Biomedical subjects

G K Wang

Publications and source records attributed to G K Wang.

At least 55 records · Page 3Linked to original sources

PubMed1995

Irreversible inhibition of sodium current and batrachotoxin binding by a photoaffinity-derivatized local anesthetic.

We have synthesized a model local anesthetic (LA), N-(2-di-N-butyl-aminoethyl)-4-azidobenzamide (DNB-AB), containing the photoactivatable aryl azido moiety, which is known to form a covalent bond to adjacent molecules when exposed to UV light (Fleet, G.W., J.R. Knowles, and R.R. Porter. 1972. Biochemical Journal. 128:499-508. Ji, T.H. 1979. Biochimica et Biophysica Acta. 559:39-69). We studied the effects of DNB-AB on the sodium current (INa) under whole-cell voltage clamp in clonal mammalian GH3 cells and on 3[H]-BTX-B binding to sheep brain synaptoneurosomes. In the absence of UV illumination, DNB-AB behaved similarly to known LAs, producing both reversible block of peak INa (IC50 = 26 microM, 20 degrees C) and reversible inhibition of 3[H]-BTX-B (50 nM in the presence of 0.12 microgram/liter Leiurus quinquestriatus scorpion venom) binding (IC50 = 3.3 microM, 37 degrees C), implying a noncovalent association between DNB-AB and its receptor(s). After exposure to UV light, both block of INa and inhibition of 3[H]-BTX-B binding were only partially reversible (INa = 42% of control; 3[H]-BTX-B binding = 23% of control) showing evidence of a light-dependent, covalent association between DNB-AB and its receptor(s). In the absence of drug, UV light had less effect on INa (post exposure INa = 96% of control) or on 3[H]-BTX-B binding (post exposure binding = 70% of control). The irreversible block of INa was partially protected by coincubation of DNB-AB with 1 mM bupivacaine (IC50 = 45 microM, for INa inhibition at 20 degrees C, Wang, G.K., and S.Y. Wang. 1992. Journal of General Physiology. 100:1003-1020), (post exposure INa = 73% of control). The irreversible inhibition of 3[H]-BTX-B binding also was partially protected by coincubation with bupivacaine (500 microM, 37 degrees C) (post exposure binding = 51% of control), suggesting that the site of irreversible inhibition of both INa and 3[H]-BTX-B binding is shared with the clinical LA bupivacaine.

Affinity Labels↗

PubMed1995

Quaternary ammonium derivative of lidocaine as a long-acting local anesthetic.

BACKGROUND: Use of long-acting local anesthetics that elicit complete neural blockade for more than 3 h often is desirable in pain management. Unfortunately, clinically available local anesthetics are in general not suitable for prolonged analgesia. This report describes the organic synthesis and functional testing of a lidocaine derivative that appears to fulfill the criteria of long-acting local anesthetics. METHODS: A lidocaine derivative, N-beta-phenylethyl lidocaine quaternary ammonium bromide, was synthesized, and its ability to inhibit Na+ currents in cultured rat neuronal GH3 cells was tested in vitro under whole-cell voltage clamp conditions. Neurologic evaluation of sciatic nerve block of sensory and motor functions in vivo was subsequently performed in rats. RESULTS: N-beta-phenylethyl lidocaine was found to be a potent Na+ channel blocker in vitro. It produced both tonic and use-dependent blocks of Na+ currents that exceeded lidocaine's effects by a factor of > 2 (P < 0.05). In vivo, N-beta-phenylethyl lidocaine elicited a prolonged and complete sciatic nerve block of the motor function and the withdrawal response to noxious pinch that was 3.6- and 9.3-fold longer than that of lidocaine (P < 0.001), respectively. CONCLUSIONS: In an attempt to elicit prolonged local anesthesia, a quaternary ammonium derivative of lidocaine containing a permanent charge and an additional hydrophobic component was synthesized. Complete sciatic neural blockade of more than 3 h was achieved with this derivative. Of note, sensory blockade was prolonged to a greater extent than motor blockade. The approach used in this study may prove useful for developing new drugs applicable in pain management.

Anesthetics, Local↗

PubMed1994

Modification of cloned brain Na+ channels by batrachotoxin.

The effects of batrachotoxin (BTX) on cloned alpha-subunit Na+ channels were examined in CHO-K1 cells (a chinese hamster ovary cell line) transfected with rat brain NaIIA cDNA. Under whole-cell patch clamp conditions, BTX shifted the voltage dependence of the activation process by about 45 mV towards the hyperpolarizing direction and eliminated the inactivating phase of Na+ currents. Repetitive depolarizations greatly facilitated the binding of BTX with NaIIA channels while the membrane was held at -100 mV. In chloramine-T-pretreated cells, the association rate of BTX binding with the NaIIA channel was 6.5-fold faster than that in untreated cells. The estimated association rate constant for BTX binding with the open form of NaIIA channel was 1.11 x 10(6) mol-1.s-1 at room temperature. BTX-modified NaIIA channels were blocked by tetrodotoxin (TTX) in a complicated manner. First, the TTX binding to the closed state of BTX-modified NaIIA channels was not voltage dependent. The KD value of TTX was measured at 8.9 nM, which was similar to that of unmodified channels (KD = 14.2 nM). Second, the block of the open state of BTX-modified NaIIA channels by TTX was voltage dependent; depolarization reduced the potency of TTX block between -20 mV to +50 mV. Below -30 mV, the TTX affinity began to level off, probably because of the increased presence of the closed state. Unexpectedly, steady-state inactivation of BTX-modified NaIIA channels was minimal as measured by the two-pulse protocol, a phenomenon distinctly different from that found in GH3 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

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.

Animals↗

PubMed1994

Binding of benzocaine in batrachotoxin-modified Na+ channels. State-dependent interactions.

Hille (1977. Journal of General Physiology. 69:497-515) first proposed a modulated receptor hypothesis (MRH) to explain the action of benzocaine in voltage-gated Na+ channels. Using the MRH as a framework, we examined benzocaine binding in batrachotoxin (BTX)-modified Na+ channels under voltage-clamp conditions using either step or ramp command signals. We found that benzocaine binding is strongly voltage dependent. At -70 mV, the concentration of benzocaine that inhibits 50% of BTX-modified Na+ currents in GH3 cells (IC50) is 0.2 mM, whereas at +50 mV, the IC50 is 1.3 mM. Dose-response curves indicate that only one molecule of benzocaine is required to bind with one BTX-modified Na+ channel at -70 mV, whereas approximately two molecules are needed at +50 mV. Upon treatment with the inactivation modifier chloramine-T, the binding affinity of benzocaine is reduced significantly at -70 mV, probably as a result of the removal of the inactivated state of BTX-modified Na+ channels. The same treatment, however, enhances the binding affinity of cocaine near this voltage. External Na+ ions appear to have little effect on benzocaine binding, although they do affect cocaine binding. We conclude that two mechanisms underlie the action of local anesthetics in BTX-modified Na+ channels. Unlike open-channel blockers such as cocaine and bupivacaine, neutral benzocaine binds preferentially with BTX-modified Na+ channels in a closed state. Furthermore, benzocaine can be modified chemically so that it behaves like an open-channel blocker. This compound also elicits a use-dependent block in unmodified Na+ channels after repetitive depolarizations, whereas benzocaine does not. The implications of these findings for the MRH theory will be discussed.

Animals↗

PubMed1994

4-Aminopyridine binding and slow inactivation are mutually exclusive in rat Kv1.1 and Shaker potassium channels.

In the present study we have used two-electrode voltage-clamping of Xenopus oocytes expressing either Kv1.1 or Shaker B (ShB) delta 6-46 K+ channels to examine the effects of 4-aminopyridine (4-AP) on the process of slow inactivation. Neither of these channels exhibits fast inactivation. Channel activation was required for block by 4-AP in both channel types. In the absence of drug, inactivation of Kv1.1 and ShB delta 6-46 channels at 0 mV was biexponential [tau fast = 7.8 +/- 0.3 sec, tau slow = 33.9 +/- 0.9 sec, and Aslow/(Afast + Aslow) = 0.79 +/- 0.02 (n = 10) for Kv1.1 and tau fast = 3.5 +/- 0.4 sec, tau slow = 13.1 +/- 1.8 sec, and Aslow/(Afast + Aslow) = 0.35 +/- 0.06 (n = 3) for ShB delta 6-46]. In the presence of 4-AP, the rates of inactivation of Kv1.1 and ShB delta 6-46 were markedly slowed, resulting in a crossover phenomenon where, in the presence of drug, the outward current was smaller than control at the beginning of the depolarizing pulse but crossed over during the pulse to become larger than the control. The most obvious change induced by 0.2 mM 4-AP was a 2-fold slowing of the slow phase of inactivation [tau fast = 3.9 +/- 1.1 sec, tau slow = 67.1 +/- 3.6 sec, and Aslow/(Afast + Aslow) = 0.85 +/- 0.04 (n = 4) for Kv1.1 and tau fast = 3.5 +/- 0.4 sec, tau slow = 23.7 +/- 2.6 sec, and Aslow/(Afast + Aslow) = 0.75 +/- 0.02 (n = 3) for ShB delta 6-46, in the presence of 0.2 mM 4-AP]. In addition, there was a significant increase in the contribution of the slower phase of inactivation of ShB delta 6-46 channels in the presence of 4-AP. The slowed inactivation in the presence of 4-AP was accompanied by removal of 4-AP block. These results are consistent with the processes of 4-AP block and slow inactivation of Kv1.1 and ShB delta 6-46 channels being mutually exclusive.

4-Aminopyridine↗

PubMed1994

Aminopyridine block of Kv1.1 potassium channels expressed in mammalian cells and Xenopus oocytes.

The mechanism by which aminopyridines (APs) block cloned Kv1.1 K+ channels expressed either in the mammalian Sol-8 muscle cell line or in Xenopus oocytes was investigated using whole-cell patch-clamp and two-electrode voltage-clamp techniques. When Sol-8 cells were exposed to 4-AP (30 microM to 1 mM) or 3-AP (300 microM to 10 mM) for 1-2 min at a holding potential of -80 mV, delayed rectifier K+ currents activated by the first depolarization to +40 mV showed a small reduction in peak amplitude but exhibited a rapid decay phase that was absent in control records. However, currents elicited by subsequent pulses showed maximal block throughout the pulse. These results suggest that 4-AP requires the channel to be activated before block can occur readily. After a 10-min washout of 4-AP in the absence of channel activation, the current elicited at the beginning of the first depolarizing pulse was similar to that observed during maximal block. However, during the pulse the current increased in an exponential manner, to an amplitude similar to that seen in the absence of 4-AP. Subsequent pulses elicited currents with profiles similar to that observed in controls. These results suggest that channel activation is also required for unblock (i.e., 4-AP is trapped when the channel closes). Block of Kv1.1 channels by 4-AP was voltage dependent. The magnitude of block decreased progressively as the membrane potential was depolarized to voltages more positive than -20 mV. The concentration producing half-maximal inhibition (IC50) of Kv1.1 currents in Sol-8 cells at +40 mV at physiological pH (7.2) was 89 microM for 4-AP (pKa = 9.2) and 2.2 mM for 3-AP (pKa = 6.0). However, when the intracellular pH was lowered to 6.0 the IC50 for 3-AP decreased to 290 microM. These results are consistent with the charged form of the APs being the active species.

Aminopyridines↗

PubMed1993

Structural determinants of quaternary ammonium blockers for batrachotoxin-modified Na+ channels.

Amphipathic quaternary ammonium (QA) compounds are potent blockers of batrachotoxin (BTX)-modified Na+ channels incorporated into planar lipid bilayers. To examine the topology of the QA binding site, we selected two series of QA compounds as structural probes. One series contains two separate hydrophobic moieties but with a common hydrophilic dimethyl QA ion. Most of the QAs within this group bind to BTX-modified Na+ channels with relatively high affinities. For example, benzyldimethyldodecyl ammonium ions, when applied internally, block single, muscle, BTX-modified Na+ channels in bilayers with a one-to-one relationship and display an equilibrium dissociation constant (Kd) of 0.2 microM at +50 mV. Furthermore, the QA dwell times appear to correlate with QA hydrophobic interactions with the channel. These results indicate that there are two large hydrophobic binding domains within the QA binding site. The QAs in the second series contain a hydrophilic head group (trialkylammonium) of variable size but with a common dodecyl hydrophobic tail. Tripropyldodecyl QAs block BTX-modified Na+ channels more effectively (Kd = 0.4 microM at +50 mV) than do trimethyl- and triethyldodecyl QAs, suggesting that the internal Na+ permeation pathway is at least 9 A wide. However, tributyl- and tripentyldodecyl QAs show much lower affinities for BTX-modified Na+ channels at comparable concentrations. These drugs are cut off from binding, probably as a result of the size of their hydrophilic heads (> 10 A), which may be too large to fit in the QA binding site and too bulky to travel freely within the internal permeation pathway. Under whole-cell voltage-clamp conditions, we have further found that BTX-modified Na+ currents in clonal GH3 cells can be blocked by these two series of QA ions, albeit only when the activation gate is open. Closed channels at rest do not bind appreciably with these QA ions. Binding of QA ions is reduced by external Na+ ions in GH3 cells in a manner indicating that external Na+ ions can clear the bound QA ions from the Na+ pore. These results from GH3 cells mirror those obtained with QA blockers in K+ channels of squid axons and suggest that the QA binding domains in BTX-modified Na+ channels and K+ channels may be structurally conserved.

Animals↗

PubMed1992

Identification of RBK1 potassium channels in C6 astrocytoma cells.

Ionic currents in C6 astrocytoma cells were studied using the patch clamp technique under the whole cell configuration. A delayed rectifier K+ current with an amplitude of approximately 1 nA at +50 mV was observed in 86% (92/107) of the cells examined. This K+ current resembled the delayed rectifier present in type-1 and type-2 astrocytes in vitro and could be inhibited by a variety of K+ channel blockers, including TEA (IC50:0.5 mM), 4-aminopyridine (IC50:0.2 mM), MCD peptide (IC50:52 nM), dendrotoxin I (IC50:9 nM), and charybdotoxin (74% inhibition at 50 nM). Northern blot analysis, cloning of cDNA and subsequent sequencing showed that the C6 cell delayed rectifier K+ channel is equivalent to the RBK1 K+ channel derived from a rat brain cDNA library. The level of RBK1 transcripts in C6 cells was comparable to that reported in rat brain. The C6 delayed rectifier K+ channel is probably a homomeric RBK1 K+ channel judging from its pharmacological properties which are similar to the RBK1 channel expressed in Xenopus oocytes. Some C6 cells also expressed a transiently activated outward K+ current (IA). This current was found in less than 50% of the cells and in general contributed no more than 8% of the total outward current. No voltage-dependent inward Na+ or Ca2+ currents or inwardly rectifying K+ currents were observed in over 100 C6 cells examined. The present results show that the dominant voltage gated ionic current in C6 cells is the RBK1 delayed rectifier K+ channel.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

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)

Batrachotoxins↗

PubMed1992

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.

Batrachotoxins↗

PubMed1992

Stereoselective inhibition of neuronal sodium channels by local anesthetics. Evidence for two sites of action?

The objective of this study was to determine if the "tonic," resting inhibition of Na+ channels by local anesthetics results from binding at a site different from that for "phasic," use-dependent inhibition. Stereoselective actions of four local anesthetics were examined in isolated frog peripheral nerve and single Na+ channels. Using the sucrose-gap method on desheathed nerves, four actions of local anesthetics were assayed: 1) tonic depression of compound action potentials at low stimulation frequency (one per minute); 2) phasic depression of the compound action potential during trains of stimulation at 5, 10, and 20 Hz; 3) competitive antagonism of the reversible Na+ channel activator veratridine assayed through the depolarization of the compound resting membrane potential; and 4) depression of the depolarization of the compound resting membrane potential initially induced by the irreversible channel activator batrachotoxin. For assays 1, 2, and 3, all local anesthetics showed a stereoselectivity, where rectus, or (+), enantiomers were more potent than sinister, or (-), enantiomers. In contrast, for the noncompetitive antagonism of veratridine's action and the depression of batrachotoxin-induced depolarization, also a noncompetitive interaction between anesthetic and activator, the (-) enantiomer was more potent than the corresponding (+) enantiomer. Blockade of single Na+ channels activated by batrachotoxin in planar lipid bilayers was also stereoselective for the (-) enantiomer. These findings, along with previously reported voltage-clamp results, can be applied to infer properties of a local anesthetic binding site in activator-free channels. Local anesthetic molecules with more sharply angled shapes have stronger stereoselectivities than less angled, more planar drugs. The inversion of the stereopotency induced by the activators can be explained by either of two mechanisms. There may be two binding sites for local anesthetics, one of high and one of low affinity and of opposite stereoselectivity; activators may change the conformation at the high affinity site, reducing its local anesthetic affinity below that of the usual low affinity site and thereby revealing the pharmacology of the weaker site. Alternatively, only a single binding site may exist and be conformationally altered by activators such that both anesthetic affinity and stereopotency are modified. In activator-free channels, however, a single, high-affinity binding site with a constant stereoselectivity can account for both tonic and phasic inhibition by local anesthetics.

Action Potentials↗

PubMed1991

Alterations in sodium channel gating produced by the venom of the marine mollusc Conus striatus.

The action of the venom from the marine mollusc Conus striatus was studied using the voltage-clamp technique on myelinated nerve. Conus venom applied to an isolated node of Ranvier at 1.1 micrograms protein/ml produced repetitive firing of action potentials when the node was depolarized under current-clamp conditions. Venom application produced a leftword (depolarizing) shift in both the peak sodium current-voltage and the permeability-voltage relationships. A concomitant decrease in maximum peak current and permeability also occurred. The time course of sodium current decline (inactivation) was slowed at all voltages by the presence of venom. Venom treatment caused only a slight depolarizing shift (5 mV) in the voltage-dependence of steady-state Na inactivation. The closing to the resting state of previously activated Na channels, "deactivation", was judged from Na "tail" currents following membrane repolarization, and was slowed more than four-fold by venom treatment. The changes in Na channel gating produced by Conus striatus venom can best be described as a stabilization of the open state of the Na channel and a shift in the voltage dependence of the opening of Na channels. The slowing of both inactivation and deactivation of Na channels can be simulated by alterations in the rate constants of a five state Markov model.

Action Potentials↗

PubMed1991

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.

Animals↗

PubMed1991

Block of single batrachotoxin-activated Na+ channels by clofilium.

The effects of clofilium on single batrachotoxin-activated Na+ channels from rabbit skeletal muscle, incorporated into planar bilayers, were studied under symmetrical 200 mM NaCl conditions. Internally applied clofilium (0.3-30 microM) induced long lasting closures, with a mean duration of approximately 450 msec at +50 mV. The fraction of time spent in the clofilium-induced closed state was concentration dependent, with an equilibrium dissociation constant (Kd) of 3.4 microM at +50 mV. Kinetic analysis showed that both open and closed time distributions were well described by single exponentials, with respective time constants of tau o and tau C. As expected for an open channel blocker, 1/tau o increased linearly with increasing clofilium concentration, whereas 1/tau c remained relatively constant. Inhibition of batrachotoxin-activated Na+ channels by clofilium exhibited a strong voltage dependence. The binding affinity of clofilium increased about 10-fold upon depolarization from -50 mV to 50 mV. Competition studies using quaternary and tertiary local anesthetics showed that clofilium and local anesthetics probably share a common receptor site located about halfway across the membrane electrical field. Together, our results demonstrate that clofilium is a potent Na+ channel blocker in bilayers and its action is similar to that of other local anesthetics characterized previously.

Animals↗

PubMed1990

pH-dependent binding of local anesthetics in single batrachotoxin-activated Na+ channels. Cocaine vs. quaternary compounds.

The effects of internal and external pH on the binding kinetics of local anesthetics (LAs) were studied in single batrachotoxin-activated Na+ channels incorporated into planar bilayers. With internal quaternary QX-314 and RAC421-II drugs, the binding interactions were little affected by either external or internal pH. With tertiary cocaine, the binding kinetics were drastically altered by pH. A decrease in the internal pH from 9.3 to 6.2 decreased the apparent equilibrium dissociation constant (Kd) of internal cocaine by more than 100-fold. This increase in the binding affinity was mostly accounted for by an increase in the apparent cocaine on-rate constant (kon) of approximately 80-fold. The cocaine off-rate constant (koff) was little changed (between 3-4 s-1). These results demonstrate quantitatively that the charged form of cocaine is the active form for BTX-activated Na+ channels. Surprisingly, the apparent pKa of cocaine near its binding site was estimated to be 1.4 units lower than that in bulk solution (7.1 vs. 8.5), indicating that the LA drug encounters a relatively hydrophobic environment. Opposite to the internal pH effect, a decrease of external pH from 8.4 to 6.2 increased the Kd value of internally and externally applied cocaine by approximately 8- and approximately 25-fold, respectively. External pH effect was primarily mediated by modulation of kon; koff was again relatively unaffected. Our findings support a model in which neutral cocaine can readily cross the membrane barrier, but needs to be protonated internally to bind to its binding site.

Anesthetics, Local↗

PubMed1990

Binding affinity and stereoselectivity of local anesthetics in single batrachotoxin-activated Na+ channels.

Several local anesthetics (LA) have been previously shown to block muscle batrachotoxin (BTX)-activated Na+ channels in planar bilayers. The mean dwell time of different LA drugs, however, varies widely, from less than 10 ms to longer than several seconds. In this study, we have examined the structural determinants that govern the dwell time, the binding affinity, and the stereoselectivity of LA drugs using cocaine and bupivacaine homologues, RAC compounds, and their available stereoisomers. Our results from the structure-activity experiments reveal that (a) there are two apparent hydrophobic binding domains present in the LA binding site; one interacts with the aromatic moiety of the LA drugs, and the other interacts with the alkyl group attached to the tertiary amine of the LA drugs; (b) the LA mean dwell time and the binding affinity are largely determined by the hydrophobic interactions; (c) the LA binding site is highly stereoselective, with a difference in KD values over 50- and 6-fold for (+/-) cocaine and (+/-) bupivacaine, respectively; (d) the cocaine stereoselectivity is comparable among muscle, brain, and heart BTX-activated Na+ channels; and finally and most unexpectedly, (e) the stereoselectivity of LA drugs in BTX-activated Na+ channels appears greatly different from that reported in normal Na+ channels. Possible explanations for this difference are discussed.

Anesthetics, Local↗

PubMed1988

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.

Anesthetics, Local↗