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ABSTRACT: We investigated the contribution of the putative inactivation lid in voltage-gated sodium channels to gating charge immobilization (i.e., the slow return of gating charge during repolarization) by studying a lid-modified mutant of the human heart sodium channel (hH1a) that had the phenylalanine at position 1485 in the isoleucine, phenylalanine, and methionine (IFM) region of the domain III-IV linker mutated to a cysteine (ICM-hH1a). Residual fast inactivation of ICM-hH1a in fused tsA201 cells was abolished by intracellular perfusion with 2.5 mM 2-(trimethylammonium)ethyl methanethiosulfonate (MTSET). The time constants of gating current relaxations in response to step depolarizations and gating charge-voltage relationships were not different between wild-type hH1a and ICM-hH1a(MTSET). The time constant of the development of charge immobilization assayed at -180 mV after depolarization to 0 mV was similar to the time constant of inactivation of I(Na) at 0 mV for hH1a. By 44 ms, 53% of the gating charge during repolarization returned slowly; i.e., became immobilized. In ICM-hH1a(MTSET), immobilization occurred with a similar time course, although only 31% of gating charge upon repolarization (OFF charge) immobilized. After modification of hH1a and ICM-hH1a(MTSET) with Anthopleurin-A toxin, a site-3 peptide toxin that inhibits movement of the domain IV-S4, charge immobilization did not occur for conditioning durations up to 44 ms. OFF charge for both hH1a and ICM-hH1a(MTSET) modified with Anthopleurin-A toxin were similar in time course and in magnitude to the fast component of OFF charge in ICM-hH1a(MTSET) in control. We conclude that movement of domain IV-S4 is the rate-limiting step during repolarization, and it contributes to charge immobilization regardless of whether the inactivation lid is bound. Taken together with previous reports, these data also suggest that S4 in domain III contributes to charge immobilization only after binding of the inactivation lid.
The Journal of General Physiology 06/2000; 115(5):609-20. · 3.84 Impact Factor
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ABSTRACT: The class IB antiarrhythmic drug, lidocaine, interacts strongly with depolarized sodium (Na) channels, an action that is thought to underlie its clinical efficacy. Previously, we have reported Na channel gating current (Ig) experiments with a quaternary form of lidocaine, QX-222, which binds preferentially to open Na channels and modifies the gating-charge/voltage (Q/V) relationship of cardiac Na channels by reducing maximal gating charge (Qmax) and lessening its voltage dependence. We report here investigations with lidocaine itself on Ig of native canine and cloned human cardiac Na channels. Although the state dependence of lidocaine binding to Na channels differs from that of quaternary drugs, Ig measurements demonstrated that lidocaine produced changes in the Q/V relationships similar to those elicited by QX-222, with a reduction in Qmax by 33% and a corresponding decrease in the slope factor. Concentration/response curves for the reduction in gating charge by lidocaine matched those for the block of sodium current (I(Na)), as would be expected if modification of Na channel voltage sensors by lidocaine underlied its action. The application of site-3 toxins, which inhibit movement of the voltage sensor associated with inactivation, to lidocaine-bound Na channels elicits an additional reduction in Qmax suggesting that lidocaine does not "stabilize" the Na channel in an inactivated state. We conclude that lidocaine blocks I(Na) by modification of the Na channel's voltage sensors predominately associated with channel activation leading to channel opening.
Pflügers Archiv - European Journal of Physiology 05/2000; 439(6):814-21. · 4.46 Impact Factor
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ABSTRACT: The class IB antiarrhythmic drug, lidocaine, interacts strongly with depolarized sodium (Na) channels, an action that is thought to underlie its clinical efficacy. Previously, we have reported Na channel gating current (Ig) experiments with a quaternary form of lidocaine, QX-222, which binds preferentially to open Na channels and modifies the gating-charge/voltage (Q/V) relationship of cardiac Na channels by reducing maximal gating charge (Qmax) and lessening its voltage dependence. We report here investigations with lidocaine itself on Ig of native canine and cloned human cardiac Na channels. Although the state dependence of lidocaine binding to Na channels differs from that of quaternary drugs, Ig measurements demonstrated that lidocaine produced changes in the Q/V relationships similar to those elicited by QX-222, with a reduction in Qmax by 33% and a corresponding decrease in the slope factor. Concentration/response curves for the reduction in gating charge by lidocaine matched those for the block of sodium current (INa), as would be expected if modification of Na channel voltage sensors by lidocaine underlied its action. The application of site-3 toxins, which inhibit movement of the voltage sensor associated with inactivation, to lidocaine-bound Na channels elicits an additional reduction in Qmax suggesting that lidocaine does not "stabilize" the Na channel in an inactivated state. We conclude that lidocaine blocks INa by modification of the Na channel's voltage sensors predominately associated with channel activation leading to channel opening.
Pflügers Archiv - European Journal of Physiology 02/2000; 439(6):814-821. · 4.46 Impact Factor
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ABSTRACT: Site-3 toxins have been shown to inhibit a component of gating charge (33% of maximum gating charge, Q(max)) in native cardiac Na channels that has been identified with the open-to-inactivated state kinetic transition. To investigate the role of the three outermost arginine amino acid residues in segment 4 domain IV (R1, R2, R3) in gating charge inhibited by site-3 toxins, we recorded ionic and gating currents from human heart Na channels with mutations of the outermost arginines (R1C, R1Q, R2C, and R3C) expressed in fused, mammalian tsA201 cells. All four mutations had ionic currents that activated over the same voltage range with slope factors of their peak conductance-voltage (G-V) relationships similar to those of wild-type channels, although decay of I(Na) was slowest for R1C and R1Q mutant channels and fastest for R3C mutant channels. After Na channel modification by Ap-A toxin, decays of I(Na) were slowed to similar values for all four channel mutants. Toxin modification produced a graded effect on gating charge (Q) of mutant channels, reducing Q(max) by 12% for the R1C and R1Q mutants, by 22% for the R2C mutant, and by 27% for the R3C mutant, only slightly less than the 31% reduction seen for wild-type currents. Consistent with these findings, the relationship of Q(max) to G(max) was significantly shallower for R1 mutants than for R2C and R3C mutant Na channels. These data suggest that site-3 toxins primarily inhibit gating charge associated with movement of the S4 in domain IV, and that the outermost arginine contributes the largest amount to channel gating, with other arginines contributing less.
Biophysical Journal 09/1999; 77(2):747-57. · 3.65 Impact Factor
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ABSTRACT: 1. Sodium channel ionic current (INa) and gating current (Ig) were compared for rat skeletal (rSkM1) and human heart Na+ channels (hH1a) heterologously expressed in cultured mammalian cells at approximately 13 C before and after modification by site-3 toxins (Anthopleurin A and Anthopleurin B). 2. For hH1a Na+ channels there was a concordance between the half-points (V ) of the peak conductance-voltage (G-V) relationship and the gating charge-voltage (Q-V) relationship with no significant difference in half-points. In contrast, the half-point of the Q-V relationship for rSkM1 Na+ channels was shifted to more negative potentials compared with its G-V relationship with a significant difference in the half-points of -8 mV. 3. Site-3 toxins slowed the decay of INa in response to step depolarizations for both rSkM1 and hH1a Na+ channels. The half-point of the G-V relationship in rSkM1 Na+ channels was shifted by -8.0 mV while toxin modification of hH1a Na+ channels produced a smaller hyperpolarizing shift of the V by -3.7 mV. 4. Site-3 toxins reduced maximal gating charge (Qmax ) by 33% in rSkM1 and by 31% in hH1a, but produced only minor changes in the half-points and slope factors of their Q-V relationships. In contrast to measurements in control solutions, after modification by site-3 toxin the half-points of the G-V and the Q-V relationships for rSkM1 Na+ channels demonstrated a concordance similar to that for hH1a. 5. Qmax vs. Gmax for rSkM1 and hH1a Na+ channels exhibited linear relationships with almost identical slopes, as would be expected if the number of electronic charges (e-) per channel was comparable. 6. We conclude that the faster kinetics in rSkM1 channels compared with hH1a channels may arise from inherently faster rate transitions in skeletal muscle Na+ channels, and not from major differences in the voltage dependence of the channel transitions.
The Journal of Physiology 02/1999; 514 ( Pt 2):425-36. · 4.72 Impact Factor
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ABSTRACT: We describe a new mammalian expression system that optimizes conditions for the measurement of Na channel gating currents (Ig). The small magnitude of Ig limits their study to preparations with high numbers of Na channels to improve signal-to-noise ratios. To increase Na channel Ig signals, single tsA201 cells (approximately 20 microns in diameter) were fused into large, multinucleated cells by treatment with polyethylene glycol. After being placed in cell culture for 48-72 h, fused tsA201 cells develop a spherical geometry with diameters up to 200 microns. Because of the large plasma membrane surface area, fused tsA201 cells are able to express high levels of Na channels after transient transfection with Na channel cDNAs using Lipofectamine. Typically, 5 days after transfection, fused tsA201 cells that are 60-100 microns in diameter are selected for voltage clamp with a large suction pipette (a pore size of 20-30 microns) that allows for both a low series resistance and internal perfusion. Approximately two-thirds of transfected fused tsA201 cells express Na current, with nearly one-third of transfected cells expressing sufficient numbers of Na channels to allow for the ready measurement of Ig. In addition to fused tsA201 cells being a preparation well suited for the study of Ig, they should also be useful for measurement of electrical signals from other voltage-gated channels and transporters that generate small electrical signals.
The American journal of physiology 10/1996; 271(3 Pt 1):C1001-6.
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ABSTRACT: The site 3 toxin, Anthopleurin-A (Ap-A), was used to modify inactivation of sodium channels in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Although Ap-A toxin markedly prolonged decay of sodium current (INa) in response to step depolarizations, there was only a minor hyperpolarizing shift by 2.5 +/- 1.7 mV (n = 13) of the half-point of the peak conductance-voltage relationship with a slight steepening of the relationship from -8.2 +/- 0.8 mV to -7.2 +/- 0.8 mV (n = 13). Increases in Gmax were dependent on the choice of cation used as a Na substitute intracellularly and ranged between 26 +/- 15% (Cs, n = 5) to 77 +/- 19% (TMA, n = 8). Associated with Ap-A toxin modification time to peak INa occurred later, but analysis of the time course INa at multiple potentials showed that the largest effects were on inactivation with only a small effect on activation. Consistent with little change in Na channel activation by Ap-A toxin, INa tail current relaxations at very negative potentials, where the dominant process of current relaxation is deactivation, were similar in control and after toxin modification. The time course of the development of inactivation after Ap-A toxin modification was dramatically prolonged at positive potentials where Na channels open. However, it was not prolonged after Ap-A toxin at negative potentials, where channels predominately inactivate directly from closed states. Steady state voltage-dependent availability (h infinity or steady state inactivation), which predominately reflects the voltage dependence of closed-closed transitions equilibrating with closed-inactivated transitions was shifted in the depolarizing direction by only 1.9 +/- 0.8 mV (n = 8) after toxin modification. The slope factor changed from 7.2 +/- 0.8 to 9.9 +/- 0.9 mV (n = 8), consistent with a prolongation of inactivation from the open state of Ap-A toxin modified channels at more depolarized potentials. We conclude that Ap-A selectively modifies Na channel inactivation from the open state with little effect on channel activation or on inactivation from closed state(s).
The Journal of General Physiology 11/1995; 106(4):601-16. · 3.84 Impact Factor
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ABSTRACT: The gating charge and voltage dependence of the open state to the inactivated state (O-->I) transition was measured for the voltage-dependent mammalian cardiac Na channel. Using the site 3 toxin, Anthopleurin-A (Ap-A), which selectively modifies the O-->I transition (see Hanck, D. A., and M. F. Sheets. 1995. Journal of General Physiology. 106:601-616), we studied Na channel gating currents (Ig) in voltage-clamped single canine cardiac Purkinje cells at approximately 12 degrees C. Comparison of Ig recorded in response to step depolarizations before and after modification by Ap-A toxin showed that toxin-modified gating currents decayed faster and had decreased initial amplitudes. The predominate change in the charge-voltage (Q-V) relationship was a reduction in gating charge at positive potentials such that Qmax was reduced by 33%, and the difference between charge measured in Ap-A toxin and in control represented the gating charge associated with Na channels undergoing inactivation by O-->I. By comparing the time course of channel activation (represented by the gating charge measured in Ap-A toxin) and gating charge associated with the O-->I transition (difference between control and Ap-A charge), the influence of activation on the time course of inactivation could be accounted for and the inherent voltage dependence of the O-->I transition determined. The O-->I transition for cardiac Na channels had a valence of 0.75 e-. The total charge of the cardiac voltage-gated Na channel was estimated to be 5 e-. Because charge is concentrated near the opening transition for this isoform of the channel, the time constant of the O-->I transition at 0 mV could also be estimated (0.53 ms, approximately 12 degrees C). Prediction of the mean channel open time-voltage relationship based upon the magnitude and valence of the O-->C and O-->I rate constants from INa and Ig data matched data previously reported from single Na channel studies in heart at the same temperature.
The Journal of General Physiology 10/1995; 106(4):617-40. · 3.84 Impact Factor
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ABSTRACT: To determine the response of cardiac Na current (INa) in adult cardiac ventricular myocytes to culture, single isolated ventricular myocytes from collagenase-perfused adult cat hearts were placed in primary culture for up to 2 wk on a two-dimensional (2D) surface (laminin-coated coverslips), which allowed the morphology of the myocytes to change markedly, or in a three-dimensional matrix (3D) of alginate, in which cell shape changed only minimally. Action potentials and INa were recorded from groups of 1) freshly isolated myocytes serving as the control (day 0),2) cells maintained in 2D culture for 9-14 days (2D, day 9-14), and 3) cells cultured in alginate for 9-14 days (3D, day 9-14) with use of a conventional whole cell patch technique. Maximal upstroke velocity (Vmax) of the action potential was reduced by approximately 50% in 2D- and 3D-cultured cells relative to controls. INa in 2D- and 3D-cultured cells was strikingly different from that in control myocytes. Half-maximal voltage (V 1/2) for the chord conductance-voltage relationship was shifted approximately 15 mV negatively to that for controls in 2D- and 3D-cultured cells. INa steady-state availability curve also shifted negatively relative to controls in 2D- and 3D-cultured myocytes, but the magnitude of this shift (approximately 16-20 mV) was greater than that for the chord conductance-voltage curve.(ABSTRACT TRUNCATED AT 250 WORDS)
The American journal of physiology 05/1995; 268(4 Pt 1):C993-1001.
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ABSTRACT: The interaction of antiarrhythmic drugs with ion channels is often described within the context of the modulated receptor hypothesis, which explains the action of drugs by proposing that the binding site has a variable affinity for drugs, depending upon whether the channel is closed, open, or inactivated. Lack of direct evidence for altered gating of cardiac Na channels allowed for the suggestion of an alternative model for drug interaction with cardiac channels, which postulated a fixed affinity receptor with access limited by the conformation of the channel (guarded receptor hypothesis). We report measurement of the gating currents of Na channels in canine cardiac Purkinje cells in the absence and presence of QX-222, a quaternary derivative of lidocaine, applied intracellularly, and benzocaine, a neutral local anesthetic. These data demonstrate that the cardiac Na channel behaves as a modulated rather than a guarded receptor in that drug-bound channels gate with altered kinetics. In addition, the results suggest a new interpretation of the modulated receptor hypothesis whereby drug occupancy reduces the overall voltage-dependence of gating, preventing full movement of the voltage sensor.
The Journal of General Physiology 02/1994; 103(1):19-43. · 3.84 Impact Factor
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ABSTRACT: Studies of tetrodotoxin-sensitive sodium current (INa) after modification of inactivation by intracellular enzymes in mammalian cells have demonstrated a marked increase in peak INa at test potentials near current threshold causing a large, negative shift of the peak INa conductance-voltage relationship by approximately -20 mV. These findings support a kinetic model in which the unmodified Na channel has rapid and voltage-independent inactivation from the open state. However, the kinetics of cardiac Na channels differ from those of mammalian neuronal Na channels. In particular, inactivation of cardiac Na channels has been proposed to be more voltage dependent than that of tetrodotoxin-sensitive Na channels. To help understand the role of inactivation in cardiac Na channel kinetic behavior, we studied Na currents before and after modification of inactivation by the proteolytic enzyme, alpha-chymotrypsin.
Whole cell INa was measured in single canine cardiac Purkinje cells that were voltage clamped and internally perfused with a large-bore suction pipette. The decay of INa in response to step depolarizations was dramatically slowed after perfusion with intracellular alpha-chymotrypsin consistent with modification of inactivation. In contrast to mammalian tetrodotoxin-sensitive Na current, Boltzmann distribution fits to peak INa conductance-voltage (GNa-V) relationships after alpha-chymotrypsin showed no change in either the potential at half maximum conductance (V 1/2), after correction for the spontaneous background shift of INa kinetics, or in the voltage-dependence of conductance (i.e., slope factor of GNa-V relationships). Maximal peak INa conductance increased by 18%. INa tail-current relaxations at potentials < or = -110 mV, after correction for spontaneous shifts in Na channel kinetics, were also similar before and after modification by alpha-chymotrypsin.
alpha-chymotrypsin modified inactivation of cardiac INa with little or no change in activation, and cardiac Na channel inactivation was slow near threshold and played little role in determining V1/2 for peak INa conductance-voltage relationships.
Journal of Cardiovascular Electrophysiology 12/1993; 4(6):686-94. · 3.06 Impact Factor
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ABSTRACT: 1. The effects of the extracellular divalent cations barium, calcium, cadmium, cobalt, magnesium, manganese, nickel and zinc and the trivalent cation lanthanum on macroscopic sodium current (INa) were characterized in enzymatically isolated single canine cardiac Purkinje cells under voltage clamp at 9-14 degrees C. 2. All di(tri)valent cations produced depolarizing shifts in the conductance-voltage relationship. The order of efficacy, taken as the concentration required to produce a 5 mV shift in the mid-point of peak INa conductance, from least to most effective was (mM): Ca2+ (2.97) approximately Mg2+ (2.67) approximately Ba2+ (1.93) > CO2+ (1.02) approximately Mn2+ (0.88) > Ni2+ (0.54) > La3+ (0.095) approximately Cd2+ (0.083) approximately Zn2+ (0.076). 3. Addition of di(tri)valent cations also produced depolarizing shifts in voltage-dependent availability. The order of efficacy from the least to most effective was (mM): Cd2+ (7.70) approximately Mg2+ (6.86) approximately Ba2+ (4.50) > Ca2+ (2.47) approximately CO2+ (1.87) approximately Mn2+ (1.24) approximately Ni2+ (1.20) > Zn2+ (0.300) > La3+ (0.060). 4. The Gouy-Chapman-Stern equations were used to evaluate di(tri)valent cation efficacy in binding to surface charges. Surface charge density was estimated as 0.72 sites nm-2, and it was assumed that Mg2+, the divalent cation that produced the smallest shift, screened but did not bind to surface charges. Based on voltage-dependent availability, KD from lowest to highest affinity were (mM): Ba2+ (2500) > CO2+ (1670) approximately Mn2+ (1430) approximately Ca2+ = Cd2+ = Ni2+ (1200) > Zn2+ (250) > La3+ (30). 5. All di(tri)valent cations also produced a concentration-dependent acceleration of INa tail current relaxation. The addition of Ca2+ and La3+ produced acceleration of tail current relaxations that could be accounted for by the surface charge effects predicted from the shift in voltage-dependent availability. Cd2+, which produced almost no change in voltage-dependent availability, dramatically accelerated tail current relaxation. Zn2+, Ni2+, Mn2+ and CO2+ also produced greater acceleration of tail current relaxation than could be accounted for by surface charge effects. 6. Di(tri)valent cations delayed time to peak INa in a concentration-dependent manner. The time to peak INa-voltage relationship was well described by an exponential plus a constant, and di(tri)valent cations did not affect the slope factor or constant but shifted the relationship in the depolarizing direction. Similar to their effect on tail currents, addition of some di(tri)valent cations produced larger effects on time to peak INa than expected from the shift of voltage-dependent availability.(ABSTRACT TRUNCATED AT 400 WORDS)
The Journal of Physiology 09/1992; 454:267-98. · 4.72 Impact Factor
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ABSTRACT: 1. Single canine cardiac Purkinje cells were internally perfused and voltage clamped with a large-bore perfusion pipette for measurement of sodium ionic current (INa) in the absence and presence of extracellular group IIA divalent cations (Mg2+, Ba2+ and Ca2+), transition divalent cations (CO2+, Mn2+ and Ni2+), group IIB divalent cations (Cd2+ and Zn2+), and the trivalent cation La3+. 2. Open channel block of cardiac INa by external Ca2+, assessed from instantaneous INa-voltage (I-V) relationships, has been well described by a two-barrier, one-well model with a dissociation constant at 0 mV, KB(0), of 37 mM and an electrical distance, z' = delta, of 0.34. At the most negative test potentials there was less block of INa than predicted by the model, but correction of INa for the contribution of Na+ channel gating current (Ig) to the peak current improved the fit by the model. 3. The divalent cations Ba2+, Mg2+, CO2+ and Mn2+ produced voltage-dependent, open channel block of INa, which by the two-barrier, one-well model predicted a similar z' about one-third into the membrane field. The relative efficacy for voltage-dependent block was CO2+ > Mn2+ > Ca2+ > Mg2+ > Ba2+ with respective KB(0)s of 11, 13, 37, 43 and 61 mM. 4. Cd2+, Zn2+ and La3+ produced block of INa at low concentrations that was nearly voltage independent with z' < or = 0.13. Fits of single-site binding curves to peak INa in response to step depolarizations at positive test potentials gave the following apparent KD values: Zn2+ 0.14 mM, Cd2+ 0.27 mM and La3+ 0.50 mM. 5. In the presence of Cd2+, INa tail current relaxations were much faster than could be accounted for by Cd2+ binding to and/or screening of extracellular surface charges. Fits of the data to a model that assumed voltage-dependent open channel block during the tail current relaxations estimated the KB(0) for Cd2+ to be 0.80 mM. 6. Both z' and KB(0) for Ni2+ from fits of the two-barrier, one-well model to instantaneous I-V relationships varied as a function of [Ni2+], consistent with the hypothesis that Ni2+ blocked with similar affinity at a voltage-dependent and a voltage-independent site. At [Ni2+] > or = 5 mM, KB(0) was 7.6 mM and z' was 0.21.(ABSTRACT TRUNCATED AT 400 WORDS)
The Journal of Physiology 09/1992; 454:299-320. · 4.72 Impact Factor
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ABSTRACT: The spontaneous hyperpolarizing shift in Na+ channel kinetics that occurs during a series of voltage-clamp recordings was characterized in single canine cardiac Purkinje cells at 10-13.5 degrees C. The change in the half-point of voltage-dependent availability, in the half-point of peak conductance, in the voltage dependence of deactivation and time to peak Na+ channel current (INa), and in the time constants of INa decay in response to step depolarizations were examined. The half points of availability and conductance shifted similarly, -0.41 +/- 0.13 and -0.47 +/- 0.19 mV/min, respectively (n = 14). These were directly correlated (slope 1.14 +/- 0.06, R2 = 0.81) with conductance shifting on average only -0.05 mV/min faster than availability. The deactivation time constant-voltage relationship shifted similarly to availability and conductance. Tail current decay time constants predicted the voltage dependence of the open to closed transition to be 0.9e-. Time to peak INa in response to step depolarizations changed e-fold for 25 mV but plateaued at positive potentials (531 microseconds, n = 22). INa decay was multiexponential between -40 and 80 mV. Decay time constants changed little as a function of voltage at positive potentials. The contribution of the second time constant to decay amplitude was 15-20% over the entire voltage range. Time to peak INa shifted in a curvilinear fashion, changing less late in an experiment. We conclude that the channel-voltage sensor responds to a changing fraction of the applied voltage during an experiment, producing similar rates of shift of voltage-dependent availability, conductance, and deactivation time constants.
The American journal of physiology 05/1992; 262(4 Pt 2):H1197-207.
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Science 05/1991; 252(5004):449-52. · 31.20 Impact Factor
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ABSTRACT: Gating currents (Ig) were recorded in single canine cardiac Purkinje cells at 10-12 degrees C. Ig characteristics corresponded closely to macroscopic INa characteristics and appeared to exhibit little contamination from other voltage-gated channels. Charge density predicted by peak INa was 0.14-0.22 fC micron -2 and this compared well with the measured value of 0.19 +/- 0.10 fC micron -2 (SD; n = 28). The charge-voltage relationship rose over a voltage similar to the peak INa conductance curve. The midpoints of the two relationships were not significantly different although the conductance curve was 1.5 +/- 0.3 (SD; n = 9) times steeper. Consistent with this observation, which predicted that a large amount of the gating charge would be associated with transitions close to the open state, an analysis of activation from Hodgkin-Huxley fits to the macroscopic currents showed that tau m corresponded well with a prominent component of Ig. Ig relaxations fitted two exponentials better than one over the range of voltages in which Na channels were activated. When the holding potential was hyperpolarized, relaxation of Ig during step depolarizations to 0 mV was prolonged but there was no substantial increase in charge, further suggesting that early closed-state transitions are less in charge, further suggesting that early closed-state transitions are less voltage dependent. The single cardiac Purkinje cell appears to be a good candidate for combining Ig and single-channel measurements to obtain a kinetic description of the cardiac Na channel.
The Journal of General Physiology 04/1990; 95(3):439-57. · 3.84 Impact Factor
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ABSTRACT: We studied the relation of the maximal upstroke velocity (Vmax) of action potentials to the peak sodium current (INa) under voltage clamp in single, internally perfused, canine cardiac Purkinje cells under conditions that ensured membrane action potentials due only to INa. Three different methods of altering sodium channel availability were investigated: voltage-dependent inactivation, tetrodotoxin (TTX) block, and use-dependent block by quinidine. Under all three conditions, the relation of Vmax to INa was nonlinear, and no relation was found that would allow prediction of INa results from Vmax measurements. With voltage-dependent inactivation or TTX block, sodium channel availability measured by Vmax was reduced less than availability measured by peak INa, so that Vmax overestimated sodium channel availability. This overestimation of sodium channel availability by Vmax could be attributed to greater sodium channel mobilization during the slowed action potential upstrokes. The overestimation varied with experimental temperature as a consequence of changes in sodium channel kinetics. Vmax also overestimated sodium channel availability during TTX exposure so that the Kd for TTX block was 4.5 micron from Vmax measurements but only 1.6 microM from INa measurements. Use-dependent block of INa by quinidine had a striking voltage-dependent component under voltage clamp that could not be appreciated from action potentials. Consequently, block could be underestimated or overestimated by Vmax measurements. We conclude that Vmax measurements represent a convenient index for INa, but Vmax is not a reliable method for quantitative studies of sodium channel behavior.
Circulation Research 09/1988; 63(2):386-98. · 9.49 Impact Factor
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ABSTRACT: Block of sodium current (INa) by ethmozin (moricizine), an antiarrhythmic drug, was investigated in isolated, voltage-clamped, canine cardiac Purkinje cells. Initial block of INa by ethmozin (2 microM) in noninactivated cells (held at -150 mV) was 9.3 +/- 1.2% (S.D.). Additional "use-dependent" block developed in response to repetitive depolarization. This block was both frequency-dependent and dose-dependent with the fall in peak INa greater at increasing depolarization frequencies (0.625 to 4 Hz) and with increasing dose (2 microM to 20 microM). Use-dependent block was modeled according to the guarded receptor hypothesis assuming ingress to the channel binding site during the open state of the channel, and egress from the channel independent of the kinetic state of the channel. The rate constants (on-rate = 2100 +/- 100 (S.D.)/M/ms and off-rate = 1.7 +/- 0.3 (S.D.) 10(-5)/ms) were used to predict the time course of INa block in response to repeated depolarizations and the dose-response relationship of steady-state used-dependent block measured in independent experiments. We conclude that ethmozin blocks INa in Purkinje cells in both a non-use-dependent and a use-dependent manner and that the guarded receptor model is useful in describing the use-dependent block.
Journal of Molecular and Cellular Cardiology 04/1988; 20(3):255-65. · 5.17 Impact Factor
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ABSTRACT: Block of sodium current (INa) by ethmozin (moricizine), an antiarrhythmic drug, was investigated in isolated, voltage-clamped, canine cardiac Purkinje cells. Initial block of INa by ethmozin (2μm) in non-inactivated cells (held at −150 mV) was 9.3±1.2% (s.d.). Additional “use-dependent” block developed in response to repetitive depolarizations. This block was both frequency-dependent and dose-dependent with the fall in peak INa greater at increasing depolarization frequencies (0.625 to 4 Hz) and with increasing dose (2 μm to 20 μm). Use-dependent block was modeled according to the guarded receptor hypothesis assuming ingress to the channel binding site during the open state of the channel, and egress from the channel independent of the kinetic state of the channel. The rate constants (on-rate=2100±100 (s.d.)/m/ms and off-rate=1.7±0.3 (s.d.) 10−5/ms) were used to predict the time course of INa block in response to repeated depolarizations and the dose-response relationship of steady-state used-dependent block measured in independent experiments. We conclude that ethmozin blocks INa in Purkinje cells in both a non-use-dependent and a use-dependent manner and that the guarded receptor model is useful in describing the use-dependent block.
Journal of Molecular and Cellular Cardiology 01/1988; · 5.17 Impact Factor
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ABSTRACT: Sodium (Na+) currents are responsible for excitation and conduction in most cardiac cells, but their study has been hampered by the lack of a satisfactory method for voltage clamp. We report a new method for low resistance access to single freshly isolated canine cardiac Purkinje cells that permits good control of voltage and intracellular ionic solutions. The series resistance was usually less than 3 omega cm2, similar to that of the squid giant axon. Cardiac Na+ currents resemble those of nerve. However, Na+ current decay is multiexponential. The basis for this was further studied with cell-attached patch clamp recording of single Na+ channel properties. A prominent characteristic of the single channels was their ability to reopen after closure. There was also a long opening state that may be the basis for a small very slowly decaying Na+ current. This rare long opening state may contribute to the Na+ current during the action potential plateau.
Experientia 01/1988; 43(11-12):1162-8.
