Mechanisms of Transition from Normal to Reentrant Electrical Activity in a Model of Rabbit Atrial Tissue: Interaction of Tissue Heterogeneity and Anisotropy

Experimental evidence suggests that regional differences in action potential (AP) morphology can provide a substrate for initiation and maintenance of reentrant arrhythmias in the right atrium (RA), but the relationships between the complex electrophysiological and anatomical organization of the RA and the genesis of reentry are unclear. In this study, a biophysically detailed three-dimensional computer model of the right atrial tissue was constructed to study the role of tissue heterogeneity and anisotropy in arrhythmogenesis. The model of Lindblad et al. for a rabbit atrial cell was modified to incorporate experimental data on regional differences in several ionic currents (primarily, I_Na, I_CaL, I_K1, I_to, and I_sus) between the crista terminalis and pectinate muscle cells. The modified model was validated by its ability to reproduce the AP properties measured experimentally. The anatomical model of the rabbit RA (including tissue geometry and fiber orientation) was based on a recent histological reconstruction. Simulations with the resultant electrophysiologically and anatomically detailed three-dimensional model show that complex organization of the RA tissue causes breakdown of regular AP conduction patterns at high pacing rates (>11.75 Hz): as the AP in the crista terminalis cells is longer, and electrotonic coupling transverse to fibers of the crista terminalis is weak, high-frequency pacing at the border between the crista terminalis and pectinate muscles results in a unidirectional conduction block toward the crista terminalis and generation of reentry. Contributions of the tissue heterogeneity and anisotropy to reentry initiation mechanisms are quantified by measuring action potential duration (APD) gradients at the border between the crista terminalis and pectinate muscles: the APD gradients are high in areas where both heterogeneity and anisotropy are high, such that intrinsic APD differences are not diminished by electrotonic interactions. Thus, our detailed computer model reconstructs complex electrical activity in the RA, and provides new insights into the mechanisms of transition from focal atrial tachycardia into reentry.     

Reexcitation mechanisms in epicardial tissue: Role of Ito density heterogeneities and INa inactivation kinetics

Dispersion of action potential repolarization is known to be an important arrhythmogenic factor in cardiopathies such as Brugada syndrome. In this work, we analyze the effect of a variation in sodium current (I_Na) inactivation and a heterogeneous rise of transient outward current (I_to) in the probability of reentry in epicardial tissue. We use the Luo-Rudy model of epicardial ventricular action potential to study wave propagation in a one-dimensional fiber. Spatial dispersion in repolarization is introduced by splitting the fiber into zones with different strength of I_to. We then analyze the pro-arrhythmic effect of a variation in the relaxation time and steady-state of the sodium channel fast inactivating gate h. We quantify the probability of reentry measuring the percentage of reexcitations that occurs in 200 beats. We find that, for high stimulation rates, this percentage is negligible, but increases notably for pacing periods above 700 ms. Surprisingly, with decreasing I_Na inactivation time, the percentage of reexcitations does not grow monotonically, but presents vulnerable windows, separated by values of the I_Na inactivation speed-up where reexcitation does not occur. By increasing the strength of L-type calcium current I_CaL above a certain threshold, reexcitation disappears. Finally, we show the formation of reentry in stimulated two-dimensional epicardial tissue with modified I_Na kinetics and I_to heterogeneity. Thus, we confirm that while I_to dispersion is necessary for phase-2 reentry, altered sodium inactivation kinetics influences the probability of reexcitation in a highly nonlinear fashion.     

Anisotropic conduction block and reentry in neonatal rat ventricular myocyte monolayers

Anisotropy can lead to unidirectional conduction block that initiates reentry. We analyzed the mechanisms in patterned anisotropic neonatal rat ventricular myocyte monolayers. Voltage and intracellular Ca (Ca(i)) were optically mapped under the following conditions: extrastimulus (S1S2) testing and/or tetrodotoxin (TTX) to suppress Na current availability; heptanol to reduce gap junction conductance; and incremental rapid pacing. In anisotropic monolayers paced at 2 Hz, conduction velocity (CV) was faster longitudinally than transversely, with an anisotropy ratio [AR = CV(L)/CV(T), where CV(L) and CV(T) are CV in the longitudinal and transverse directions, respectively], averaging 2.1 ± 0.8. Interventions decreasing Na current availability, such as S1S2 pacing and TTX, slowed CV(L) and CV(T) proportionately, without changing the AR. Conduction block preferentially occurred longitudinal to fiber direction, commonly initiating reentry. Interventions that decreased gap junction conductance, such as heptanol, decreased CV(T) more than CV(L), increasing the AR and causing preferential transverse conduction block and reentry. Rapid pacing resembled the latter, increasing the AR and promoting transverse conduction block and reentry, which was prevented by the Ca(i) chelator 1,2-bis oaminophenoxy ethane-N,N,N',N'-tetraacetic acid (BAPTA). In contrast to isotropic and uniformly anisotropic monolayers, in which reentrant rotors drifted and self-terminated, bidirectional anisotropy (i.e., an abrupt change in fiber direction exceeding 45°) caused reentry to anchor near the zone of fiber direction change in 77% of monolayers. In anisotropic monolayers, unidirectional conduction block initiating reentry can occur longitudinal or transverse to fiber direction, depending on whether the experimental intervention reduces Na current availability or decreases gap junction conductance, agreeing with theoretical predictions.     

A computational study of invariant I5 in a nearly incompressible transversely isotropic model for white matter

The aligned axonal fiber bundles in white matter make it suitable to be modeled as a transversely isotropic material. Recent experimental studies have shown that a minimal form, nearly incompressible transversely isotropic (MITI) material model, is capable of describing mechanical anisotropy of white matter. Here, we used a finite element (FE) computational approach to demonstrate the significance of the fifth invariant (I₅) when modeling the anisotropic behavior of white matter in the large-strain regime. We first implemented and validated the MITI model in an FE simulation framework for large deformations. Next, we applied the model to a plate-hole structural problem to highlight the significance of the invariant I₅ by comparing with the standard fiber reinforcement (SFR) model. We also compared the two models by fitting the experiment data of asymmetric indentation, shear test, and uniaxial stretch of white matter. Our results demonstrated the significance of I₅ in describing shear deformation/anisotropy, and illustrated the potential of the MITI model to characterize transversely isotropic white matter tissues in the large-strain regime.     

Block of voltage-dependent K+ channels in insect muscle by the diacylhydrazine insecticide RH-5849, 4-aminopyridine,and quinidine

In calcium-free saline, voltage-clamped ventral longitudinal muscles of housefly larvae have maintained (I_K) and transient (I_A) voltage-dependent K⁺ currents. With 500 ms conditioning pulses, inactivation of I_A had a midpoint at ?53 mV and changed e-fold in 3.46 mV. I_A inactivated completely at ?40 mV, with a time constant of 71 ms, allowing the effects of various K⁺ channel blockers to be studied on I_K in isolation. RH-5849 (1,2-dibenzoyl-1-tert-butylhydrazine), a novel insect growth regulator, induces a lethal premature molt in insect larvae by mimicking the action of the molting hormone at ecdysone receptors. RH-5849 also causes acute neurotoxicity in some insects by selectively blocking of I_K in nerve and muscle. While most channel blockers have a Hill coefficient near 1, consistent with a simple one molecule per channel block mechanism, RH-5849 and the analog RH-1266 were found in the present study to block I_K channels in insect muscle with a Hill coefficient of 1.5. The lC₅₀ (concentration that caused 50% block) for block of I_K was 59 μM for RH-5849 and 40 μM for RH-1266. While tetraethylammonium blocked I_K by only 20% at 100 mM, 4-aminopyridine blocked the current with an lC₅₀ of 1.2 mM and a Hill coefficient of 0.97. Quinidine was the most potent blocker of I_K in this study, with an lC₅₀ of 20 μM. Block of I_K by either RH-5849 or 4-aminopyridine was independent of test pulse potential, but block by quinidine increased with depolarization. Block of I_K by RH-5849 and quinidine was time dependent, suggesting an open channel block mechanism, but the time course was too fast relative to channel activation for kinetic analysis. The lC₅₀ for block of I_K by RH-5849 decreased with temperature, with a Q₁₀ of 0.52. I_A was also blocked by RH-5849, but was less sensitive than I_K. The lC₅₀ for block of I_A by RH-5849 was 775 μM, 13-fold higher than the lC₅₀ for block of I_K. © 1992 Wiley-Liss, Inc.     

Interaction of galantamine with ionic channels in molluscan neurons

Galantamine is widely used for the treatment of Alzheimer’s disease. According to the generally accepted viewpoint, its therapeutic effect is based on inhibition of acetylcholinesterase (AChE) and potentiation of nicotinic receptors. Alternative molecular targets for galanatamine, namely, voltage-gated Ca²⁺ and K⁺ channels of the neuronal membrane, are also widely discussed in the current literature. The present study is devoted to the analysis of effects of galantamine on high-threshold Ca²⁺ currents (I _Ca) and three different kinds of highthreshold K⁺ current, viz.: Ca²⁺-dependent K⁺ current (I _C), delayed rectifier (I _DR), and fast-inactivating K₊ current (I _Adepol). Experiments were conducted on molluscan neurons with the help of two-microelectrode voltageclamp technique. It was found that galantamine caused a fast, reversible and dose-dependent suppression of all types of high-threshold ionic currents. The maximal blocking effect of the alkaloid for I _Ca, I _C, and I _DR, was 100%, while for I Adepol the maximal suppression was only 60%. The mean values of IC ₅₀ for I _C, I _DR, I _Adepol, and I _Ca were 109, 237, 66, and 515 μ M, respectively, i.e., substantially higher than the corresponding values for the alkaloid-induced inhibition of AChE and potentiation of nicotinic receptors. It is concluded that the blockade of Ca₂₊ and K₊ channels has little or no contribution to the therapeutic activity of galantamine.     

Inhibitory Effects of Capsaicin on Voltage-Gated Potassium Channels by TRPV1-Independent Pathway

Previously we observed that capsaicin, a transient receptor potential vanilloid 1 (TRPV1) receptor activator, inhibited transient potassium current (I_A) in capsaicin-sensitive and capsaicin-insensitive trigeminal ganglion (TG) neurons from rats. It suggested that the inhibitory effects of capsaicin on I_A have two different mechanisms: TRPV1-dependent and TRPV1-independent pathways. The main purpose of this study is to further investigate the TRPV1-independent effects of capsaicin on voltage-gated potassium channels (VGPCs). Whole cell patch-clamp technique was used to record I_A and sustained potassium current (I_K) in cultured TG neurons from trpv1 knockout (TRPV1^?/?) mice. We found that capsaicin reversibly inhibited I_A and I_K in a dose-dependent manner. Capsaicin (30 μM) did not alter the activation curve of I_A and I_K but shifted the inactivation–voltage curve to hyperpolarizing direction, thereby increasing the number of inactivated VGPCs at the resting potential. Administrations of high concentrations capsaicin, no use-dependent block, and delay of recovery time course were found on I_K and I_A. Moreover, forskolin, an adenylate cyclase agonist, selectively decreased the inhibitory effects of I_K by capsaicin, whereas none influenced the inhibitions of I_A. These results suggest that capsaicin inhibits the VGPCs through TRPV1-independent and PKA-dependent mechanisms, which may contribute to the capsaicin-induced nociception.     

Impaired Inactivation of L-Type Ca2+ Current as a Potential Mechanism for Variable Arrhythmogenic Liability of HERG K+ Channel Blocking Drugs

The proarrhythmic effects of new drugs have been assessed by measuring rapidly activating delayed-rectifier K⁺ current (I_Kr) antagonist potency. However, recent data suggest that even drugs thought to be highly specific I_Kr blockers can be arrhythmogenic via a separate, time-dependent pathway such as late Na⁺ current augmentation. Here, we report a mechanism for a quinolone antibiotic, sparfloxacin-induced action potential duration (APD) prolongation that involves increase in late L-type Ca²⁺ current (I_CaL) caused by a decrease in Ca²⁺-dependent inactivation (CDI). Acute exposure to sparfloxacin, an I_Kr blocker with prolongation of QT interval and torsades de pointes (TdP) produced a significant APD prolongation in rat ventricular myocytes, which lack I_Kr due to E4031 pretreatment. Sparfloxacin reduced peak I_CaL but increased late I_CaL by slowing its inactivation. In contrast, ketoconazole, an I_Kr blocker without prolongation of QT interval and TdP produced reduction of both peak and late I_CaL, suggesting the role of increased late I_CaL in arrhythmogenic effect. Further analysis showed that sparfloxacin reduced CDI. Consistently, replacement of extracellular Ca²⁺ with Ba²⁺ abolished the sparfloxacin effects on I_CaL. In addition, sparfloxacin modulated I_CaL in a use-dependent manner. Cardiomyocytes from adult mouse, which is lack of native I_Kr, demonstrated similar increase in late I_CaL and afterdepolarizations. The present findings show that sparfloxacin can prolong APD by augmenting late I_CaL. Thus, drugs that cause delayed I_CaL inactivation and I_Kr blockage may have more adverse effects than those that selectively block I_Kr. This mechanism may explain the reason for discrepancies between clinically reported proarrhythmic effects and I_Kr antagonist potencies.     

Adaptation and accommodation in the squid axon

Current clamp data of the squid axon indicate that there is a qualitative change in the adaptive response as the magnitude of the current step is increased. Large stimulus currents have a strong inhibitory effect on spike generation and on active responses in general. Such currents always lead to only one action-potential and to the elimination of post-spike subthreshold oscillation. In view of a direct connection between stimulus current and potassium current I _K, the potassium channel of the Hodgkin-Huxley model is reinterpreted in a natural way such that the K⁺ conductance is directly dependent on I _K in addition to a voltage dependence. The I-_Kdependence seems to dominate whenever the stimulus current is greater than approximately 35 μA/cm². For current ramps, and large current steps, such a current formulation leads to good agreement with the data.     

Nonselective Suppression of Voltage-gated Currents by Odorants in the Newt Olfactory Receptor Cells

Effects of odorants on voltage-gated ionic channels were investigated in isolated newt olfactory receptor cells by using the whole cell version of the patch–clamp technique. Under voltage clamp, membrane depolarization to voltages between −90 mV and +40 mV from a holding potential (V_h) of −100 mV generated time- and voltage-dependent current responses; a rapidly (< 15 ms) decaying initial inward current and a late outward current. When odorants (1 mM amyl acetate, 1 mM acetophenone, and 1 mM limonene) were applied to the recorded cell, the voltage-gated currents were significantly reduced. The dose-suppression relations of amyl acetate for individual current components (Na⁺ current: I_Na, T-type Ca²⁺ current: I_Ca,T, L-type Ca²⁺ current: I_Ca,L, delayed rectifier K⁺ current: I_Kv and Ca²⁺-activated K⁺ current: I_K(Ca)) could be fitted by the Hill equation. Half-blocking concentrations for each current were 0.11 mM (I_Na), 0.15 mM (I_Ca,T), 0.14 mM (I_Ca,L), 1.7 mM (I_Kv), and 0.17 mM (I_K(Ca)), and Hill coefficient was 1.4 (I_Na), 1.0 (I_Ca,T), 1.1 (I_Ca,L), 1.0 (I_Kv), and 1.1 (I_K(Ca)), suggesting that the inward current is affected more strongly than the outward current. The activation curve of I_Na was not changed significantly by amyl acetate, while the inactivation curve was shifted to negative voltages; half-activation voltages were −53 mV at control, −66 mV at 0.01 mM, and −84 mV at 0.1 mM. These phenomena are similar to the suppressive effects of local anesthetics (lidocaine and benzocaine) on I_Na in various preparations, suggesting that both types of suppression are caused by the same mechanism. The nonselective blockage of ionic channels observed here is consistent with the previous notion that the suppression of the transduction current by odorants is due to the direst blockage of transduction channels.     

Protonography and anion inhibition profile of the α-carbonic anhydrase (CruCA4) identified in the Mediterranean red coral Corallium rubrum

CruCA4 is a secreted isoform of the α-carbonic anhydrase (CA, EC 4.2.1.1) family, which has been identified in the octocoral Corallium rubrum. This enzyme is involved in the calcification process leading to the formation of the coral calcium carbonate skeleton. We report here experiments performed on the recombinant CruCA4 with the technique of protonography that can be used to detect in a simple way the enzyme activity. We have also investigated the inhibition profile of CruCA4 with one major class of CA inhibitors, the inorganic anions. A range of weak and moderate inhibitors have been identified having K_I in the range of 1–100 mM, among which the halides, pseudohalides, bicarbonate, sulfate, nitrate, nitrite, and many complex inorganic anions. Stronger inhibitors were sulfamide, sulfamate, phenylboronic acid, phenylarsonic acid, and diethylditiocarbamate, which showed a better affinity for this enzyme, with K_I in the range of 75 μM–0.60 mM. All these anions/small molecules probably coordinate to the Zn(II) ion within the CA active site as enzyme inhibition mechanism.     

Involvement of Caveolin in Low K+-induced Endocytic Degradation of Cell-surface Human Ether-a-go-go-related Gene (hERG) Channels

Reduction in the rapidly activating delayed rectifier K⁺ channel current (I_Kr) due to either mutations in the human ether-a-go-go-related gene (hERG) or drug block causes inherited or drug-induced long QT syndrome. A reduction in extracellular K⁺ concentration ([K⁺]_o) exacerbates long QT syndrome. Recently, we demonstrated that lowering [K⁺]_o promotes degradation of I_Kr in rabbit ventricular myocytes and of the hERG channel stably expressed in HEK 293 cells. In this study, we investigated the degradation pathways of hERG channels under low K⁺ conditions. We demonstrate that under low K⁺ conditions, mature hERG channels and caveolin-1 (Cav1) displayed a parallel time-dependent reduction. Mature hERG channels coprecipitated with Cav1 in co-immunoprecipitation analysis, and internalized hERG channels colocalized with Cav1 in immunocytochemistry analysis. Overexpression of Cav1 accelerated internalization of mature hERG channels in 0 mm K⁺_o, whereas knockdown of Cav1 impeded this process. In addition, knockdown of dynamin 2 using siRNA transfection significantly impeded hERG internalization and degradation under low K⁺_o conditions. In cultured neonatal rat ventricular myocytes, knockdown of caveolin-3 significantly impeded low K⁺_o-induced reduction of I_Kr. Our data indicate that a caveolin-dependent endocytic route is involved in low K⁺_o-induced degradation of mature hERG channels.     

Succinylacetone pyrrole,a powerful inhibitor of vitamin B12 biosynthesis: Effect on δ-aminolevulinic acid dehydratase

Succinylacetone, a competitive inhibitor (K_I = 400 μM) of δ-aminolevulinic acid dehydratase of $\text{Clostridium}\text{tetanomorphum}$, is converted non-enzymatically upon incubation with δ-aminolevulinic acid to succinylacetone pyrrole, a much stronger competitive inhibitor (K_I = 5 μM) of the enzyme. A similar effect is seen in vivo: when present in the growth medium at concentrations of about 1 μM, the pyrrole decreases the level of corrinoids produced by this organism by half, while succinylacetone at 200 μM causes only 19 per cent inhibition of corrinoid formation. Levulinic acid is a much weaker inhibitor in vitro and in vivo. The inhibition by succinylacetone pyrrole is considered to be due to its structural resemblance to δ-aminolevulinic acid rather than to porphobilinogen, the reaction product of δ-aminolevulinic acid dehydratase: succinylacetone, succinylacetone pyrrole, and levulinic acid all contain a succinyl group.     

Gender disparities in torsade de pointes ventricular tachycardia

Background Gender disparities in the incidence of torsade de pointes (TdP) ventricular tachycardia exist, but the mechanisms in humans are unresolved. We addressed this issue using a mathematical model of a human ventricular cell. MethodsWe implemented gender differences in the Priebe-Beuckelmann model cell by modifying the amplitudes of the L-type Ca²⁺ current (I_Ca,L), transient outward K₊ current (I_to), and rapid component of the delayed rectifier K₊current (I_Kr), according to experimental data from animal male and female hearts. Gender disparities in electrical heterogeneity between transmural layers (subepicardium, midmyocardium, subendocardium) were implemented by modifying various ion currents according to experimental data. ResultsAction potentials in female cells have longer durations and steeper duration versus frequency relationships than male cells. In the female cells, electrical heterogeneity between transmural layers is larger and the susceptibility to early afterdepolarisations is higher than in male cells. ConclusionGender-related differences in I_Ca,L, I_to, and I_Kr may explain the gender disparities in human cardiac electrophysiology. Female cells have an increased susceptibility to early afterdepolarisations following mild reductions in net repolarising forces. Combined with their greater electrical heterogeneity, this renders them more vulnerable to TdP. (Neth Heart J 2007;15:405-11.)     

Potassium Currents in Freshly Dissociated Uterine Myocytes from Nonpregnant and Late-Pregnant Rats

In freshly dissociated uterine myocytes, the outward current is carried by K⁺ through channels highly selective for K⁺. Typically, nonpregnant myocytes have rather noisy K⁺ currents; half of them also have a fast-inactivating transient outward current (I_TO). In contrast, the current records are not noisy in late pregnant myocytes, and I_TO densities are low. The whole-cell I_K of nonpregnant myocytes respond strongly to changes in [Ca²⁺]_o or changes in [Ca²⁺]_i caused by photolysis of caged Ca²⁺ compounds, nitr 5 or DM-nitrophene, but that of late-pregnant myocytes respond weakly or not at all. The Ca²⁺ insensitivity of the latter is present before any exposure to dissociating enzymes. By holding at −80, −40, or 0 mV and digital subtractions, the whole-cell I_K of each type of myocyte can be separated into one noninactivating and two inactivating components with half-inactivation at approximately −61 and −22 mV. The noninactivating components, which consist mainly of iberiotoxin-susceptible large-conductance Ca²⁺-activated K⁺ currents, are half-activated at 39 mV in nonpregnant myocytes, but at 63 mV in late-pregnant myocytes. In detached membrane patches from the latter, identified 139 pS, Ca²⁺-sensitive K⁺ channels also have a half-open probability at 68 mV, and are less sensitive to Ca²⁺ than similar channels in taenia coli myocytes. Ca²⁺-activated K⁺ currents, susceptible to tetraethylammonium, charybdotoxin, and iberiotoxin contribute 30–35% of the total I_K in nonpregnant myocytes, but <20% in late-pregnant myocytes. Dendrotoxin-susceptible, small-conductance delayed rectifier currents are not seen in nonpregnant myocytes, but contribute ∼20% of total I_K in late-pregnant myocytes. Thus, in late-pregnancy, myometrial excitability is increased by changes in K⁺ currents that include a suppression of the I_TO, a redistribution of I_K expression from large-conductance Ca²⁺-activated channels to smaller-conductance delayed rectifier channels, a lowered Ca²⁺ sensitivity, and a positive shift of the activation of some large-conductance Ca²⁺-activated channels.     

Selective block by α-dendrotoxin of the K+ inward rectifier at the Vicia guard cell plasma membrane

The efficacy and mechanism of -dendrotoxin (DTX) block of K⁺ channel currents in Vicia stomatal guard cells was examined. Currents carried by inward- and outward-rectifying K⁺ channels were determined under voltage clamp in intact guard cells, and block was characterized as a function of DTX and external K⁺ (K⁺) concentrations. Added to the bath, 0.1-30 nM DTX blocked the inward-rectifying K⁺ current (I_K,in), but was ineffective in blocking current through the outward-rectifying K⁺ channels (I_K,out) even at concentrations of 30 nM. DTX block was independent of clamp voltage and had no significant effect on the voltage-dependent kinetics for I_K,in, neither altering its activation at voltages negative of –120 mV nor its deactivation at more positive voltages. No evidence was found for a use dependence to DTX action. Block of I_K,in followed a simple titration function with an apparent K_1/2 for block of 2.2 nM in 3 mm K _o ⁺ . However, DTX block was dependent on the external K⁺ concentration. Raising K⁺ from 3 to 30 mm slowed block and resulted in a 60–70% reduction in its efficacy (apparent K _i = 10 mm in 10 nm DTX). The effect of K⁺ in protecting I _K,in was competitive with DTX and specific for permeant cations. A joint analysis of I_K,in block with DTX and K⁺ concentration was consistent with a single class of binding sites with a K _d for DTX of 240 pm. A K _d of 410 m for extracellular K⁺ was also indicated. These results complement previous studies implicating a binding site requiring extracellular K⁺ (K_1/2 1 mm) for I_K,in activation; they parallel features of K⁺ channel block by DTX and related peptide toxins in many animal cells, demonstrating the sensitivity of plant plasma membrane K⁺ channels to nanomolar toxin concentrations under physiological conditions; the data also highlight one main difference: in the guard cells, DTX action appears specific to the K⁺ inward rectifier.We thank J.O. Dolly (Imperial, London) and S.M. Jarvis (University of Kent, Canterbury) for several helpful discussions. This work was supported by SERC grant GR/H07696 and was aided by equipment grants from the Gatsby Foundation, the Royal Society and the University of London Central Research Fund. G.O. was supported by an Ausbildungsstipendium (OB 85/1-1) from the Deutsche Forschungsgemeinschaft. F.A. holds a Sainsbury Studentship.     

Mechanism of Ion Permeation in Skeletal Muscle Chloride Channels

Voltage-gated Cl⁻ channels belonging to the ClC family exhibit unique properties of ion permeation and gating. We functionally probed the conduction pathway of a recombinant human skeletal muscle Cl⁻ channel (hClC-1) expressed both in Xenopus oocytes and in a mammalian cell line by investigating block by extracellular or intracellular I⁻ and related anions. Extracellular and intracellular I⁻ exert blocking actions on hClC-1 currents that are both concentration and voltage dependent. Similar actions were observed for a variety of other halide (Br⁻) and polyatomic (SCN⁻, NO₃ ⁻, CH₃SO₃ ⁻) anions. In addition, I⁻ block is accompanied by gating alterations that differ depending on which side of the membrane the blocker is applied. External I⁻ causes a shift in the voltage-dependent probability that channels exist in three definable kinetic states (fast deactivating, slow deactivating, nondeactivating), while internal I⁻ slows deactivation. These different effects on gating properties can be used to distinguish two functional ion binding sites within the hClC-1 pore. We determined K _D values for I⁻ block in three distinct kinetic states and found that binding of I⁻ to hClC-1 is modulated by the gating state of the channel. Furthermore, estimates of electrical distance for I⁻ binding suggest that conformational changes affecting the two ion binding sites occur during gating transitions. These results have implications for understanding mechanisms of ion selectivity in hClC-1, and for defining the intimate relationship between gating and permeation in ClC channels.