Intrinsic versus extrinsic voltage sensitivity of blocker interaction with an ion channel pore

Many physiological and synthetic agents act by occluding the ion conduction pore of ion channels. A hallmark of charged blockers is that their apparent affinity for the pore usually varies with membrane voltage. Two models have been proposed to explain this voltage sensitivity. One model assumes that the charged blocker itself directly senses the transmembrane electric field, i.e., that blocker binding is intrinsically voltage dependent. In the alternative model, the blocker does not directly interact with the electric field; instead, blocker binding acquires voltage dependence solely through the concurrent movement of permeant ions across the field. This latter model may better explain voltage dependence of channel block by large organic compounds that are too bulky to fit into the narrow (usually ion-selective) part of the pore where the electric field is steep. To date, no systematic investigation has been performed to distinguish between these voltage-dependent mechanisms of channel block. The most fundamental characteristic of the extrinsic mechanism, i.e., that block can be rendered voltage independent, remains to be established and formally analyzed for the case of organic blockers. Here, we observe that the voltage dependence of block of a cyclic nucleotide–gated channel by a series of intracellular quaternary ammonium blockers, which are too bulky to traverse the narrow ion selectivity filter, gradually vanishes with extreme depolarization, a predicted feature of the extrinsic voltage dependence model. In contrast, the voltage dependence of block by an amine blocker, which has a smaller “diameter” and can therefore penetrate into the selectivity filter, follows a Boltzmann function, a predicted feature of the intrinsic voltage dependence model. Additionally, a blocker generates (at least) two blocked states, which, if related serially, may preclude meaningful application of a commonly used approach for investigating channel gating, namely, inferring the properties of the activation gate from the kinetics of channel block.     

Spermine and arcaine block and permeate N-methyl-D-aspartate receptor channels.

Polyamines such as spermine are thought to be endogenous regulators of NMDA (N-methyl-D-aspartate)-type glutamate receptors. Polyamine block of NMDA receptors was studied in excised outside-out patches from rat hippocampal neurons and Xenopus oocytes expressing recombinant receptors. Extracellular spermine and arcaine reduced NMDA single-channel conductance in a voltage-dependent manner, with partial relief of block evident at large inside negative membrane potentials. Reducing extracellular Na+ concentration increased the apparent affinities for spermine and arcaine, indicating strong interaction between spermine and permeant ions. Internal spermine also blocked NMDA channels in a voltage-dependent manner, with relief of block evident at large inside positive potentials. The Woodhull model of channel block by an impermeant ion adequately described the actions of external spermine from -60 to +60 mV, but failed for more negative potentials. Eyring rate theory for a permeable blocker with two barriers and one binding site adequately described the voltage-dependent block and relief from block by both external and internal spermine over the range of -120 to +60 mV. These findings indicate that polyamines block and permeate neuronal NMDA receptor channels from the extracellular and intracellular sides, although sensitivity to internal spermine is probably too low to be physiologically relevant.     

Ionic permeability characteristics of the N-methyl-D-aspartate receptor channel

N-methyl-D-aspartate (NMDA) receptor channels in cultured CA1 hippocampal neurons were studied using patch-clamp techniques. The purpose of the research was to determine the occupancy of the channel by permeant cations and to determine the influence of charged residues in or near the pore. The concentration dependence of permeability ratios, the mole-fraction dependence of permeability ratios, the concentration dependence of the single-channel conductance, and a single-channel analysis of Mg2+ block all independently indicated that the NMDA receptor behaves as a singly-occupied channel. More precisely, there is one permeant cation at a time occupying the site or sites that are in the narrow region of the pore directly in the permeation pathway. Permeability-ratio measurements in mixtures of monovalent and divalent cations indicated that local charges in or near the pore do not produce a large local surface potential in physiologic solutions. In low ionic strength solutions, a local negative surface potential does influence the ionic environment near the pore, but in normal physiologic solutions the surface potential appears too small to significantly influence ion permeation. The results indicate that the mechanism for the high Ca2+ conductance of the NMDA receptor channel is not the same as for the voltage-dependent Ca2+ channel (VDCC). The VDCC has two high affinity, interacting binding sites that provide high Ca2+ selectivity and conductance. The binding site of the NMDA receptor is of lower affinity. Therefore, the selectivity for Ca2+ is not as high, but the lower affinity of binding provides a faster off rate so that interacting sites are not required for high conductance.     

The pore helix dipole has a minor role in inward rectifier channel function

Ion channels lower the energetic barrier for ion passage across cell membranes and enable the generation of bioelectricity. Electrostatic interactions between permeant ions and channel pore helix dipoles have been proposed as a general mechanism for facilitating ion passage. Here, using genetic selections to probe interactions of an exemplar potassium channel blocker, barium, with the inward rectifier Kir2.1, we identify mutants bearing positively charged residues in the potassium channel signature sequence at the pore helix C terminus. We show that these channels are functional, selective, resistant to barium block, and have minimally altered conductance properties. Both the experimental data and model calculations indicate that barium resistance originates from electrostatics. We demonstrate that potassium channel function is remarkably unperturbed when positive charges occur near the permeant ions at a location that should counteract pore helix electrostatic effects. Thus, contrary to accepted models, the pore helix dipole seems to be a minor factor in potassium channel permeation.     

External barium affects the gating of KCNQ1 potassium channels and produces a pore block via two discrete sites

The pore properties and the reciprocal interactions between permeant ions and the gating of KCNQ channels are poorly understood. Here we used external barium to investigate the permeation characteristics of homomeric KCNQ1 channels. We assessed the Ba(2+) binding kinetics and the concentration and voltage dependence of Ba(2+) steady-state block. Our results indicate that extracellular Ba(2+) exerts a series of complex effects, including a voltage-dependent pore blockade as well as unique gating alterations. External barium interacts with the permeation pathway of KCNQ1 at two discrete and nonsequential sites. (a) A slow deep Ba(2+) site that occludes the channel pore and could be simulated by a model of voltage-dependent block. (b) A fast superficial Ba(2+) site that barely contributes to channel block and mostly affects channel gating by shifting rightward the voltage dependence of activation, slowing activation, speeding up deactivation kinetics, and inhibiting channel inactivation. A model of voltage-dependent block cannot predict the complex impact of Ba(2+) on channel gating in low external K(+) solutions. Ba(2+) binding to this superficial site likely modifies the gating transitions states of KCNQ1. Both sites appear to reside in the permeation pathway as high external K(+) attenuates Ba(2+) inhibition of channel conductance and abolishes its impact on channel gating. Our data suggest that despite the high degree of homology of the pore region among the various K(+) channels, KCNQ1 channels display significant structural and functional uniqueness.     

Ryanoid modification of the cardiac muscle ryanodine receptor channel results in relocation of the tetraethylammonium binding site

The interaction of ryanodine and derivatives of ryanodine with the high affinity binding site on the ryanodine receptor (RyR) channel brings about a characteristic modification of channel function. In all cases, channel open probability increases dramatically and single-channel current amplitude is reduced. The amplitude of the ryanoid-modified conductance state is determined by structural features of the ligand. An investigation of ion handling in the ryanodine-modified conductance state has established that reduced conductance results from changes in both the affinity of the channel for permeant ions and the relative permeability of ions within the channel (Lindsay, A.R.G., A. Tinker, and A.J. Williams. 1994. J. Gen. Physiol. 104:425-447). It has been proposed that these alterations result from a reorganization of channel structure induced by the binding of the ryanoid. The experiments reported here provide direct evidence for ryanoid-induced restructuring of RyR. TEA+ is a concentration- and voltage-dependent blocker of RyR in the absence of ryanoids. We have investigated block of K+ current by TEA+ in the unmodified open state and modified conductance states of RyR induced by 21-amino-9alpha-hydroxyryanodine, 21-azido-9alpha-hydroxyryanodine, ryanodol, and 21-p-nitrobenzoylamino-9alpha-hydroxyryanodine. Analysis of the voltage dependence of block indicates that the interaction of ryanoids with RyR leads to an alteration in this parameter with an apparent relocation of the TEA+ blocking site within the voltage drop across the channel and an alteration in the affinity of the channel for the blocker. The degree of change of these parameters correlates broadly with the change in conductance of permeant cations induced by the ryanoids, indicating that modification of RyR channel structure by ryanoids is likely to underlie both phenomena.     

Disulfonic stilbene permeation and block of the anion channel from the sarcoplasmic reticulum of rabbit skeletal muscle

Block of a sarcoplasmic reticulum anion channel (SCl channel) by disulfonic stilbene derivatives [DIDS, dibenzamidostilbene-2,2'-disulfonic acid (DBDS), and 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS)] was investigated in planar bilayers using SO4(2-) as the conducting ion. All molecules caused reversible voltage-dependent channel block when applied to either side of the membrane. DIDS also produced nonreversible channel block from both sides within 1-3 min. Reversible inhibition was associated with a decrease in channel open probability and mean open duration but not with any change in channel conductance. The half inhibitory concentration for cis- and trans-inhibition had voltage dependencies with minima of 190 nM and 33 microM for DBDS and 3.4 and 55 microM for DNDS. Our data supports a permeant blocker mechanism, in which stilbenes block SCl channels by lodging in the permeation pathway, where they may dissociate to either side of the membrane and thus permeate the channel. The stilbenes acted as open channel blockers where the binding of a single molecule occludes the channel. DBDS and DNDS, from opposite sides of the membrane, competed for common sites on the channel. Dissociation rates exhibited biphasic voltage dependence, indicative of two dissociation processes associated with ion movement in opposite directions within the trans-membrane electric field. The kinetics of DNDS and DBDS inhibition predict that there are two stilbene sites in the channel that are separated by 14-24 A and that the pore constriction is approximately 10 A in diameter.     

Mechanism of the voltage sensitivity of IRK1 inward-rectifier K+ channel block by the polyamine spermine

IRK1 (Kir2.1) inward-rectifier K+ channels exhibit exceedingly steep rectification, which reflects strong voltage dependence of channel block by intracellular cations such as the polyamine spermine. On the basis of studies of IRK1 block by various amine blockers, it was proposed that the observed voltage dependence (valence approximately 5) of IRK1 block by spermine results primarily from K+ ions, not spermine itself, traversing the transmembrane electrical field that drops mostly across the narrow ion selectivity filter, as spermine and K+ ions displace one another during channel block and unblock. If indeed spermine itself only rarely penetrates deep into the ion selectivity filter, then a long blocker with head groups much wider than the selectivity filter should exhibit comparably strong voltage dependence. We confirm here that channel block by two molecules of comparable length, decane-bis-trimethylammonium (bis-QA(C10)) and spermine, exhibit practically identical overall voltage dependence even though the head groups of the former are much wider ( approximately 6 A) than the ion selectivity filter ( approximately 3 A). For both blockers, the overall equilibrium dissociation constant differs from the ratio of apparent rate constants of channel unblock and block. Also, although steady-state IRK1 block by both cations is strongly voltage dependent, their apparent channel-blocking rate constant exhibits minimal voltage dependence, which suggests that the pore becomes blocked as soon as the blocker encounters the innermost K+ ion. These findings strongly suggest the existence of at least two (potentially identifiable) sequentially related blocked states with increasing numbers of K+ ions displaced. Consequently, the steady-state voltage dependence of IRK1 block by spermine or bis-QA(C10) should increase with membrane depolarization, a prediction indeed observed. Further kinetic analysis identifies two blocked states, and shows that most of the observed steady-state voltage dependence is associated with the transition between blocked states, consistent with the view that the mutual displacement of blocker and K+ ions must occur mainly as the blocker travels along the long inner pore.     

Interactions of protons with single open L-type calcium channels. pH dependence of proton-induced current fluctuations with Cs+, K+, and Na+ as permeant ions

We studied the pH dependence of the proton-induced current fluctuations that appear in single open L-type Ca channels when monovalent ions are the charge carriers. We used different methods of analysis to obtain kinetic measurements even under conditions where the individual transitions were too fast to be resolved directly as discrete current steps between two conductance levels. The reciprocal of the dwell times at the high conductance level increased linearly with the pipette proton activity, with a slope that was similar for Cs, K, and Na as permeant ions. Contrary to the expectation for a simple model in which the high and low conductances represent the unprotonated and protonated states of the channel, respectively, the dwell times at the low conductance level were also pH dependent and lengthened with increasing proton activity. At all pH values the dwell times at the low conductance level were longest with Cs as permeant ion and shortened in the order Cs greater than K greater than Na. We introduce a more general model of the protonation cycle in which the channel is represented by four states and can be protonated and deprotonated both at the high and low conductance levels. The conductance change is represented by a conformational change of the channel protein. We discuss the validity of this model and its implications for the mechanism by which protons interact with ion permeation through L-type Ca channels.     

State-dependent block of CNG channels by dequalinium

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.     

Mutation-induced blocker permeability and multiion block of the CFTR chloride channel pore

Chloride permeation through the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel is blocked by a broad range of anions that bind tightly within the pore. Here we show that the divalent anion Pt(NO2)42- acts as an impermeant voltage-dependent blocker of the CFTR pore when added to the intracellular face of excised membrane patches. Block was of modest affinity (apparent Kd 556 microM), kinetically fast, and weakened by extracellular Cl- ions. A mutation in the pore region that alters anion selectivity, F337A, but not another mutation at the same site that has no effect on selectivity (F337Y), had a complex effect on channel block by intracellular Pt(NO2)42- ions. Relative to wild-type, block of F337A-CFTR was weakened at depolarized voltages but strengthened at hyperpolarized voltages. Current in the presence of Pt(NO2)42- increased at very negative voltages in F337A but not wild-type or F337Y, apparently due to relief of block by permeation of Pt(NO2)42- ions to the extracellular solution. This "punchthrough" was prevented by extracellular Cl- ions, reminiscent of a "lock-in" effect. Relief of block in F337A by Pt(NO2)42- permeation was only observed for blocker concentrations above 300 microM; as a result, block at very negative voltages showed an anomalous concentration dependence, with an increase in blocker concentration causing a significant weakening of block and an increase in Cl- current. We interpret this effect as reflecting concentration-dependent permeability of Pt(NO2)42- in F337A, an apparent manifestation of an anomalous mole fraction effect. We suggest that the F337A mutation allows intracellular Pt(NO2)42- to enter deeply into the CFTR pore where it interacts with multiple binding sites, and that simultaneous binding of multiple Pt(NO2)42- ions within the pore promotes their permeation to the extracellular solution.     

Dequalinium: a novel,high-affinity blocker of CNGA1 channels

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.     

Slow permeation of organic cations in acetylcholine receptor channels

Block, permeation, and agonist action of small organic amine compounds were studied in acetylcholine receptor (AChR) channels. Single channel conductances were calculated from fluctuation analysis at the frog neuromuscular junction and measured by patch clamp of cultured rat myotubes. The conductance was depressed by a few millimolar external dimethylammonium, arginine, dimethyldiethanolammonium, and Tris. Except with dimethylammonium, the block was intensified with hyperpolarization. A two-barrier Eyring model describes the slowed permeation and voltage dependence well for the three less permeant test cations. The cations were assumed to pause at a site halfway across the electric field of the channel while passing through it. For the voltage-independent action of highly permeant dimethylammonium, a more appropriate model might be a superficial binding site that did not prevent the flow of other ions, but depressed it. Solutions of several amine compounds were found to have agonist activity at millimolar concentrations, inducing brief openings of AChR channels on rat myotubes in the absence of ACh.     

On the origin of asymmetric interactions between permeant anions and the cystic fibrosis transmembrane conductance regulator chloride channel pore

Single channel and macroscopic current recording was used to investigate block of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel pore by the permeant anion Au(CN)2(-). Block was 1-2 orders of magnitude stronger when Au(CN)2(-) was added to the intracellular versus the extracellular solution, depending on membrane potential. A point mutation within the pore, T-338A, strongly decreased the asymmetry of block, by weakening block by intracellular Au(CN)2(-) and at the same time strengthening block by external Au(CN)2(-). Block of T-338A, but not wild-type, was strongest at the current reversal potential and weakened by either depolarization or hyperpolarization. In contrast to these effects, the T-338A mutation had no impact on block by the impermeant Pt(NO2)4(2-) ion. We suggest that the CFTR pore has at least two anion binding sites at which Au(CN)2(-) and Pt(NO2)4(2-) block Cl- permeation. The T-338A mutation decreases a barrier for Au(CN)2(-) movement between different sites, leading to significant changes in its blocking action. Our finding that apparent blocker binding affinity can be altered by mutagenesis of a residue which does not contribute to a blocker binding site has important implications for interpreting the effects of mutagenesis on channel blocker effects.     

Anion permeation in an apical membrane chloride channel of a secretory epithelial cell

Single channel currents though apical membrane Cl channels of the secretory epithelial cell line T84 were measured to determine the anionic selectivity and concentration dependence of permeation. The current-voltage relation was rectified with single channel conductance increasing at positive potentials. At 0 mV the single channel conductance was 41 +/- 2 pS. Permeability, determined from reversal potentials, was optimal for anions with diameters between 0.4 and 0.5 nm. Anions of larger diameter had low permeability, consistent with a minimum pore diameter of 0.55 nm. Permeability for anions of similar size was largest for those ions with a more symmetrical charge distribution. Both HCO3 and H2PO4 had lower permeability than the similar-sized symmetrical anions, NO3 and ClO4. The permeability sequence was SCN greater than I approximately NO3 approximately ClO4 greater than Br greater than Cl greater than PF6 greater than HCO3 approximately F much greater than H2PO4. Highly permeant anions had lower relative single channel conductance, consistent with longer times of residence in the channel for these ions. The conductance sequence for anion efflux was NO3 greater than SCN approximately ClO4 greater than Cl approximately I approximately Br greater than PF6 greater than F approximately HCO3 much greater than H2PO4. At high internal concentrations, anions with low permeability and conductance reduced Cl influx consistent with block of the pore. The dependence of current on Cl concentration indicated that Cl can also occupy the channel long enough to limit current flow. Interaction of Cl and SCN within the conduction pathway is supported by the presence of a minimum in the conductance vs. mole fraction relation. These results indicate that this 40-pS Cl channel behaves as a multi-ion pathway in which other permeant anions could alter Cl flow across the apical membrane.     

Physico-chemical model of a voltage-gated sodium channel

A permeant ion is known to create in the channel pore a local electrical field, the intensity of which exceeds the intensity of an electrical field produced by the membrane potential. In our study, we consider a sodium channel model, in which the effects of a permeant ion, an inactivating particle, and pharmacological agents on mobile charged groups of the channel are semi-phenomenologically taken into account by using motion equations for a generalized structural variable. Stationary solutions for the equation correspond to “open,” “closed,” and “inactivated” channel states. Because of this, the channel free energy profile, as a function of the structural variable, has three local minima. The three energy values of these states depend both on the electrical field applied externally and on the near-membrane concentrations of permeant ions and acting pharmacological agents. Sodium channel activation and inactivation kinetics are considered resulting from relative changes of the free energy typical of the above three states of the channel. The results we obtained in the course of channel activation and inactivation modeling and their voltage dependence are qualitatively consistent with the commonly known experimental data. The proposed model allows one to qualitatively predict the dependence of the sodium channel kinetic characteristics on the concentrations of permeant ions and pharmacological agents.     

Rapid intracellular TEA block of the KcsA potassium channel

Intracellular tetraethylammonium (TEA) inhibition was studied at the single-channel level in the KcsA potassium channel reconstituted in planar lipid bilayers. TEA acts as a fast blocker (resulting in decreased current amplitude) with an affinity in the 75 mM range even at high bandwidth. Studies over a wide voltage range reveal that TEA block has a complex voltage-dependence that also depends on the ionic conditions. These observations are examined in the context of permeation models to extend our understanding of the coupling between permeant ions and TEA blockade.     

Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+

We investigated the mechanism whereby ions cross dihydropyridine- sensitive (L-type) Ca channels in guinea pig ventricular myocytes. At the single-channel level, we found no evidence of an anomalous mole- fraction effect like that reported previously for whole-cell currents in mixtures of Ba and Ca. With the total concentration of Ba + Ca kept constant at 10 (or 110) mM, neither conductance nor absolute unitary current exhibits a paradoxical decrease when Ba and Ca are mixed, thereby weakening the evidence for a multi-ion permeation scheme. We therefore sought independent evidence to support or reject the multi- ion nature of the L-type Ca channel by measuring conductance at various permeant ion concentrations. Contrary to the predictions of models with only one binding site in the permeation pathway, single-channel conductance does not follow Michaelis-Menten kinetics as Ba activity is increased over three orders of magnitude. Two-fold variation in the Debye length of permeant ion solutions has little effect on conductance, making it unlikely that local surface charge effects could account for these results. Instead, the marked deviation from Michaelis- Menten behavior was best explained by supposing that the permeation pathway contains three or more binding sites that can be occupied simultaneously. The presence of three sites helps explain both a continued rise in conductance as [Ba2+] is increased above 110 mM, and the high single-channel conductance (approximately 7 pS) with 1 mM [Ba2+] as the charge carrier; the latter feature enables the L-type channel to carry surprisingly large currents at physiological divalent cation concentrations. Thus, despite the absence of an anomalous mole- fraction effect between Ba and Ca, we suggest that the L-type Ca channel in heart cells supports ion flux by a single-file, multi-ion permeation mechanism.     

New insights on the voltage dependence of the KCa3.1 channel block by internal TBA

We present in this work a structural model of the open IKCa (KCa3.1) channel derived by homology modeling from the MthK channel structure, and used this model to compute the transmembrane potential profile along the channel pore. This analysis showed that the selectivity filter and the region extending from the channel inner cavity to the internal medium should respectively account for 81% and 16% of the transmembrane potential difference. We found however that the voltage dependence of the IKCa block by the quaternary ammonium ion TBA applied internally is compatible with an apparent electrical distance delta of 0.49 +/- 0.02 (n = 6) for negative potentials. To reconcile this observation with the electrostatic potential profile predicted for the channel pore, we modeled the IKCa block by TBA assuming that the voltage dependence of the block is governed by both the difference in potential between the channel cavity and the internal medium, and the potential profile along the selectivity filter region through an effect on the filter ion occupancy states. The resulting model predicts that delta should be voltage dependent, being larger at negative than positive potentials. The model also indicates that raising the internal K+ concentration should decrease the value of delta measured at negative potentials independently of the external K+ concentration, whereas raising the external K+ concentration should minimally affect delta for concentrations >50 mM. All these predictions are born out by our current experimental results. Finally, we found that the substitutions V275C and V275A increased the voltage sensitivity of the TBA block, suggesting that TBA could move further into the pore, thus leading to stronger interactions between TBA and the ions in the selectivity filter. Globally, these results support a model whereby the voltage dependence of the TBA block in IKCa is mainly governed by the voltage dependence of the ion occupancy states of the selectivity filter.     

Anion competition for a volume-regulated current.

We have examined whether the anionic amino acids, glutamate and aspartate, permeate through the same volume-regulated conductance permeant to Cl- ions. Cell swelling was initiated in response to establishing a whole-cell configuration in the presence of a hyposmotic gradient. Volume-regulated anion currents carried by Cl-, glutamate, or aspartate developed with similar time courses and showed similar voltage-dependent inactivation. Permeability ratios (Paa/PCl) calculated from measured reversal potentials were dependent on the mole fraction ratio (MFR) of the permeant anions ([aa]/([aa] + [Cl-])). MFR was varied from 0.00 to 0.97. As the fraction of amino acid increased, Paa/PCl decreased. Current amplitude was similarly dependent on MFR. These results show that the permeation of anionic amino acids and that of Cl- ions are not independent of each other, indicating that the ion channel underlying the volume-regulated conductance can be occupied by more than one ion at a time. Application of Eyring rate theory indicated that the major barrier to Cl- ion permeation is at the intracellular side of the membrane, and that the major barrier to amino acid permeation is at the extracellular side of the membrane. The interactions between these permeant ions may have a physiological modulatory role in volume regulation through a volume-regulated anion conductance.