Characterization of the isoform-specific differences in the gating of neuronal and muscle sodium channels

1998 ◽  
Vol 76 (10-11) ◽  
pp. 1041-1050 ◽  
Author(s):  
Michael E O'Leary
Keyword(s):  
Skeletal Muscle ◽  
Steady State ◽  
Cultured Cells ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Rate Of Recovery

Human heart (hH1), human skeletal muscle (hSkM1), and rat brain (rIIA) Na channels were expressed in cultured cells and the activation and inactivation of the whole-cell Na currents measured using the patch clamp technique. hH1 Na channels were found to activate and inactivate at more hyperpolarized voltages than hSkM1 and rIIA. The conductance versus voltage and steady state inactivation relationships have midpoints of -48 and -92 mV (hH1), -28 and -72 mV (hSkM1), and -22 and -61 mV (rIIA). At depolarized voltages, where Na channels predominately inactivate from the open state, the inactivation of hH1 is 2-fold slower than that of hSkM1 and rIIA. The recovery from fast inactivation of all three isoforms is well described by a single rapid component with time constants at -100 mV of 44 ms (hH1), 4.7 ms (hSkM1), and 7.6 ms (rIIA). After accounting for differences in voltage dependence, the kinetics of activation, inactivation, and recovery of hH1 were found to be generally slower than those of hSkM1 and rIIA. Modeling of Na channel gating at hyperpolarized voltages where the channel does not open suggests that the slow rate of recovery from inactivation of hH1 accounts for most of the differences in the steady-state inactivation of these Na channels.Key words: cardiac, neuronal, skeletal muscle, sodium channel.

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

1992 ◽  
Vol 99 (1) ◽  
pp. 1-20 ◽  
Author(s):  
G K Wang ◽  
S Y Wang
Keyword(s):  
Time Course ◽  
Cell Voltage ◽  
Gh3 Cells ◽  
Na Channel ◽  
Na Channels ◽  
H Infinity ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Steady State Inactivation ◽  
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.

scholarly journals Block of Neuronal Na+Channels by Antidepressant Duloxetine in a State-dependent Manner

2010 ◽  
Vol 113 (3) ◽  
pp. 655-665 ◽  
Author(s):  
Sho-Ya Wang ◽  
Joanna Calderon ◽  
Ging Kuo Wang
Keyword(s):  
Open Channel ◽  
Site Directed Mutagenesis ◽  
Na Channel ◽  
Dependent Manner ◽  
Channel Block ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Channel Block ◽  
Human Embryonic Kidney Cells ◽  
Na Currents

Background Duloxetine is a mixed serotonin-norepinephrine reuptake inhibitor used for major depressive disorder. Duloxetine is also beneficial for patients with diabetic peripheral neuropathy and with fibromyalgia, but how it works remains unclear. Methods We used the whole cell, patch clamp technique to test whether duloxetine interacts with the neuronal Nav1.7 Na+ channel as a potential target. Resting and inactivated Nav1.7 Na+ channel block by duloxetine were measured by conventional pulse protocols in transfected human embryonic kidney cells. The open-channel block was determined directly using inactivation-deficient mutant Nav1.7 Na+ channels. Results The 50% inhibitory concentration (IC50) of duloxetine for the resting and inactivated wild-type hNav1.7 Na+ channel were 22.1+/-0.4 and 1.79+/-0.10 microM, respectively (mean+/-SE, n=5). The IC50 for the open Na+ channel was 0.25+/-0.02 microM (n=5), as determined by the block of persistent late Nav1.7 Na+ currents. Similar open-channel block by duloxetine was found in the muscle Nav1.4 isoform (IC50=0.51+/-0.05 microM; n=5). Block by duloxetine appeared via the conserved local anesthetic receptor as determined by site-directed mutagenesis. Finally, duloxetine elicited strong use-dependent block of neuronal transient Nav1.7 Na+ currents during repetitive stimulations. Conclusions Duloxetine blocks persistent late Nav1.7 Na+ currents preferentially, which may in part account for its analgesic action.

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

1992 ◽  
Vol 100 (6) ◽  
pp. 1003-1020 ◽  
Author(s):  
G K Wang ◽  
S Y Wang
Keyword(s):  
Gh3 Cells ◽  
Na Channel ◽  
Optical Isomers ◽  
Na Channels ◽  
Na Current ◽  
Response Curves ◽  
Whole Cell Patch Clamp ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Order Of Magnitude

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)

Differential Effects of Anesthetic and Nonanesthetic Cyclobutanes on Neuronal Voltage-gated Sodium Channels

2000 ◽  
Vol 92 (2) ◽  
pp. 529-529 ◽  
Author(s):  
Lingamaneni Ratnakumari ◽  
Tatyana N. Vysotskaya ◽  
Daniel S. Duch ◽  
Hugh C. Hemmings
Keyword(s):  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Voltage Dependence ◽  
Glutamate Release ◽  
Dorsal Root Ganglion Neurons ◽  
Na Channel ◽  
Na Channels ◽  
Na Currents ◽  
Steady State Inactivation ◽  
Ganglion Neurons

Background Despite their key role in the generation and propagation of action potentials in excitable cells, voltage-gated sodium (Na+) channels have been considered to be insensitive to general anesthetics. The authors tested the sensitivity of neuronal Na+ channels to structurally similar anesthetic (1-chloro-1,2,2-trifluorocyclobutane; F3) and nonanesthetic (1,2-dichlorohexafluorocyclobutane; F6) polyhalogenated cyclobutanes by neurochemical and electrophysiologic methods. Methods Synaptosomes (pinched-off nerve terminals) from adult rat cerebral cortex were used to determine the effects of F3 and F6 on 4-aminopyridine- or veratridine-evoked (Na+ channel-dependent) glutamate release (using an enzyme-coupled spectrofluorimetric assay) and increases in intracellular Ca2+ ([Ca2+]i) (using ion-specific spectrofluorimetry). Effects of F3 and F6 on Na+ currents were evaluated directly in rat lumbar dorsal root ganglion neurons by whole-cell patch-clamp recording. Results F3 inhibited glutamate release evoked by 4-aminopyridine (inhibitory concentration of 50% [IC50] = 0.77 mM [approximately 0.8 minimum alveolar concentration (MAC)] or veratridine (IC50 = 0.42 mM [approximately 0.4 MAC]), and veratridine-evoked increases in [Ca2+]i (IC50 = 0.5 mM [approximately 0.5 MAC]) in synaptosomes; F6 had no significant effects up to 0.05 mM (approximately twice the predicted MAC). F3 caused reversible membrane potential-independent inhibition of peak Na+ currents (70+/-9% block at 0.6 mM [approximately 0.6 MAC]), and a hyperpolarizing shift in the voltage-dependence of steady state inactivation in dorsal root ganglion neurons (-21+/-9.3 mV at 0.6 mM). F6 inhibited peak Na+ currents to a lesser extent (16+/-2% block at 0.018 mM [predicted MAC]) and had minimal effects on steady state inactivation. Conclusions The anesthetic cyclobutane F3 significantly inhibited Na+ channel-mediated glutamate release and increases in [Ca2+]i. In contrast, the nonanesthetic cyclobutane F6 had no significant effects at predicted anesthetic concentrations. F3 inhibited dorsal root ganglion neuron Na+ channels with a potency and by mechanisms similar to those of conventional volatile anesthetics; F6 was less effective and did not produce voltage-dependent block. This concordance between anesthetic activity and Na+ channel inhibition supports a role for presynaptic Na+ channels as targets for general anesthetic effects and suggests that shifting the voltage-dependence of Na+ channel inactivation is an important property of volatile anesthetic compounds.

Ion channels in spinal cord astrocytes in vitro. III. Modulation of channel expression by coculture with neurons and neuron-conditioned medium

1993 ◽  
Vol 69 (3) ◽  
pp. 819-831 ◽  
Author(s):  
C. L. Thio ◽  
S. G. Waxman ◽  
H. Sontheimer
Keyword(s):  
Spinal Cord ◽  
Steady State ◽  
Conditioned Media ◽  
Na Channel ◽  
Na Channels ◽  
Drg Neurons ◽  
Channel Density ◽  
Na Currents ◽  
Channel Expression

1. Astrocytes cultured from rat spinal cord express voltage-activated Na+ channels in high densities (up to 8 channels per microns2). Stellate astrocytes express Na+ currents at all times in vitro. In pancake astrocytes, Na+ channel expression shows a distinct temporal pattern, an absence of channel expression at 1–3 days in vitro (DIV), and peak Na+ channel density at 7–8 DIV. 2. Coculture of spinal cord astrocytes with dorsal root ganglion (DRG) neurons substantially reduces the expression of voltage-activated Na+ channels in both spinal cord astrocyte types. In pancake spinal cord astrocytes, both the percentage of cells expressing Na+ channels and the channel density in Na+ channel-expressing cells are markedly reduced. In stellate spinal cord astrocytes, the percentage of Na+ channel-expressing cells is unchanged, but the Na+ channel density per cell is markedly reduced in coculture. 3. Culturing spinal cord astrocytes in neuron-conditioned media reduces Na+ channel expression in both spinal cord astrocyte types to levels intermediate between coculture and control, suggesting that, at least in part, neuronal effects on Na+ channel expression are mediated by a soluble factor secreted into the media by neurons. 4. As with the expression of voltage-activated Na+ channels, the expression of voltage-activated K+ channels is reduced in both spinal cord astrocyte types cocultured with DRG neurons. The effect is not mimicked by culturing cells in neuron-conditioned media, suggesting that effects on K+ channel expression are mediated by a less stable and more readily degradable factor. 5. Coculture with DRG neurons or culture in neuron-conditioned media do not alter the biophysical properties of voltage-activated Na+ currents in pancake spinal cord astrocytes. Thus steady-state activation, steady-state inactivation, and the time constants of activation and inactivation are virtually unchanged under the various culture conditions.

scholarly journals Properties of single cardiac Na channels at 35 degrees C.

1994 ◽  
Vol 104 (5) ◽  
pp. 801-820 ◽  
Author(s):  
K Benndorf
Keyword(s):  
Single Channel ◽  
Ventricular Myocytes ◽  
Band Width ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Probability ◽  
Total Noise ◽  
Na Currents ◽  
Transition Points

Single Na channel currents were recorded in cell-attached patches of mouse ventricular myocytes with an improved patch clamp technique. Using patch pipettes with a pore diameter in the range of 200 nm, seals with a resistance of up to 4 T omega were obtained. Under those conditions, total noise could be reduced to levels as low as 0.590 pA rms at 20 kHz band width. At this band width, properties of single-channel Na currents were studied at 35 degrees C. Six out of a total of 23 patches with teraohm seals contained channel activity and five of these patches contained one and only one active channel. Amplitude histograms excluding transition points showed heterogenous distributions of levels. In one patch, part of the openings was approximately Gaussian distributed at different potentials yielding a slope conductance of 27 pS. The respective peak open probability at -10 mV was 0.26. The mean open time was determined at voltages between -60 and -10 mV by evaluation of the distribution of the event-related gaps in the center of the baseline noise to be approximately 40 microseconds at -60 mV and 50-74 microseconds between -50 and -10 mV. It is concluded that single cardiac Na channels open at 35 degrees C frequently with multiple levels and with open times in the range of several tens of microseconds.

scholarly journals Two modes of gating during late Na+ channel currents in frog sartorius muscle.

1986 ◽  
Vol 87 (2) ◽  
pp. 305-326 ◽  
Author(s):  
J B Patlak ◽  
M Ortiz
Keyword(s):  
Reversal Potential ◽  
Current Amplitude ◽  
Na Channel ◽  
Sartorius Muscle ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Na Currents ◽  
Inward Currents ◽  
Channel Currents

Na+ currents were measured during 0.4-s depolarizing pulses using the cell-attached variation of the patch-clamp technique. Patches on Cs-dialyzed segments of sartorius muscle of Rana pipiens contained an estimated 25-500 Na+ channels. Three distinct types of current were observed after the pulse onset: a large initial surge of inward current that decayed within 10 ms (early currents), a steady "drizzle" of isolated, brief, inward unitary currents (background currents), and occasional "cloudbursts" of tens to hundreds of sequential unitary inward currents (bursts). Average late currents (background plus bursts) were 0.12% of peak early current amplitude at -20 mV. 85% of the late currents were carried by bursting channels. The unit current amplitude was the same for all three types of current, with a conductance of 10.5 pS and a reversal potential of +74 mV. The magnitudes of the three current components were correlated from patch to patch, and all were eliminated by slow inactivation. We conclude that all three components were due to Na+ channel activity. The mean open time of the background currents was approximately 0.25 ms, and the channels averaged 1.2 openings for each event. Neither the open time nor the number of openings of background currents was strongly sensitive to membrane potential. We estimated that background openings occurred at a rate of 0.25 Hz for each channel. Bursts occurred once each 2,000 pulses for each channel (assuming identical channels). The open time during bursts increased with depolarization to 1-2 ms at -20 mV, whereas the closed time decreased to less than 20 ms. The fractional open time during bursts was fitted with m infinity 3 using standard Na+ channel models. We conclude that background currents are caused by a return of normal Na+ channels from inactivation, while bursts are instances where the channel's inactivation gate spontaneously loses its function for prolonged periods.

Meperidine and Lidocaine Block of Recombinant Voltage-Dependent Na+Channels 

1999 ◽  
Vol 91 (5) ◽  
pp. 1481-1481 ◽  
Author(s):  
Larry E. Wagner ◽  
Michael Eaton ◽  
Salas S. Sabnis ◽  
Kevin J. Gingrich
Keyword(s):  
Steady State ◽  
Local Anesthesia ◽  
Local Anesthetic ◽  
Voltage Dependence ◽  
Tonic Inhibition ◽  
Na Channel ◽  
Mutant Form ◽  
Na Channels ◽  
Voltage Dependent ◽  
Steady State Inactivation

Background The opioid meperidine induces spinal anesthesia and blocks nerve action potentials, suggesting it is a local anesthetic. However, whether it produces effective clinical local anesthesia in peripheral nerves remains unclear. Classification as a local anesthetic requires clinical local anesthesia but also blockade of voltage-dependent Na+ channels with characteristic features (tonic and phasic blockade and a negative shift in the voltage-dependence of steady-state inactivation) involving an intrapore receptor. The authors tested for these molecular pharmacologic features to explore whether meperidine is a local anesthetic. Methods The authors studied rat skeletal muscle mu1 (RSkM1) voltage-dependent Na+ channels or a mutant form heterologously coexpressed with rat brain Na+ channel accessory beta1, subunit in Xenopus oocytes. Polymerase chain reaction was used for mutagenesis, and mutations were confirmed by sequencing. Na+ currents were measured using a two-microelectrode voltage clamp. Meperidine and the commonly used local anesthetic lidocaine were applied to oocytes in saline solution at room temperature. Results Meperidine and lidocaine produced tonic current inhibition with comparable concentration dependence. Meperidine caused phasic current inhibition in which the concentration-response relationship was shifted to fivefold greater concentration relative to lidocaine. Meperidine and lidocaine negatively shifted the voltage dependence of steady-state inactivation. Mutation of a putative local anesthetic receptor reduced phasic inhibition by meperidine and lidocaine and tonic inhibition by lidocaine, but not meperidine tonic inhibition. Conclusions Meperidine blocks Na+ channels with molecular pharmacologic features of a local anesthetic. The findings support classification of meperidine as a local anesthetic but with less overall potency than lidocaine.

scholarly journals Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes.

1995 ◽  
Vol 106 (6) ◽  
pp. 1171-1191 ◽  
Author(s):  
H B Nuss ◽  
N Chiamvimonvat ◽  
M T Pérez-García ◽  
G F Tomaselli ◽  
E Marbán
Keyword(s):  
Skeletal Muscle ◽  
Xenopus Oocytes ◽  
Normal Function ◽  
Alpha Subunit ◽  
Na Channels ◽  
Rat Skeletal Muscle ◽  
Time Constants ◽  
Alpha Subunits ◽  
Na Currents ◽  
Steady State Inactivation

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1-deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use-dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.

scholarly journals Inactivation defects caused by myotonia-associated mutations in the sodium channel III-IV linker.

1996 ◽  
Vol 107 (5) ◽  
pp. 559-576 ◽  
Author(s):  
L J Hayward ◽  
R H Brown ◽  
S C Cannon
Keyword(s):  
Periodic Paralysis ◽  
Na Channel ◽  
Functional Difference ◽  
Missense Mutations ◽  
Na Current ◽  
Recovery From Inactivation ◽  
Na Currents ◽  
Well Model ◽  
Steady State Inactivation ◽  
Order Of Magnitude

Missense mutations in the skeletal muscle Na+ channel alpha subunit occur in several heritable forms of myotonia and periodic paralysis. Distinct phenotypes arise from mutations at two sites within the III-IV cytoplasmic loop: myotonia without weakness due to substitutions at glycine 1306, and myotonia plus weakness caused by a mutation at threonine 1313. Heterologous expression in HEK cells showed that substitutions at either site disrupted inactivation, as reflected by slower inactivation rates, shifts in steady-state inactivation, and larger persistent Na+ currents. For T1313M, however, the changes were an order of magnitude larger than any of three substitutions at G1306, and recovery from inactivation was hastened as well. Model simulations demonstrate that these functional difference have distinct phenotypic consequences. In particular, a large persistent Na+ current predisposes to paralysis due to depolarization-induced block of action potential generation.

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