Biomedical subjects

S N Wright

Publications and source records attributed to S N Wright.

10 recordsLinked to original sources

PubMed2001

Irreversible block of human heart (hH1) sodium channels by the plant alkaloid lappaconitine.

The roots from Aconitum sp. plants have long been used in Chinese herbal medicine for treating pain and various heart conditions. The principal component of Aconitum remedies is usually aconitine, a site 2 neurotoxin that may induce severe neurological symptoms and cardiovascular collapse. Some Aconitum species also contain lappaconitine, the structure of which is remarkably similar to that of aconitine. In contrast to aconitine, a sodium channel agonist, lappaconitine reportedly blocks voltage-gated sodium channels in heart tissue. The results in the present study demonstrate that lappaconitine blocks cloned human heart (hH1) sodium channels under whole-cell, voltage-clamp conditions. Lappaconitine binding has several characteristics in common with the binding of site 2 neurotoxins, such as aconitine and batrachotoxin. For example, lappaconitine binds almost exclusively to open channels, but has little affect on resting or inactivated channels. Moreover, lappaconitine binding is inhibited by bupivacaine, a tertiary amine local anesthetic. Whereas site 2 neurotoxins often irreversibly modify channel kinetics, lappaconitine irreversibly blocks the channels. Finally, channels containing lysine substitutions within the local anesthetic receptor region at residues F1760 or N1765 are resistant to block by bupivacaine or lappaconitine. Given that site 2 neurotoxins and local anesthetics have nonidentical but overlapping binding regions, these data suggest that lappaconitine irreversibly blocks hH1 channels by binding to the site 2 receptor.

Aconitine↗

PubMed2000

Coexpression with beta(1)-subunit modifies the kinetics and fatty acid block of hH1(alpha) Na(+) channels.

Voltage-gated cardiac Na(+) channels are composed of alpha- and beta(1)-subunits. In this study beta(1)-subunit was cotransfected with the alpha-subunit of the human cardiac Na(+) channel (hH1(alpha)) in human embryonic kidney (HEK293t) cells. The effects of this coexpression on the kinetics and fatty acid-induced suppression of Na(+) currents were assessed. Current density was significantly greater in HEK293t cells coexpressing alpha- and beta(1)-subunits (I(Na,alpha beta)) than in HEK293t cells expressing alpha-subunit alone (I(Na,alpha)). Compared with I(Na,alpha), the voltage-dependent inactivation and activation of I(Na,alpha beta) were significantly shifted in the depolarizing direction. In addition, coexpression with beta(1)-subunit prolonged the duration of recovery from inactivation. Eicosapentaenoic acid [EPA, C20:5(n-3)] significantly reduced I(Na,alpha beta) in a concentration-dependent manner and at 5 microM shifted the midpoint voltage of the steady-state inactivation by -22 +/- 1 mV. EPA also significantly accelerated channel transition from the resting state to the inactivated state and prolonged the recovery time from inactivation. Docosahexaenoic acid [C22:6(n-3)], alpha-linolenic acid [C18:3(n-3)], and conjugated linoleic acid [C18:2(n-6)] at 5 microM significantly inhibited both I(Na,alpha beta) and I(Na,alpha.) In contrast, saturated and monounsaturated fatty acids had no effects on I(Na,alpha beta). This finding differs from the results for I(Na,alpha), which was significantly inhibited by both saturated and unsaturated fatty acids. Our data demonstrate that functional association of beta(1)-subunit with hH1(alpha) modifies the kinetics and fatty acid block of the Na(+) channel.

Cell Line↗

PubMed1999

State-dependent cocaine block of sodium channel isoforms, chimeras, and channels coexpressed with the beta1 subunit.

Cocaine block of human cardiac (hH1) and rat skeletal (mu1) muscle sodium channels was examined under whole-cell voltage clamp in transiently transfected HEK293t cells. Low affinity block of resting mu1 and hH1 channels at -180 mV was the same, and high affinity block of inactivated channels at -70 mV was the same. Cocaine block of hH1 channels was greater than block of mu1 channels at voltages between -120 mV and -90 mV, suggesting that greater steady-state inactivation of hH1 channels in this voltage range makes them more susceptible to cocaine block. We induced shifts in the voltage dependence of steady-state inactivation at mu1 and hH1 channels by constructing mu1/hH1 channel chimeras or by coexpressing the wild-type channels with the rat brain beta1 subunit. In contrast to several previous reports, coexpression of the rat brain beta1 subunit with mu1 or hH1 produced large positive shifts in steady-state inactivation. Shifts in the voltage dependence of steady-state inactivation elicited linear shifts in steady-state cocaine block, yet these manipulations did not affect the cocaine affinity of resting or inactivated channels. These data, as well as simulations used to predict block, indicate that state-dependent cocaine block depends on both steady-state inactivation and channel activation, although inactivation appears to have the predominant role.

Animals↗

PubMed1998

Fatty acids suppress voltage-gated Na+ currents in HEK293t cells transfected with the alpha-subunit of the human cardiac Na+ channel.

Studies have shown that fish oils, containing n-3 fatty acids, have protective effects against ischemia-induced, fatal cardiac arrhythmias in animals and perhaps in humans. In this study we used the whole-cell voltage-clamp technique to assess the effects of dietary, free long-chain fatty acids on the Na+ current (INa,alpha) in human embryonic kidney (HEK293t) cells transfected with the alpha-subunit of the human cardiac Na+ channel (hH1alpha). Extracellular application of 0.01 to 30 microM eicosapentaenoic acid (EPA, C20:5n-3) significantly reduced INa,alpha with an IC50 of 0.51 +/- 0.06 microM. The EPA-induced suppression of INa,alpha was concentration- and voltage-dependent. EPA at 5 microM significantly shifted the steady-state inactivation relationship by -27.8 +/- 1.2 mV (n = 6, P < 0.0001) at the V1/2 point. In addition, EPA blocked INa,alpha with a higher "binding affinity" to hH1alpha channels in the inactivated state than in the resting state. The transition from the resting state to the inactivated state was markedly accelerated in the presence of 5 microM EPA. The time for 50% recovery from the inactivation state was significantly slower in the presence of 5 microM EPA, from 2.1 +/- 0.8 ms for control to 34.8 +/- 2.1 ms (n = 5, P < 0.001). The effects of EPA on INa,alpha were reversible. Furthermore, docosahexaenoic acid (C22:6n-3), alpha-linolenic acid (C18:3n-3), conjugated linoleic acid (C18:2n-7), and oleic acid (C18:1n-9) at 5 microM and all-trans-retinoic acid at 10 microM had similar effects on INa,alpha as EPA. Even 5 microM of stearic acid (C18:0) or palmitic acid (C16:0) also significantly inhibited INa, alpha. In contrast, 5 microM EPA ethyl ester did not alter INa,alpha (8 +/- 4%, n = 8, P > 0.05). The present data demonstrate that free fatty acids suppress INa,alpha with high "binding affinity" to hH1alpha channels in the inactivated state and prolong the duration of recovery from inactivation.

Cell Line↗

PubMed1998

Lysine point mutations in Na+ channel D4-S6 reduce inactivated channel block by local anesthetics.

Voltage-gated Na+ channels are a primary target for local anesthetics (LAs). Open or inactivated Na+ channels usually have a severalfold higher affinity for LAs than do resting channels. Hille's modulated receptor hypothesis attributed the changes in LA affinity to state-dependent alterations in the conformation of the LA receptor. We expressed wild-type and mutant rat skeletal muscle (mu1) Na+ channels in human embryonic kidney cells to investigate the state-dependent modulation of LA receptor affinity. As an alternative approach to using alanine for point mutation, we substituted lysine (a hydrophilic residue) for native residues in the putative LA receptor located in D4-S6 of the mu1 Na+ channel. Lysine mutation at Y1586 did not alter resting channel affinity for cocaine but did reduce resting affinity at F1579K and N1584K by 2- and 3-fold, respectively. Compared with mu1, resting benzocaine block did not change at F1579K, decreased at N1584K, and increased at Y1586K. These effects on resting block could largely be accounted for by either steric/charge interference or cation-pi electron interactions between particular moieties on the LA and lysine. Surprisingly, lysine substitution at these residues allowed the channels to undergo steady state fast inactivation yet reduced inactivated channel block by cocaine by up to 27-fold and reduced the benzocaine-induced leftward shift in the h(infinity) curve by up to 22 mV. Our data suggest that transitions in channel state indeed invoke conformational changes in the LA receptor and that lysine mutations in the LA receptor region alter such conformational changes during the transition to the inactivated state.

Anesthetics, Local↗

PubMed1997

Differences in steady-state inactivation between Na channel isoforms affect local anesthetic binding affinity.

Cocaine and lidocaine are local anesthetics (LAs) that block Na currents in excitable tissues. Cocaine is also a cardiotoxic agent and can induce cardiac arrhythmia and ventricular fibrillation. Lidocaine is commonly used as a postinfarction antiarrhythmic agent. These LAs exert clinically relevant effects at concentrations that do not obviously affect the normal function of either nerve or skeletal muscle. We compared the cocaine and lidocaine affinities of human cardiac (hH1) and rat skeletal (mu 1) muscle Na channels that were transiently expressed in HEK 293t cells. The affinities of resting mu 1 and hH1 channels were similar for cocaine (269 and 235 microM, respectively) and for lidocaine (491 and 440 microM, respectively). In addition, the affinities of inactivated mu 1 and hH1 channels were also similar for cocaine (12 and 10 microM, respectively) and for lidocaine (19 and 12 microM, respectively). In contrast to previous studies, our results indicate that the greater sensitivity of cardiac tissue to cocaine or lidocaine is not due to a higher affinity of the LA receptor in cardiac Na channels, but that at physiological resting potentials (-100 to -90 mV), a greater percentage of hH1 channels than mu 1 channels are in the inactivated (i.e., high-affinity) state.

Anesthetics, Local↗

PubMed1996

Presynaptic calcium currents at voltage-clamped excitor and inhibitor nerve terminals of crayfish.

1. A two-electrode voltage clamp was used to record calcium currents from the excitatory and inhibitory nerve terminals that innervate the crayfish (Procambarus spp.) opener muscle. Other voltage-dependent currents were blocked with tetrodotoxin, 3,4-diaminopyridine, 4-aminopyridine and tetraethylammonium. 2. The presynaptic calcium current at both excitatory and inhibitory synapses was blocked by cadmium and omega-agatoxin IVA but was not affected by omega-conotoxin GVIA, omega-conotoxin MVIIC or nifedipine, suggesting that the calcium currents flow through P-type calcium channels. 3. Current-voltage (I-V) relations at both excitatory and inhibitory synapses are similar, with current activation near -40 mV, peak current near -10 mV and current reversal at membrane potentials greater than +25 mV. I-V relations were scaled along the current axis by partial calcium current blockade with cobalt, suggesting that series resistance and space-clamp errors were small. 4. A subset of terminals on one muscle fibre was locally superfused with a physiological saline containing barium; the rest of the preparation was superfused with a physiological saline containing calcium channel antagonists. Under such conditions the characteristics of the I-V relation were very similar to the I-V relations recorded when the entire preparation was bathed in physiological levels of calcium, suggesting that the space clamp was adequate. 5. Calcium channel activation, as determined from tail current analyses, was similar when the entire preparation was bathed in physiological levels of calcium or if terminals on one muscle fibre were locally superfused with barium. 6. During a 30 ms depolarization, calcium currents inactivated to a greater extent in inhibitory than in excitatory terminals. The inactivation was of small magnitude (< 20%) and was eliminated by intracellular injection of the calcium chelator BAPTA, suggesting that the inactivation was calcium dependent. 7. These data show that biophysical and pharmacological properties of calcium currents at crayfish neuromuscular junctions resemble those found at stellate synapses in squid.

Animals↗

PubMed1996

Calcium currents, transmitter release and facilitation of release at voltage-clamped crayfish nerve terminals.

1. The presynaptic terminals at crayfish (Procambarus spp.) opener neuromuscular junctions were voltage clamped. Calcium currents were measured during (ICa) and following (tail ICa) presynaptic depolarizations; EPSPs or IPSPs were simultaneously recorded from the (postsynaptic) muscle fibre directly beneath the presynaptic impalement. 2. For short (< or = 6 ms) presynaptic depolarizations, most of the transmitter release occurred during the tail ICa. EPSP or IPSP amplitudes at the end of the 6 ms pulse (end EPSP or end IPSP) increased monotonically with the integral of the ICa ([symbol: see text]ICa). The suppression potential for transmitter release was near the apparent reversal potential for ICa. 3. When the end EPSP or end IPSP amplitude was plotted against the peak ICa elicited during a presynaptic pulse (peak ICa), large and small depolarizations which evoked the same peak ICa evoked different amounts of transmitter release. The differences in transmitter release were eliminated when end EPSP amplitude was plotted against [symbol: see text] ICa, suggesting that transmitter release during a depolarization depends only upon calcium current and not upon a subsequent voltage-dependent step. 4. The synaptic transfer function of various measurements of EPSP or IPSP amplitude vs. [symbol: see text]ICa evoked during a presynaptic depolarization was a power function having an exponent of about 3. Similar measurements of EPSP amplitude vs. [symbol: see text]tail ICa evoked following a presynaptic depolarization had an exponent of about 2. 5. Facilitation of an EPSP or IPSP was not due to increases in calcium current at the test depolarization. 6. When the conditioning depolarization was increased and the test depolarization remained constant, EPSP amplitude at the test depolarization and facilitation increased . When the conditioning depolarization remained constant and the test depolarization was increased, EPSP amplitude at the test depolarization increased, while facilitation decreased. 7. Our data suggested that transmitter release at crayfish neuromuscular junctions is a non-linear function of calcium influx, and that facilitated release utilizes intracellular calcium differently from non-facilitated release. These data contradict simple models of facilitation which combine the residual calcium hypothesis with the calcium co-operativity hypothesis of non-facilitated release.

Animals↗

PubMed1995

Presynaptic calcium-activated potassium channels and calcium channels at a crayfish neuromuscular junction.

1. We used a two-microelectrode current clamp to investigate various characteristics of the Ca(2+)-activated K+ conductance [gK(Ca)] and Ca2+ conductance (gCa), and transmitter release in presynaptic terminals of excitatory neuromuscular junctions in the crayfish walking leg. 2. Voltage-activated Na+ conductances (gNa) and K+ conductances [gK(v)] were blocked with tetrodotoxin and 3,4-diaminopyridine, respectively. Under these conditions, presynaptic depolarization produced by a first (conditioning) pulse admitted Ca2+ into the presynaptic terminals and activated gK(Ca), which modulated the amplitude of the depolarization produced by a second (test) pulse. The relative amount of gK(Ca) measured at the test pulse increased with increased magnitude or duration of the conditioning pulse. 3. A brief hyperpolarization immediately after a conditioning pulse substantially reduced gK(Ca). 4. gK(Ca) activation was blocked by funnel web spider toxin (a Ca2+ channel blocker) or by injection of the presynaptic terminal region with a calcium chelator, bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). Under current-clamp conditions, gK(Ca) was not blocked by charybdotoxin or iberiotoxin [specific gK(Ca) blockers]. 5. When gK(Ca) was blocked or reduced, the amplitude of the depolarizing afterpotential of action potentials was increased. When gK(v) was blocked or reduced, the duration of action potentials was increased. 6. Intracellular injection of BAPTA into the presynaptic terminal region eliminated evoked neurotransmitter release before test pulse modulation was affected, suggesting that the K(Ca) channel had a greater sensitivity (greater affinity or lower stoichiometry) for Ca2+ than did the transmitter release machinery. BAPTA reduced neurotransmitter release by 66-78%, but did not affect facilitation of neurotransmitter release. 7. When gNa, gK(v), and gK(Ca) were blocked, we detected a membrane depolarization produced by an increase in presynaptic gCa that was eliminated by 2 mM Cd2+ or 0 mM Ca2+.

Action Potentials↗

PubMed1993

Residual free calcium is not responsible for facilitation of neurotransmitter release.

An increase in internal free calcium ([Ca2+]i) in the presynaptic terminal is often assumed to directly produce facilitation of neurotransmitter release. Using a Ca(2+)-activated potassium conductance as a bioassay for free [Ca2+]i in the presynaptic terminal of the crayfish (Procambarus clarkii) opener neuromuscular junction, we now demonstrate that free [Ca2+]i has a decay time constant (tau) of 1-4 msec, whereas facilitation of neurotransmitter release has a decay tau of 7-43 msec. In addition, facilitation of neurotransmitter release can be markedly different at times when free [Ca2+]i values and presynaptic membrane voltages are equal. We conclude that free [Ca2+]i in the presynaptic terminal is not directly responsible for facilitation of neurotransmitter release. Our data suggest that facilitation results from bound Ca2+ or some long-lived consequence of bound Ca2+.

Animals↗