability of finding a patch of membrane with an inward rectifier channel was high (66%) and many membrane patches contained more than one inward rectifier channel. The open state probability (with no applied potential) was high for both inward rectifier channel classes so that 70% of the time there was a channel open. Seventy-three percent of the membrane patches with ACh receptor channels (n = 11) also had at least one inward rectifier channel present when the patch electrode contained 0.1 μM ACh. Inward rectifier channels were also found at 71% of the sites of high ACh receptor density (n = 14), which were identified with rhodamine-conjugated α-bungarotoxin. The results indicate that the density of inward rectifier channels in this embryonic skeletal muscle membrane was relatively high and includes sites of membrane that have synaptic specializations. © 1996 John Wiley & Sons, Inc.     

K-current fluctuations in inward-rectifying channels of frog skeletal muscle

Summary K currents and K-current fluctuations were recorded in inwardly rectifying K channels of frog skeletal muscle under voltage-clamp conditions. External application of 0.2 to 10mm Cs reduces the inward mean K current but produces a distinct increase of the spectral density of K-current fluctuations. The additional fluctuations arise from the random blocking by Cs ions. From the variance of current fluctuations, the steady-state current and the probability of the open unblocked channel an effective single-channel conductance ^* was calculated. ^* strongly depends on the external Cs concentration (7.8 pS at 0.2mm Cs, 2.1 pS at 10mm Cs). This dependence is interpreted in terms of a two-step blocking process: (1) a fast exchange of Cs ions between the external solution and a first binding site inside the channel which leads to the Cs-modulated effective single-channel conductance, and (2) a slow Cs binding to a second site deeper in the channel which produces the observed current fluctuations. With this hypothesis we obtained a real single-channel conductance of 10 pS and a real density ofn4 inwardly rectifying channels per m² of muscle surface area.     

The influence of amino-reactive substances on contraction threshold of frog skeletal muscle

Summary The action of the amino-reactive substances pyridoxal phosphate, 4-acetamido-4-isothiocyanato-stilbene-2,2-disulfonic acid and 2,4,6-trinitrobenzene sulfonic acid on the contraction threshold, taken as parameter for the initiation of contraction, was investigated in fibers of the sartorius muscle of the frog. The contraction threshold was shifted by 1 to 11 mV tomore negative potentials with 1 to 20mm PDP. Similar shifts from 2 to 17 mV were produced by 0.66 to 20mm SITS. The threshold shift was only partially reversible. The shift of the contraction threshold obtained with 2mm SITS was nearly constant at different [Ca²⁺]_o and [Mg²⁺]_o from 1.5 to 50mm with a tendency to increase at higher divalent cation concentration. TNBS had no effect on the contraction threshold.The action of PDP and SITS on the contraction threshold was successfully described by the surface charge model used earlier to explain the effect of lanthanum, neuraminidase and ruthenium red on the contraction threshold (M. Dörrscheidt-Käfer,Pfluegers Arch. 380:171–179, 181–187, 1979;J. Membrane Biol. 62:95–103, 1981). Here it was assumed that PDP and SITS bind to positive fixed charges on the surface of the T-tubular wall. This results in a shift of the calculated surface potential to more negative values which is thought to account for the measured shift of the contraction threshold.     

提高胞外钾引起的蛙骨骼肌咖啡因挛缩增强

用蛙胫前肌小束为材料, 研究了提高胞外钾[K+]O对咖啡因挛缩的作用.[K+]O从2 mmol/L提高到10或25 mmol/L, 由3 mmol/L咖啡因引起的挛缩明显增强.以PKC/PC (PKC和PC分别为在高钾和正常钾条件下的咖啡因挛缩)表示的咖啡因挛缩增强, 依赖[K+]O和高钾作用时间.随着10 mmol/L [K+]O作用时间延长, 直至10 min, 增强逐渐增加.但是, 25 mmol/L [K+]O作用1 min时增强达到最大, 然后下降到对照.PKC/PC变化时程不能用高钾引起的去极化解释, 而与由相似[K+]O引起的胞浆自由钙变化时程相符.提示, 至少在蛙骨骼肌, 高钾引起的咖啡因挛缩增强主要是由胞浆自由钙升高引起的.     

Two open states or two different Na channels in skeletal muscle fibers: Markov models of the decay of Na currents in frog skeletal muscle

The rate of sodium current decay at –140 mV was studied as a function of the duration and amplitude of the activating voltage pulse. These sodium current decays or tails of current showed a biexponential decline in amplitude which depended upon the duration of the activating pulse. At 12°C, the two exponential components of the Na tail currents exhibited time constants of 72 and 534 s. As the duration of an activating pulse was lengthened, the relative amplitude of the slow component of the decay increased compared to the fast component, without any changes in the fast and slow time constants. This slowing of the decay of current as a function of the duration of the activating pulse is found only in fibers with inactivation intact.A number of Markov models were tested for their ability to predict the biexponential decays found in muscle fibers with inactivation intact and removed. A homogeneous population of channels having only a single open state fails to predict the behavior. A homogeneous population of channels having two open states predicts the behavior. The behavior can also be predicted by two different types of single open-state sodium channels, with one ensemble of channels carrying a minority of the current and exhibiting a much slower closing rate. If a homogeneous population of channels is present, the simulations show that the observed changes in decay rates are driven by inactivation.     

Radial spread of aequorin Ca2+ signal in single frog skeletal muscle fibers

The Ca²⁺-sensitive photoprotein aequorin was injected into single frog skeletal muscle fibers, and the intracellular aequorin light intensity during muscle activation with different maneuvers was mapped with digital imaging microscopy. During 50 Hz electrical activation (tetanus), the aequorin light intensity from different locations in the muscle fiber rose with very similar time course. Caffeine (10 mM) application, on the other hand, caused aequorin light signals to show significantly different time courses, with an earlier increase in Ca²⁺ concentration near the surface of the fiber than near the core. The non-uniform rise of intracellular Ca²⁺ concentration with caffeine treatment is consistent with the slow inward diffusion of caffeine and subsequent Ca²⁺ release from sarcoplasmic reticulum.     

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Summary— Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I_Ba could be detected (exogenous I_Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I_Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I_Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.     

Prolonged high-pressure treatments in mammalian skeletal muscle result in loss of functional sodium channels and altered calcium channel kinetics

Activation and inactivation of ion channels involve volume changes from conformational rearrangements of channel proteins. These volume changes are highly susceptible to changes in ambient pressure. Depending on the pressure level, channel function may be irreversibly altered by pressure. The corresponding structural changes persist through the post-decompression phase. High-pressure applications are a useful tool to evaluate the pressure dependence as well as pressure limits for reversibility of such alterations. Mammalian cells are only able to tolerate much lower pressures than microorganisms. Although some limits for pressure tolerance in mammalian cells have been evaluated, the mechanisms of pressure-induced alteration of membrane physiology, in particular of channel function, are unknown. To address this question, we recorded fast inward sodium (I(Na)) and slowly activating L-type calcium (I(Ca)) currents in single mammalian muscle fibers in the post-decompression phase after a prolonged 3-h, high-pressure treatment of up to 20 MPa. I(Na) and I(Ca) peak amplitudes were markedly reduced after pressure treatment at 20 MPa. This was not from a general breakdown of membrane integrity as judged from in situ high-pressure fluorescence microscopy. Membrane integrity was preserved even for pressures as high as 35 MPa at least for pressure applications of shorter durations. Therefore, the underlying mechanisms for the observed amplitude reductions have to be determined from the activation (time-to-peak [TTP]) and inactivation (tau(dec)) kinetics of I(Na) and I(Ca). No major changes in I(Na) kinetics, but marked increases, both in TTP and tau(dec) for I(Ca), were detected after 20 MPa. The apparent molecular volume changes (activation volumes) deltaV(double dagger) for the pressure-dependent irreversible alteration of channel gating approached zero for Na+ channels. For Ca2+ channels, deltaV(double dagger) was very large, with approx 2.5-fold greater values for channel activation than inactivation (approx 210 A3). We conclude, that in skeletal muscle, high pressure differentially and irreversibly affects the gating properties and the density of functional Na+ and Ca2+ channels. Based on these results, a model of high pressure-induced alterations to the channel conformation is proposed.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M⁻¹s⁻¹ and k_off = 1609 sec⁻¹ with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Evidence TRPV4 contributes to mechanosensitive ion channels in mouse skeletal muscle fibers

We recorded the activity of single mechanosensitive (MS) ion channels from membrane patches on single muscle fibers isolated from mice. We investigated the actions of various TRP (transient receptor potential) channel blockers on MS channel activity. 2-aminoethoxydiphenyl borate (2-APB) neither inhibited nor facilitated single channel activity at submillimolar concentrations. The absence of an effect of 2-APB indicates MS channels are not composed purely of TRPC or TRPV1, 2 or 3 proteins. Exposing patches to 1-oleolyl-2-acetyl-sn-glycerol (OAG), a potent activator of TRPC channels, also had no effect on MS channel activity. In addition, flufenamic acid and spermidine had no effect on the activity of single MS channels. By contrast, SKF-96365 and ruthenium red blocked single-channel currents at micromolar concentrations. SKF-96365 produced a rapid block of the open channel current. The blocking rate depended linearly on blocker concentration, while the unblocking rate was independent of concentration, consistent with a simple model of open channel block. A fit to the concentration-dependence of block gave k_on = 13 x 10⁶ M^?1s^?1 and k_off = 1609 sec^?1 with K_D = ~124 µM. Block by ruthenium red was complex, involving both reduction of the amplitude of the single-channel current and increased occupancy of subconductance levels. The reduction in current amplitude with increasing concentration of ruthenium red gave a K_D = ~49 µM. The high sensitivity of MS channels to block by ruthenium red suggests MS channels in skeletal muscle contain TRPV subunits. Recordings from skeletal muscle isolated from TRPV4 knockout mice failed to show MS channel activity, consistent with a contribution of TRPV4. In addition, exposure to hypo-osmotic solutions increases opening of MS channels in muscle. Our results provide evidence TRPV4 contributes to MS channels in skeletal muscle.     

Comparison of properties of Ca2+ release channels between rabbit and frog skeletal muscles

Biochemical investigation of Ca2+ release channel proteins has been carried out mainly with rabbit skeletal muscles, while frog skeletal muscles have been preferentially used for physiological investigation of Ca2+ release. In this review, we compared the properties of ryanodine receptors (RyR), Ca2+ release channel protein, in skeletal muscles between rabbit and frog. While the Ryr1 isoform is the main RyR of rabbit skeletal muscles, two isoforms, - and -RyR which are homologous to Ryr1 and Ryr3 isoforms in mammals, respectively, coexist as a homotetramer in a similar amount in frog skeletal muscles. The two isoforms in an isotonic medium show very similar property in [3H]ryanodine binding activity which is parallel to Ca2+-induced Ca2+ release (CICR) activity, and make independent contributions to the activities of the sarcoplasmic reticulum. CICR and [3H]ryanodine binding activities of rabbit and frog are qualitatively similar in stimulation by Ca2+, adenine nucleotide and caffeine, however, they showed the following quantitative differences. First, rabbit RyR showed higher Ca2+ affinity than the frog. Second, rabbit RyR showed higher activity in the presence of Ca2+ alone with less stimulation by adenine nucleotide than the frog. Third, rabbit RyR displayed less enhancement of [3H]ryanodine binding by caffeine in spite of having a similar magnitude of Ca2+ sensitization than the frog, which may explain the occasional difficulty by researchers to demonstrate caffeine contracture with mammalian skeletal muscles. Finally, but not least, rabbit RyR still showed marked inhibition of [3H]ryanodine binding in the presence of high Ca2+ concentrations in the 1 M NaCl medium, while frog RyR showed disinhibition. Other matters relevant to Ca2+ release were also discussed.     

Ion permeation through single channels activated by acetylcholine in denervated toad sartorius skeletal muscle fibers: Effects of alkali cations

Summary The gigaohm seal technique was used to study ion permeation through acetylcholine-activated channels in cell-attached patches of the extrajunctional membrane of chronically denervated, enzyme-treated cells from the sartorius muscle of the toadBufo marinus. The most frequently occurring channel type (>95% of channel openings), provisionally classified as extrajunctional, had a chord conductance of approximately 25 pS under normal conditions (–70 mV, 11°C, Normal Toad Ringer's). The less frequently observed channel type (<5% of channel openings), classified as a junctional type, had a conductance of 35 pS under the same conditions, and a similar null potential. In many patches, a small percentage (usually <2%) of openings of the extrajunctional channel displayed a lower conductance state. The shape of theI–V curves obtained for the extrajunctional channel dependend on the predominant extracellular cation. For Cs and K, theI–V curves were essentially linear over the voltage range +50 to –150 mV across the patch, suggesting that the potential independent component of the energy profile within the channel was symmetrical. For Li, theI–V curve was very nonlinear, displaying a significant sublinearity at hyperpolarized potentials. Both an electrodiffusion and a symmetrical uniform four-barrier, three-site rate-theory model provided reasonable fits to the data, whereas symmetrical two-barrier, single-site rate-theory models did not. For the alkali cations examined, the relative permeability sequence wasP _Cs>P _K>P _Na>P _Li—a proportional selectivity sequence. This was different from the single channel conductance sequence which was found to be _K> _Cs> _Na> _Li implying that ions do not move independently through the channel. The relative binding constant sequence for the channel sites was found to be a polarizability sequence, i.e.,K _Li>K _Cs>K _Na>K _K There was an inverse relationship for the cations examined. Under conditions when the single-channel conductance was relatively high, the conductance at depolarized potentials was lower than that predicted by both electrodiffusion and rate theory models, suggesting that there was a rate-limiting access step for ions, from the intracellular compartment into the channel.