Inactivation of batrachotoxin-modified Na+ channels in GH3 cells. Characterization and pharmacological modification

Batrachotoxin (BTX)-modified Na+ currents were characterized in GH3 cells with a reversed Na+ gradient under whole-cell voltage clamp conditions. BTX shifts the threshold of Na+ channel activation by approximately 40 mV in the hyperpolarizing direction and nearly eliminates the declining phase of Na+ currents at all voltages, suggesting that Na+ channel inactivation is removed. Paradoxically, the steady-state inactivation (h infinity) of BTX-modified Na+ channels as determined by a two-pulse protocol shows that inactivation is still present and occurs maximally near -70 mV. About 45% of BTX-modified Na+ channels are inactivated at this voltage. The development of inactivation follows a sum of two exponential functions with tau d(fast) = 10 ms and tau d(slow) = 125 ms at -70 mV. Recovery from inactivation can be achieved after hyperpolarizing the membrane to voltages more negative than -120 mV. The time course of recovery is best described by a sum of two exponentials with tau r(fast) = 6.0 ms and tau r(slow) = 240 ms at -170 mV. After reaching a minimum at -70 mV, the h infinity curve of BTX-modified Na+ channels turns upward to reach a constant plateau value of approximately 0.9 at voltages above 0 mV. Evidently, the inactivated, BTX-modified Na+ channels can be forced open at more positive potentials. The reopening kinetics of the inactivated channels follows a single exponential with a time constant of 160 ms at +50 mV. Both chloramine-T (at 0.5 mM) and alpha-scorpion toxin (at 200 nM) diminish the inactivation of BTX-modified Na+ channels. In contrast, benzocaine at 1 mM drastically enhances the inactivation of BTX-modified Na+ channels. The h infinity curve reaches minimum of less than 0.1 at -70 mV, indicating that benzocaine binds preferentially with inactivated, BTX-modified Na+ channels. Together, these results imply that BTX-modified Na+ channels are governed by an inactivation process.     

Charged tetracaine as an inactivation enhancer in batrachotoxin-modified Na+ channels.

Two distinct types of local anesthetics (LAs) have previously been found to block batrachotoxin (BTX)-modified Na+ channels: type 1 LAs such as cocaine and bupivacaine interact preferentially with open channels, whereas type 2 LAs, such as benzocaine and tricaine, with inactivated channels. Herein, we describe our studies of a third type of LA, represented by tetracaine as a dual blocker that binds strongly with closed channels but also binds to a lesser extent with open channels when the membrane is depolarized. Enhanced inactivation of BTX-modified Na+ channels by tetracaine was determined by steady-state inactivation measurement and by the dose-response curve. The 50% inhibitory concentration (IC50) was estimated to be 5.2 microM at -70 mV, where steady-state inactivation was maximal, with a Hill coefficient of 0.98 suggesting that one tetracaine molecule binds with one inactivated channel. Tetracaine also interacted efficiently with Na+ channels when the membrane was depolarized; the IC50 was estimated to be 39.5 microM at +50 mV with a Hill coefficient of 0.94. Unexpectedly, charged tetracaine was found to be the primary active form in the blocking of inactivated channels. In addition, external Na+ ions appeared to antagonize the tetracaine block of inactivated channels. Consistent with these results, N-butyl tetracaine quaternary ammonium, a permanently charged tetracaine derivative, remained a strong inactivation enhancer. Another derivative of tetracaine, 2-(di-methylamino) ethyl benzoate, which lacked a 4-butylamino functional group on the phenyl ring, elicited block that was approximately 100-fold weaker than that of tetracaine. We surmise that 1) the binding site for inactivation enhancers is within the Na+ permeation pathway, 2) external Na+ ions antagonize the block of inactivation enhancers by electrostatic repulsion, 3) the 4-butylamino functional group on the phenyl ring is critical for block and for the enhancement of inactivation, and 4) there are probably overlapping binding sites for both inactivation enhancers and open-channel blockers within the Na+ pore.     

Redox properties of local anesthetics: A structural determinant of closed channel blockers in BTX-modified Na+ channels

Single channel analyses and macroscopic current measurements have shown that benzocaine is a predominantly closed channel blocker in BTX-modified Na+ channels; cocaine is an open channel blocker; and tetracaine, a dual channel blocker (Wang & Wang, 1994; Wang et al., 1994). The reason for such a selective state-dependent block by local anesthetics in BTX-modified Na+ channels is not clear. We assessed the redox properties of tetracaine, benzocaine, cocaine, and various derivatives by their ability to donate electrons to radical intermediates of eosin dye excited by visible light. Electron-donor properties of the drugs were previously proposed to be involved in Na+ channel blockade (Marinov, 1991). Our results provide evidence that redox properties of tetracaine, benzocaine, and their homologs correlate with their ability to enhance Na+ channel inactivation in BTX-modified Na+ channels. This correlation may be explained in terms of the previously proposed redox model of ion channels.     

Altered stereoselectivity of cocaine and bupivacaine isomers in normal and batrachotoxin-modified Na+ channels

The inhibitory effects of local anesthetics (LAs) of cocaine and bupivacaine optical isomers on Na+ currents were studied in clonal GH3 cells under whole-cell patch clamp conditions. At holding potential of -100 mV, all four isomers inhibited peak Na+ currents when the cell was stimulated infrequently. The dose-response curves of this tonic block of peak Na+ currents by (-)/(+) cocaine and (-)/(+) bupivacaine were well fitted by the Langmuir isotherm, suggesting that one LA isomer blocked one Na+ channel. Each pair of isomers showed no greater than a twofold difference in stereoselectivity toward Na+ channels. Additional block of Na+ currents occurred when the cell was stimulated at 2 Hz. This use-dependent block was also observed in all four isomers, which again displayed little stereoselectivity. The voltage dependence of the use-dependent block produced by cocaine isomers did not overlap with the activation of Na+ channels but did overlap with the steady-state inactivation (h infinity), indicating that cocaine can bind directly to the inactivated state of Na+ channels before channel opening. In comparison, the peak batrachotoxin (BTX)-modified Na+ currents were little inhibited by cocaine and bupivacaine isomers. However, the maintained BTX-modified Na+ currents were highly sensitive toward the (-) form of cocaine and bupivacaine isomers during a prolonged depolarization. As a result, a profound time-dependent block of BTX-modified Na+ currents was evident in the presence of these LA isomers. The estimated values of the equilibrium dissociation constant (KD in micromolar) at +50 mV were 35.8, 661, 7.0, and 222 for (-)/(+) cocaine and (-)/(+) bupivacaine, respectively. Although chloramine-T (CT) also modified the fast inactivation of Na+ channels and gave rise to a maintained Na+ current during a prolonged depolarization, LA isomers showed no greater stereoselectivity in blocking this maintained current than in blocking the normal transient Na+ current. We conclude that (a) cocaine and bupivacaine isomers exhibit only weak stereoselectivity toward the LA receptor in normal and CT-treated Na+ channels, (b) BTX drastically modifies the configuration of the LA binding site so that the LA stereoselectivity of the open Na+ channels is altered by an order of magnitude, and (c) the (-) forms of cocaine and bupivacaine interact strongly with the open state of BTX-modified Na+ channels but only weakly, if at all, with the closed state. The last finding may explain why most LA drugs were reported to be less effective toward BTX-modified Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

The properties of batrachotoxin-modified cardiac Na channels, including state-dependent block by tetrodotoxin

Batrachotoxin (BTX) modification and tetrodotoxin (TTX) block of BTX-modified Na channels were studied in single cardiac cells of neonatal rats using the whole-cell patch-clamp recording technique. The properties of BTX-modified Na channels in heart are qualitatively similar to those in nerve. However, quantitative differences do exist between the modified channels of these two tissues. In the heart, the shift of the conductance-voltage curve for the modified channel was less pronounced, the maximal activation rate constant, (tau m)max, of modified channels was considerably slower, and the slow inactivation of the BTX-modified cardiac Na channels was only partially abolished. TTX blocked BTX-modified mammalian cardiac Na channels and the block decreased over the potential range of -80 to -40 mV. The apparent dissociation constant of TTX changed from 0.23 microM at -50 mV to 0.69 microM at 0 mV. No further reduction of block was observed at potentials greater than -40 mV. This is the potential range over which gating from closed to open states occurred. These results were explained by assuming that TTX has a higher affinity for closed BTX-modified channels than for open modified channels. Hence, the TTX-binding rate constants are considered to be state dependent rather than voltage dependent. This differs from the voltage dependence of TTX block reported for BTX-modified Na channels from membrane vesicles incorporated into lipid bilayers and from amphibian node of Ranvier.     

Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers

Batrachotoxin (BTX)-activated Na+ channels from rabbit skeletal muscle were incorporated into planar lipid bilayers. These channels appear to open most of the time at voltages greater than -60 mV. Local anesthetics, including QX-314, bupivacaine, and cocaine when applied internally, induce different durations of channel closures and can be characterized as "fast" (mean closed duration less than 10 ms at +50 mV), "intermediate" (approximately 80 ms), and "slow" (approximately 400 ms) blockers, respectively. The action of these local anesthetics on the Na+ channel is voltage dependent; larger depolarizations give rise to stronger binding interactions. Both the dose-response curve and the kinetics of the cocaine-induced closures indicate that there is a single class of cocaine-binding site. QX-314, though a quaternary-amine local anesthetic, apparently competes with the same binding site. External cocaine or bupivacaine application is almost as effective as internal application, whereas external QX-314 is ineffective. Interestingly, external Na+ ions reduce the cocaine binding affinity drastically, whereas internal Na+ ions have little effect. Both the cocaine association and dissociation rate constants are altered when external Na+ ion concentrations are raised. We conclude that (a) one cocaine molecule closes one BTX-activated Na+ channel in an all-or-none manner, (b) the binding affinity of cocaine is voltage sensitive, (c) this cocaine binding site can be reached by a hydrophilic pathway through internal surface and by a hydrophobic pathway through bilayer membrane, and (d) that this binding site interacts indirectly with the Na+ ions. A direct interaction between the receptor and Na+ ions seems minimal.     

Block of inactivation-deficient Na+ channels by local anesthetics in stably transfected mammalian cells: evidence for drug binding along the activation pathway

According to the classic modulated receptor hypothesis, local anesthetics (LAs) such as benzocaine and lidocaine bind preferentially to fast-inactivated Na(+) channels with higher affinities. However, an alternative view suggests that activation of Na(+) channels plays a crucial role in promoting high-affinity LA binding and that fast inactivation per se is not a prerequisite for LA preferential binding. We investigated the role of activation in LA action in inactivation-deficient rat muscle Na(+) channels (rNav1.4-L435W/L437C/A438W) expressed in stably transfected Hek293 cells. The 50% inhibitory concentrations (IC(50)) for the open-channel block at +30 mV by lidocaine and benzocaine were 20.9 +/- 3.3 microM (n = 5) and 81.7 +/- 10.6 microM (n = 5), respectively; both were comparable to inactivated-channel affinities. In comparison, IC(50) values for resting-channel block at -140 mV were >12-fold higher than those for open-channel block. With 300 microM benzocaine, rapid time-dependent block (tau approximately 0.8 ms) of inactivation-deficient Na(+) currents occurred at +30 mV, but such a rapid time-dependent block was not evident at -30 mV. The peak current at -30 mV, however, was reduced more severely than that at +30 mV. This phenomenon suggested that the LA block of intermediate closed states took place notably when channel activation was slow. Such closed-channel block also readily accounted for the LA-induced hyperpolarizing shift in the conventional steady-state inactivation measurement. Our data together illustrate that the Na(+) channel activation pathway, including most, if not all, transient intermediate closed states and the final open state, promotes high-affinity LA binding.     

Modification of cardiac Na+ channels by batrachotoxin: effects on gating, kinetics, and local anesthetic binding.

The purpose of the present study was to examine the characteristics of Na+ channel modification by batrachotoxin (BTX) in cardiac cells, including changes in channel gating and kinetics as well as susceptibility to block by local anesthetic agents. We used the whole cell configuration of the patch clamp technique to measure Na+ current in guinea pig myocytes. Extracellular Na+ concentration and temperature were lowered (5-10 mM, 17 degrees C) in order to maintain good voltage control. Our results demonstrated that 1) BTX modifies cardiac INa, causing a substantial steady-state (noninactivating) component of INa, 2) modification of cardiac Na+ channels by BTX shifts activation to more negative potentials and reduces both maximal gNa and selectivity for Na+; 3) binding of BTX to its receptor in the cardiac Na+ channel reduces the affinity of local anesthetics for their binding site; and 4) BTX-modified channels show use-dependent block by local anesthetics. The reduced blocking potency of local anesthetics for BTX-modified Na+ channels probably results from an allosteric interaction between BTX and local anesthetics for their respective binding sites in the Na+ channel. Our observations that use-dependent block by local anesthetics persists in BTX-modified Na+ channels suggest that this form of extra block can occur in the virtual absence of the inactivated state. Thus, the development of use-dependent block appears to rely primarily on local anesthetic binding to activated Na+ channels under these conditions.     

Divalent cation selectivity for external block of voltage-dependent Na+ channels prolonged by batrachotoxin. Zn2+ induces discrete substates in cardiac Na+ channels

The mechanism of block of voltage-dependent Na+ channels by extracellular divalent cations was investigated in a quantitative comparison of two distinct Na+ channel subtypes incorporated into planar bilayers in the presence of batrachotoxin. External Ca2+ and other divalent cations induced a fast voltage-dependent block observed as a reduction in unitary current for tetrodotoxin-sensitive Na+ channels of rat skeletal muscle and tetrodotoxin-insensitive Na+ channels of canine heart ventricular muscle. Using a simple model of voltage-dependent binding to a single site, these two distinct Na+ channel subtypes exhibited virtually the same affinity and voltage dependence for fast block by Ca2+ and a number of other divalent cations. This group of divalent cations exhibited an affinity sequence of Co congruent to Ni greater than Mn greater than Ca greater than Mg greater than Sr greater than Ba, following an inverse correlation between binding affinity and ionic radius. The voltage dependence of fast Ca2+ block was essentially independent of CaCl2 concentration; however, at constant voltage the Ca2+ concentration dependence of fast block deviated from a Langmuir isotherm in the manner expected for an effect of negative surface charge. Titration curves for fast Ca2+ block were fit to a simplified model based on a single Ca2+ binding site and the Gouy-Chapman theory of surface charge. This model gave similar estimates of negative surface charge density in the vicinity of the Ca2+ blocking site for muscle and heart Na+ channels. In contrast to other divalent cations listed above, Cd2+ and Zn2+ are more potent blockers of heart Na+ channels than muscle Na+ channels. Cd2+ induced a fast, voltage-dependent block in both Na+ channel subtypes with a 46-fold higher affinity at 0 mV for heart (KB = 0.37 mM) vs. muscle (KB = 17 mM). Zn2+ induced a fast, voltage-dependent block of muscle Na+ channels with low affinity (KB = 7.5 mM at 0 mV). In contrast, micromolar Zn2+ induced brief closures of heart Na+ channels that were resolved as discrete substate events at the single-channel level with an apparent blocking affinity of KB = 0.067 mM at 0 mV, or 110-fold higher affinity for Zn2+ compared with the muscle channel. High-affinity block of the heart channel by Cd2+ and Zn2+ exhibited approximately the same voltage dependence (e-fold per 60 mV) as low affinity block of the muscle subtype (e-fold per 54 mV), suggesting that the block occurs at structurally analogous sites in the two Na+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)     

Permeation of Na+ through a delayed rectifier K+ channel in chick dorsal root ganglion neurons

In whole-cell patch clamp recordings from chick dorsal root ganglion neurons, removal of intracellular K+ resulted in the appearance of a large, voltage-dependent inward tail current (Icat). Icat was not Ca2+ dependent and was not blocked by Cd2+, but was blocked by Ba2+. The reversal potential for Icat shifted with the Nernst potential for [Na+]. The channel responsible for Icat had a cation permeability sequence of Na+ >> Li+ >> TMA+ > NMG+ (PX/PNa = 1:0.33:0.1:0) and was impermeable to Cl-. Addition of high intracellular concentrations of K+, Cs+, or Rb+ prevented the occurrence of Icat. Inhibition of Icat by intracellular K+ was voltage dependent, with an IC50 that ranged from 3.0-8.9 mM at membrane potentials between -50 and -110 mV. This voltage- dependent shift in IC50 (e-fold per 52 mV) is consistent with a single cation binding site approximately 50% of the distance into the membrane field. Icat displayed anomolous mole fraction behavior with respect to Na+ and K+; Icat was inhibited by 5 mM extracellular K+ in the presence of 160 mM Na+ and potentiated by equimolar substitution of 80 mM K+ for Na+. The percent inhibition produced by both extracellular and intracellular K+ at 5 mM was identical. Reversal potential measurements revealed that K+ was 65-105 times more permeant than Na+ through the Icat channel. Icat exhibited the same voltage and time dependence of inactivation, the same voltage dependence of activation, and the same macroscopic conductance as the delayed rectifier K+ current in these neurons. We conclude that Icat is a Na+ current that passes through a delayed rectifier K+ channel when intracellular K+ is reduced to below 30 mM. At intracellular K+ concentrations between 1 and 30 mM, PK/PNa remained constant while the conductance at -50 mV varied from 80 to 0% of maximum. These data suggest that the high selectivity of these channels for K+ over Na+ is due to the inability of Na+ to compete with K+ for an intracellular binding site, rather than a barrier that excludes Na+ from entry into the channel or a barrier such as a selectivity filter that prevents Na+ ions from passing through the channel.     

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

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

State-dependent block of wild-type and inactivation-deficient Na+ channels by flecainide

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/DeltaKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with beta1 subunits in Hek293t cells. The flecainide-resting block at -140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 micro M when the cell was not stimulated for 1,000 s. At 100 micro M flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an approximately 70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at -140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at -50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 micro M, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 micro M-1s-1 and 12.2 s-1, respectively. Upon repolarization to -140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was approximately 20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.     

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.     

Quaternary ammonium compounds as structural probes of single batrachotoxin-activated Na+ channels

Quaternary ammonium (QA) blockers are well-known structural probes for studying the permeation pathway of voltage-gated K+ channels. In this study we have examined the effects of a series of n-alkyl-trimethylammonium compounds (Cn-QA) on batrachotoxin (BTX)-activated Na+ channels from skeletal muscle incorporated into planar lipid bilayers. We found that these amphipathic QA compounds (Cn-QA where n = 10-18) block single Na+ channels preferentially from the internal side with equilibrium dissociation constants (KD) in the submicromolar to micromolar range. External application of amphipathic QA compounds is far less effective, by a factor of greater than 200. The block can be described by a QA molecule binding to a single site in the Na+ channel permeation pathway. QA binding affinity is dependent on transmembrane voltage with an effective valence (delta) of approximately 0.5. QA dwell times (given as mean closed times, tau c) increase as a function of n-alkyl chain length, ranging from approximately 13 ms for C10-QA to 500 ms for C18-QA at +50 mV. The results imply that there is a large hydrophobic region within the Na+ channel pore which accepts up to 18 methylene groups of the Cn-QA cation. This hydrophobic domain may be of clinical significance since it also interacts with local anesthetics such as cocaine and mepivacaine. Finally, like BTX-activated Na+ channels in bilayers, unmodified Na+ channels in GH3 cells are also susceptible to QA block. Amphipathic QA cations elicit both tonic and use-dependent inhibitions of normal Na+ currents in a manner similar to that of local anesthetic cocaine. We conclude that amphipathic QA compounds are valuable structural probes to study the permeation pathway of both normal and BTX-activated Na+ channels.     

Voltage and substrate dependence of the inverse transport mode of the rabbit Na(+)/glucose cotransporter (SGLT1)

Properties of the cytoplasmic binding sites of the rabbit Na(+)/glucose cotransporter, SGLT1, expressed in Xenopus oocytes were investigated using the giant excised patch clamp technique. Voltage and substrate dependence of the outward cotransport were studied using alpha-methyl D-glucopyranoside (alphaMDG) as a substrate. The apparent affinity for alphaMDG depends on the cytoplasmic Na(+) concentration and voltage. At 0 mV the K(M) for alphaMDG is 7 mM at 110 mM Na(+) and 31 mM at 10 mM Na(+). The apparent affinity for alphaMDG and Na(+) is voltage dependent and increases at positive potentials. At 0 mV holding potential the outward current is half-maximal at about 70 mM. The results show that SGLT1 can mediate sugar transport out of the cell under appropriate concentration and voltage conditions, but under physiological conditions this transport is highly improbable due to the low affinity for sugar.     

Alkaloid-modified sodium channels from lobster walking leg nerves in planar lipid bilayers

Alkaloid-modified, voltage-dependent sodium channels from lobster walking leg nerves were studied in planar neutral lipid bilayers. In symmetrical 0.5 M NaCl the single channel conductance of veratridine (VTD) (10 pS) was less than that of batrachotoxin (BTX) (16 pS) modified channels. At positive potentials, VTD- but not BTX-modified channels remained open at a flickery substate. VTD-modified channels underwent closures on the order of milliseconds (fast process), seconds (slow process), and minutes. The channel fractional open time (f(o)) due to the fast process, the slow process, and all channel closures (overall f(o)) increased with depolarization. The fast process had a midpoint potential (V(a)) of -122 mV and an apparent gating charge (z(a)) of 2.9, and the slow process had a V(a) of -95 mV and a z(a) of 1.6. The overall f(o) was predominantly determined by closures on the order of minutes, and had a V(a) of about -24 mV and a shallow voltage dependence (z(a) approximately 0.7). Augmenting the VTD concentration increased the overall f(o) without changing the number of detectable channels. However, the occurrence of closures on the order of minutes persisted even at super-saturating concentrations of VTD. The occurrence of these long closures was nonrandom and the level of nonrandomness was usually unaffected by the number of channels, suggesting that channel behavior was nonindependent. BTX-modified channels also underwent closures on the order of milliseconds, seconds, and minutes. Their characterization, however, was complicated by the apparent low BTX binding affinity and by an apparent high binding reversibility (channel disappearance) of BTX to these channels. VTD- but not BTX-modified channels inactivated slowly at high positive potentials (greater than +30 mV). Single channel conductance versus NaCl concentrations saturated at high NaCl concentrations and was non-Langmuirian at low NaCl concentrations. At all NaCl concentrations the conductance of VTD-modified channels was lower than that of BTX-modified channels. However, this difference in conductance decreased as NaCl concentrations neared zero, approaching the same limiting value. The permeability ratio of sodium over potassium obtained under mixed ionic conditions was similar for VTD (2.46)- and BTX (2.48)-modified channels, whereas that obtained under bi-ionic conditions was lower for VTD (1.83)- than for BTX (2.70)-modified channels. Tetrodotoxin blocked these alkaloid-modified channels with an apparent binding affinity in the nanomolar range.     

Use-dependent inhibition of Na+ currents by benzocaine homologs.

Most local anesthetics (LAs) elicit use-dependent inhibition of Na+ currents when excitable membranes are stimulated repetitively. One exception to this rule is benzocaine, a neutral LA that fails to produce appreciable use-dependent inhibition. In this study, we have examined the use-dependent phenomenon of three benzocaine homologs: ethyl 4-diethylaminobenzoate, ethyl 4-ethoxybenzoate, and ethyl 4-hydroxybenzoate. Ethyl 4-hydroxybenzoate at 1 mM, like benzocaine, elicited little use-dependent inhibition of Na+ currents, whereas ethyl 4-diethylaminobenzoate at 0.15 mM and ethyl 4-ethoxybenzoate at 0.5 mM elicited substantial use-dependent inhibition--up to 55% of peak Na+ currents were inhibited by repetitive depolarizations at 5 Hz. Each of these compounds produced significant tonic block of Na+ currents at rest and shifted the steady-state inactivation curve (h infinity) toward the hyperpolarizing direction. Kinetic analyses showed that the decaying phase of Na+ currents during a depolarizing pulse was significantly accelerated by all drugs, thus suggesting that these drugs also block the activated channel. The recovery time course for the use-dependent inhibition of Na+ currents was relatively slow, with time constants of 6.8 and 4.4 s for ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate, respectively. We conclude that benzocaine and 4-hydroxybenzoate interact with the open and inactivated channels during repetitive pulses, but during the interpulse the complex dissociates too fast to accumulate sufficient use-dependent block of Na+ currents. In contrast, ethyl 4-diethylaminobenzoate and ethyl 4-ethoxybenzoate dissociate slowly from their binding site and consequently elicit significant use-dependent block. A common LA binding site suffices to explain the presence and absence of use-dependent block by benzocaine homologs during repetitive pulses.     

The block of Shaker K+ channels by kappa-conotoxin PVIIA is state dependent.

kappa-conotoxin PVIIA is the first conotoxin known to interact with voltage-gated potassium channels by inhibiting Shaker-mediated currents. We studied the mechanism of inhibition and concluded that PVIIA blocks the ion pore with a 1:1 stoichiometry and that binding to open or closed channels is very different. Open-channel properties are revealed by relaxations of partial block during step depolarizations, whereas double-pulse protocols characterize the slower reequilibration of closed-channel binding. In 2.5 mM-[K+]o, the IC50 rises from a tonic value of approximately 50 to approximately 200 nM during openings at 0 mV, and it increases e-fold for about every 40-mV increase in voltage. The change involves mainly the voltage dependence and a 20-fold increase at 0 mV of the rate of PVIIA dissociation, but also a fivefold increase of the association rate. PVIIA binding to Shaker Delta6-46 channels lacking N-type inactivation or to wild phenotypes appears similar, but inactivation partially protects the latter from open-channel unblock. Raising [K+]o to 115 mM has little effect on open-channel binding, but increases almost 10-fold the tonic IC50 of PVIIA due to a decrease by the same factor of the toxin rate of association to closed channels. In analogy with charybdotoxin block, we attribute the acceleration of PVIIA dissociation from open channels to the voltage-dependent occupancy by K+ ions of a site at the outer end of the conducting pore. We also argue that the occupancy of this site by external cations antagonizes on binding to closed channels, whereas the apparent competition disappears in open channels if the competing cation can move along the pore. It is concluded that PVIIA can also be a valuable tool for probing the state of ion permeation inside the pore.     

Barium modulates the gating of batrachotoxin-treated Na+ channels in high ionic strength solutions.

Batrachotoxin-activated rat brain Na+ channels were reconstituted in neutral planar phospholipid bilayers in high ionic strength solutions (3 M NaCl). Under these conditions, diffuse surface charges present on the channel protein are screened. Nevertheless, the addition of extracellular and/or intracellular Ba2+ caused the following alterations in the gating of Na+ channels: (a) external (or internal) Ba2+ caused a depolarizing (or hyperpolarizing) voltage shift in the gating curve (open probability versus membrane potential curve) of the channels; (b) In the concentration range of 10-120 mM, extracellular Ba2+ caused a larger voltage shift in the gating curve of Na+ channels than intracellular Ba2+; (c) voltage shifts of the gating curve of Na+ channels as a function of external or internal Ba2+ were fitted with a simple binding isotherm with the following parameters: for internal Ba2+, delta V0.5,max (maximum voltage shift) = -11.5 mV, KD = 64.7 mM; for external Ba2+, delta V0.5,max = 13.5 mV, KD = 25.8 mM; (d) the change in the open probability of the channel caused by extracellular or intracellular Ba2+ is a consequence of alterations in both the opening and closing rate constants. Extracellular and intracellular divalent cations can modify the gating kinetics of Na+ channels by a specific modulatory effect that is independent of diffuse surface potentials. External or internal divalent cations probably bind to specific charges on the Na+ channel glycoprotein that modulate channel gating.     

Effect of Na(+) flow on Cd(2+) block of tetrodotoxin-resistant Na(+) channels

Tetrodotoxin-resistant (TTX-R) Na(+) channels are 1,000-fold less sensitive to TTX than TTX-sensitive (TTX-S) Na(+) channels. On the other hand, TTX-R channels are much more susceptible to external Cd(2+) block than TTX-S channels. A cysteine (or serine) residue situated just next to the aspartate residue of the presumable selectivity filter "DEKA" ring of the TTX-R channel has been identified as the key ligand determining the binding affinity of both TTX and Cd(2+). In this study we demonstrate that the binding affinity of Cd(2+) to the TTX-R channels in neurons from dorsal root ganglia has little intrinsic voltage dependence, but is significantly influenced by the direction of Na(+) current flow. In the presence of inward Na(+) current, the apparent dissociation constant of Cd(2+) ( approximately 200 microM) is approximately 9 times smaller than that in the presence of outward Na(+) current. The Na(+) flow-dependent binding affinity change of Cd(2+) block is true no matter whether the direction of Na(+) current is secured by asymmetrical chemical gradient (e.g., 150 mM Na(+) vs. 150 mM Cs(+) on different sides of the membrane, 0 mV) or by asymmetrical electrical gradient (e.g., 150 mM Na(+) on both sides of the membrane, -20 mV vs. 20 mV). These findings suggest that Cd(2+) is a pore blocker of TTX-R channels with its binding site located in a multiion, single-file region near the external pore mouth. Quantitative analysis of the flow dependence with the flux-coupling equation reveals that at least two Na(+) ions coexist with the blocking Cd(2+) ion in this pore region in the presence of 150 mM ambient Na(+). Thus, the selectivity filter of the TTX-R Na(+) channels in dorsal root ganglion neurons might be located in or close to a multiion single-file pore segment connected externally to a wide vestibule, a molecular feature probably shared by other voltage-gated cationic channels, such as some Ca(2+) and K(+) channels.