Anomalous Effect of Permeant Ion Concentration on Peak Open Probability of Cardiac Na+ Channels

Human heart Na⁺ channels were expressed transiently in both mammalian cells and Xenopus oocytes, and Na⁺ currents measured using 150 mM intracellular Na⁺. Decreasing extracellular permeant ion concentration decreases outward Na⁺ current at positive voltages while increasing the driving force for the current. This anomalous effect of permeant ion concentration, especially obvious in a mutant (F1485Q) in which fast inactivation is partially abolished, is due to an alteration of open probability. The effect is only observed when a highly permeant cation (Na⁺, Li⁺, or hydrazinium) is substituted for a relatively impermeant cation (K⁺, Rb⁺, Cs⁺, N -methylglucamine, Tris, choline, or tetramethylammonium). With high concentrations of extracellular permeant cations, the peak open probability of Na⁺ channels increases with depolarization and then saturates at positive voltages. By contrast, with low concentrations of permeant ions, the open probability reaches a maximum at approximately 0 mV and then decreases with further depolarization. There is little effect of permeant ion concentration on activation kinetics at depolarized voltages. Furthermore, the lowered open probability caused by a brief depolarization to +60 mV recovers within 5 ms upon repolarization to −140 mV, indicative of a gating process with rapid kinetics. Tail currents at reduced temperatures reveal the rapid onset of this gating process during a large depolarization. A large depolarization may drive a permeant cation out of a site within the extracellular mouth of the pore, reducing the efficiency with which the channel opens.     

Block of Brain Sodium Channels by Peptide Mimetics of the Isoleucine,Phenylalanine, and Methionine (IFM) Motif from the Inactivation Gate

Inactivation of sodium channels is thought to be mediated by an inactivation gate formed by the intracellular loop connecting domains III and IV. A hydrophobic motif containing the amino acid sequence isoleucine, phenylalanine, and methionine (IFM) is required for the inactivation process. Peptides containing the IFM motif, when applied to the cytoplasmic side of these channels, produce two types of block: fast block, which resembles the inactivation process, and slow, use-dependent block stimulated by strong depolarizing pulses. Fast block by the peptide ac-KIFMK-NH₂, measured on sodium channels whose inactivation was slowed by the α-scorpion toxin from Leiurus quinquestriatus (LqTx), was reversed with a time constant of 0.9 ms upon repolarization. In contrast, control and LqTx-modified sodium channels were slower to recover from use-dependent block. For fast block, linear peptides of three to six amino acid residues containing the IFM motif and two positive charges were more effective than peptides with one positive charge, whereas uncharged IFM peptides were ineffective. Substitution of the IFM residues in the peptide ac-KIFMK-NH₂ with smaller, less hydrophobic residues prevented fast block. The positively charged tripeptide IFM-NH₂ did not cause appreciable fast block, but the divalent cation IFM-NH(CH₂)₂NH₂ was as effective as the pentapeptide ac-KIFMK-NH₂. The constrained peptide cyclic KIFMK containing two positive charges did not cause fast block. These results indicate that the position of the positive charges is unimportant, but flexibility or conformation of the IFM-containing peptide is important to allow fast block. Slow, use-dependent block was observed with IFM-containing peptides of three to six residues having one or two positive charges, but not with dipeptides or phenylalanine-amide. In contrast to its lack of fast block, cyclic KIFMK was an effective use-dependent blocker. Substitutions of amino acid residues in the tripeptide IFM-NH₂ showed that large hydrophobic residues are preferred in all three positions for slow, use-dependent block. However, substitution of the large hydrophobic residue diphenylalanine or the constrained residues phenylglycine or tetrahydroisoquinoline for phe decreased potency, suggesting that this phe residue must be able to enter a restricted hydrophobic pocket during the binding of IFM peptides. Together, the results on fast block and slow, use-dependent block indicate that IFM peptides form two distinct complexes of different stability and structural specificity with receptor site(s) on the sodium channel. It is proposed that fast block represents binding of these peptides to the inactivation gate receptor, while slow, use-dependent block represents deeper binding of the IFM peptides in the pore.     

Na+ Influx Inhibits Neuronal Ca2+ Channels

Experiments on isolated rat brain neurons with an elevated intracellular sodium concentration (due to tetanic stimulation) demonstrated the existence of earlier unknown negative modulation of calcium channels by intracellular sodium.     

Functional Interaction between the Scaffold Protein Kidins220/ARMS and Neuronal Voltage-Gated Na+ Channels

Kidins220 (kinase D-interacting substrate of 220 kDa)/ankyrin repeat-rich membrane spanning (ARMS) acts as a signaling platform at the plasma membrane and is implicated in a multitude of neuronal functions, including the control of neuronal activity. Here, we used the Kidins220^−/− mouse model to study the effects of Kidins220 ablation on neuronal excitability. Multielectrode array recordings showed reduced evoked spiking activity in Kidins220^−/− hippocampal networks, which was compatible with the increased excitability of GABAergic neurons determined by current-clamp recordings. Spike waveform analysis further indicated an increased sodium conductance in this neuronal subpopulation. Kidins220 association with brain voltage-gated sodium channels was shown by co-immunoprecipitation experiments and Na⁺ current recordings in transfected HEK293 cells, which revealed dramatic alterations of kinetics and voltage dependence. Finally, an in silico interneuronal model incorporating the Kidins220-induced Na⁺ current alterations reproduced the firing phenotype observed in Kidins220^−/− neurons. These results identify Kidins220 as a novel modulator of Na_v channel activity, broadening our understanding of the molecular mechanisms regulating network excitability.     

Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

Prolonged depolarization induces a slow inactivation process in some K⁺ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K⁺ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K⁺ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.     

Fast Inactivation in Shaker K+ Channels : Properties of Ionic and Gating Currents

Fast inactivating Shaker H4 potassium channels and nonconducting pore mutant Shaker H4 W434F channels have been used to correlate the installation and recovery of the fast inactivation of ionic current with changes in the kinetics of gating current known as “charge immobilization” (Armstrong, C.M., and F. Bezanilla. 1977. J. Gen. Physiol. 70:567–590.). Shaker H4 W434F gating currents are very similar to those of the conducting clone recorded in potassium-free solutions. This mutant channel allows the recording of the total gating charge return, even when returning from potentials that would largely inactivate conducting channels. As the depolarizing potential increased, the OFF gating currents decay phase at −90 mV return potential changed from a single fast component to at least two components, the slower requiring ∼200 ms for a full charge return. The charge immobilization onset and the ionic current decay have an identical time course. The recoveries of gating current (Shaker H4 W434F) and ionic current (Shaker H4) in 2 mM external potassium have at least two components. Both recoveries are similar at −120 and −90 mV. In contrast, at higher potentials (−70 and −50 mV), the gating charge recovers significantly more slowly than the ionic current. A model with a single inactivated state cannot account for all our data, which strongly support the existence of “parallel” inactivated states. In this model, a fraction of the charge can be recovered upon repolarization while the channel pore is occupied by the NH₂-terminus region.     

Identification of the Intracellular Na+ Sensor in Slo2.1 Potassium Channels

Slo2 potassium channels have a very low open probability under normal physiological conditions, but are readily activated in response to an elevated [Na⁺]_i (e.g. during ischemia). An intracellular Na⁺ coordination motif (DX(R/K)XXH) was previously identified in Kir3.2, Kir3.4, Kir5.1, and Slo2.2 channel subunits. Based loosely on this sequence, we identified five potential Na⁺ coordination motifs in the C terminus of the Slo2.1 subunit. The Asp residue in each sequence was substituted with Arg, and single mutant channels were heterologously expressed in Xenopus oocytes. The Na⁺ sensitivity of each of the mutant channels was assessed by voltage clamp of oocytes using micropipettes filled with 2 m NaCl. Wild-type channels and four of the mutant Slo2.1 channels were rapidly activated by leakage of NaCl solution into the cytoplasm. D757R Slo2.1 channels were not activated by NaCl, but were activated by the fenamate niflumic acid, confirming their functional expression. In whole cell voltage clamp recordings of HEK293 cells, wild-type but not D757R Slo2.1 channels were activated by a [NaCl]_i of 70 mm. Thus, a single Asp residue can account for the sensitivity of Slo2.1 channels to intracellular Na⁺. In excised inside-out macropatches of HEK293 cells, activation of wild-type Slo2.1 currents by 3 mm niflumic acid was 14-fold greater than activation achieved by increasing [NaCl]_i from 3 to 100 mm. Thus, relative to fenamates, intracellular Na⁺ is a poor activator of Slo2.1.     

Idiosyncratic Gating of HERG-like K+ Channels in Microglia

A simple kinetic model is presented to explain the gating of a HERG-like voltage-gated K⁺ conductance described in the accompanying paper (Zhou, W., F.S. Cayabyab, P.S. Pennefather, L.C. Schlichter, and T.E. DeCoursey. 1998. J. Gen. Physiol. 111:781–794). The model proposes two kinetically distinct closing pathways, a rapid one favored by depolarization (deactivation) and a slow one favored by hyperpolarization (inactivation). The overlap of these two processes leads to a window current between −50 and +20 mV with a peak at −36 mV of ∼12% maximal conductance. The near absence of depolarization-activated outward current in microglia, compared with HERG channels expressed in oocytes or cardiac myocytes, can be explained if activation is shifted negatively in microglia. As seen with experimental data, availability predicted by the model was more steeply voltage dependent, and the midpoint more positive when determined by making the holding potential progressively more positive at intervals of 20 s (starting at −120 mV), rather than progressively more negative (starting at 40 mV). In the model, this hysteresis was generated by postulating slow and ultra-slow components of inactivation. The ultra-slow component takes minutes to equilibrate at −40 mV but is steeply voltage dependent, leading to protocol-dependent modulation of the HERG-like current. The data suggest that “deactivation” and “inactivation” are coupled through the open state. This is particularly evident in isotonic Cs⁺, where a delayed and transient outward current develops on depolarization with a decay time constant more voltage dependent and slower than the deactivation process observed at the same potential after a brief hyperpolarization.     

Macroscopic Na+ Currents in the “Nonconducting” Shaker Potassium Channel Mutant W434F

C-type inactivation in Shaker potassium channels inhibits K⁺ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na⁺ and Li⁺ currents in K⁺-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na⁺ and Li⁺ currents (in K⁺-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na⁺ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.     

Allosteric Effects of Permeating Cations on Gating Currents during K+ Channel Deactivation

K⁺ channel gating currents are usually measured in the absence of permeating ions, when a common feature of channel closing is a rising phase of off-gating current and slow subsequent decay. Current models of gating invoke a concerted rearrangement of subunits just before the open state to explain this very slow charge return from opening potentials. We have measured gating currents from the voltage-gated K⁺ channel, Kv1.5, highly overexpressed in human embryonic kidney cells. In the presence of permeating K⁺ or Cs⁺, we show, by comparison with data obtained in the absence of permeant ions, that there is a rapid return of charge after depolarizations. Measurement of off-gating currents on repolarization before and after K⁺ dialysis from cells allowed a comparison of off-gating current amplitudes and time course in the same cells. Parallel experiments utilizing the low permeability of Cs⁺ through Kv1.5 revealed similar rapid charge return during measurements of off-gating currents at E_Cs. Such effects could not be reproduced in a nonconducting mutant (W472F) of Kv1.5, in which, by definition, ion permeation was macroscopically absent. This preservation of a fast kinetic structure of off-gating currents on return from potentials at which channels open suggests an allosteric modulation by permeant cations. This may arise from a direct action on a slow step late in the activation pathway, or via a retardation in the rate of C-type inactivation. The activation energy barrier for K⁺ channel closing is reduced, which may be important during repetitive action potential spiking where ion channels characteristically undergo continuous cyclical activation and deactivation.     

钠离子通道研究进展

细胞电活动是生命现象的基本特征之一,而离子通道是其结构和功能的基础。因此,研究离子通道作用机制具有重大的理论和现实意义。许多神经系统和心血管疾病,如多发性硬化症、癫痫、脑溢血、神经痛、Brugada综合征(BrS)、进行性心脏传导缺陷(PCCD)和原发性心室纤颤(IVF)等,都与钠离子通道氨基酸序列和结构发生的改变有关。该文对钠离子通道的研究进展进行综述。     

Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels

Fast Na⁺ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in L_III-IV of the rat brain type IIA Na⁺ channel. We have analyzed macroscopic and single channel currents from Na⁺ channels with mutations within and adjacent to hydrophobic clusters in L_III-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in L_III-IV also affected the latency to first opening, indicating coupling between L_III-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na⁺ channels.     

Slow inactivation of tetrodotoxin-insensitive Na+ channels in neurons of rat dorsal root ganglia

Summary Whole-cell patch-clamp experiments were performed with neurons cultured from rat dorsal root ganglia (DRG). Two types of Na⁺ currents were identified on the basis of sensitivity to tetrodotoxin. One type was blocked by 0.1 nm tetrodotoxin, while the other type was insensitive to 10 m tetrodotoxin. The peak amplitude of the tetrodotoxin-insensitive Na⁺ current gradually decreased after depolarization of the membrane. The steady-state value of the peak amplitude was attained several minutes after the change of holding potential. Such a slow inactivation was not observed in tetrodotoxin-sensitive Na⁺ current. The slow inactivation of the tetrodotoxin-insensitive Na⁺ current was kinetically distinct from the ordinary short-time steady-state inactivation. The voltage dependence of the slow inactivation could be described by a sigmoidal function, and its time course had a double-exponential process. A decrease of external pH partially antagonized the slow inactivation, probably through an increased diffusion potential across the membrane. However, the slow inactivation was not due to change in surface negative charges, since a shift of the kinetic parameters along the voltage axis was not observed during the slow inactivation. Due to the slow inactivation, the inactivation curves for the tetrodotoxininsensitive Na⁺ current were shifted in the negative direction as the prepulse duration was increased. Consequently, the window current activated at potentials close to the resting membrane potential was markedly reduced. Thus, the slow inactivation may be involved in the long-term regulation of the excitability of sensory neurons.We thank Prof. Hirosi Kuriyama for his support and advice and Dr. M. Yoshii for helpful discussions. This study was supported by the Japanese Ministry of Education (Scientific Research 02670090).     

A Conserved Residue Cluster That Governs Kinetics of ATP-dependent Gating of Kir6.2 Potassium Channels

ATP-sensitive potassium (K_ATP) channels are heteromultimeric complexes of an inwardly rectifying Kir channel (Kir6.x) and sulfonylurea receptors. Their regulation by intracellular ATP and ADP generates electrical signals in response to changes in cellular metabolism. We investigated channel elements that control the kinetics of ATP-dependent regulation of K_ATP (Kir6.2 + SUR1) channels using rapid concentration jumps. WT Kir6.2 channels re-open after rapid washout of ATP with a time constant of ∼60 ms. Extending similar kinetic measurements to numerous mutants revealed fairly modest effects on gating kinetics despite significant changes in ATP sensitivity and open probability. However, we identified a pair of highly conserved neighboring amino acids (Trp-68 and Lys-170) that control the rate of channel opening and inhibition in response to ATP. Paradoxically, mutations of Trp-68 or Lys-170 markedly slow the kinetics of channel opening (500 and 700 ms for W68L and K170N, respectively), while increasing channel open probability. Examining the functional effects of these residues using φ value analysis revealed a steep negative slope. This finding implies that these residues play a role in lowering the transition state energy barrier between open and closed channel states. Using unnatural amino acid incorporation, we demonstrate the requirement for a planar amino acid at Kir6.2 position 68 for normal channel gating, which is potentially necessary to localize the ϵ-amine of Lys-170 in the phosphatidylinositol 4,5-bisphosphate-binding site. Overall, our findings identify a discrete pair of highly conserved residues with an essential role for controlling gating kinetics of Kir channels.     

Molecular Analysis of Potential Hinge Residues in the Inactivation Gate of Brain Type IIA Na+ Channels

During inactivation of Na⁺ channels, the intracellular loop connecting domains III and IV is thought to fold into the channel protein and occlude the pore through interaction of the hydrophobic motif isoleucine-phenylalanine-methionine (IFM) with a receptor site. We have searched for amino acid residues flanking the IFM motif which may contribute to formation of molecular hinges that allow this motion of the inactivation gate. Site-directed mutagenesis of proline and glycine residues, which often are components of molecular hinges in proteins, revealed that G1484, G1485, P1512, P1514, and P1516 are required for normal fast inactivation. Mutations of these residues slow the time course of macroscopic inactivation. Single channel analysis of mutations G1484A, G1485A, and P1512A showed that the slowing of macroscopic inactivation is produced by increases in open duration and latency to first opening. These mutant channels also show a higher probability of entering a slow gating mode in which their inactivation is further impaired. The effects on gating transitions in the pathway to open Na⁺ channels indicate conformational coupling of activation to transitions in the inactivation gate. The results are consistent with the hypothesis that these glycine and proline residues contribute to hinge regions which allow movement of the inactivation gate during the inactivation process of Na⁺ channels.     

Separation of Gating Properties from Permeation and Block in mslo Large Conductance Ca-activated K+ Channels

In this and the following paper we have examined the kinetic and steady-state properties of macroscopic mslo Ca-activated K⁺ currents in order to interpret these currents in terms of the gating behavior of the mslo channel. To do so, however, it was necessary to first find conditions by which we could separate the effects that changes in Ca²⁺ concentration or membrane voltage have on channel permeation from the effects these stimuli have on channel gating. In this study we investigate three phenomena which are unrelated to gating but are manifest in macroscopic current records: a saturation of single channel current at high voltage, a rapid voltage-dependent Ca²⁺ block, and a slow voltage-dependent Ba²⁺ block. Where possible methods are described by which these phenomena can be separated from the effects that changes in Ca²⁺ concentration and membrane voltage have on channel gating. Where this is not possible, some assessment of the impact these effects have on gating parameters determined from macroscopic current measurements is provided. We have also found that without considering the effects of Ca²⁺ and voltage on channel permeation and block, macroscopic current measurements suggest that mslo channels do not reach the same maximum open probability at all Ca²⁺ concentrations. Taking into account permeation and blocking effects, however, we find that this is not the case. The maximum open probability of the mslo channel is the same or very similar over a Ca²⁺ concentration range spanning three orders of magnitude indicating that over this range the internal Ca²⁺ concentration does not limit the ability of the channel to be activated by voltage.     

G-protein activation modulates pseudo-periodic oscillation of Na channel

We have shown before that the duration and amplitude of both prolonged (1-160 s) and short (100-1000 ms) depolarizing prepulse altered all the steady-state and kinetic parameters of rNav1.2a voltage-gated sodium channel in a pseudo-oscillatory fashion with variable time period and amplitude, often superimposed on a linear trend. In this study, we have examined the effect of G-protein activation on pseudo-oscillatory properties of the rNav1.2a sodium channel alpha subunit, heterologously expressed in Chinese hamster ovary cells. G-protein modification caused insignificant changes in the slow pseudo-periodic oscillation of the activation properties of sodium channel; only the time period of the oscillation was altered from approximately 30 to 21s. In contrast, G-protein activation abolished the faster component of pseudo-periodic oscillation in steady-state inactivation properties of sodium channel; the conditioning duration dependence of steady-state inactivation becomes monotonic in nature.     

Inactivation Kinetics and Mechanical Gating of Piezo1 Ion Channels Depend on Subdomains within the Cap

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Block of N-type Calcium Channels in Chick Sensory Neurons by External Sodium

L-type Ca²⁺ channels select for Ca²⁺ over sodium Na⁺ by an affinity-based mechanism. The prevailing model of Ca²⁺ channel permeation describes a multi-ion pore that requires pore occupancy by at least two Ca²⁺ ions to generate a Ca²⁺ current. At [Ca²⁺] < 1 μM, Ca²⁺ channels conduct Na⁺. Due to the high affinity of the intrapore binding sites for Ca²⁺ relative to Na⁺, addition of μM concentrations of Ca²⁺ block Na⁺ conductance through the channel. There is little information, however, about the potential for interaction between Na⁺ and Ca²⁺ for the second binding site in a Ca²⁺ channel already occupied by one Ca²⁺. The two simplest possibilities, (a) that Na⁺ and Ca²⁺ compete for the second binding site or (b) that full time occupancy by one Ca²⁺ excludes Na⁺ from the pore altogether, would imply considerably different mechanisms of channel permeation. We are studying permeation mechanisms in N-type Ca²⁺ channels. Similar to L-type Ca²⁺ channels, N-type channels conduct Na⁺ well in the absence of external Ca²⁺. Addition of 10 μM Ca²⁺ inhibited Na⁺ conductance by 95%, and addition of 1 mM Mg²⁺ inhibited Na⁺ conductance by 80%. At divalent ion concentrations of 2 mM, 120 mM Na⁺ blocked both Ca²⁺ and Ba²⁺ currents. With 2 mM Ba²⁺, the IC₅₀ for block of Ba²⁺ currents by Na⁺ was 119 mM. External Li⁺ also blocked Ba²⁺ currents in a concentration-dependent manner, with an IC₅₀ of 97 mM. Na⁺ block of Ba²⁺ currents was dependent on [Ba²⁺]; increasing [Ba²⁺] progressively reduced block with an IC₅₀ of 2 mM. External Na⁺ had no effect on voltage-dependent activation or inactivation of the channel. These data suggest that at physiological concentrations, Na⁺ and Ca²⁺ compete for occupancy in a pore already occupied by a single Ca²⁺. Occupancy of the pore by Na⁺ reduced Ca²⁺ channel conductance, such that in physiological solutions, Ca²⁺ channel currents are between 50 and 70% of maximal.