scholarly journals Voltage-dependent open-state inactivation of cardiac sodium channels: gating current studies with Anthopleurin-A toxin.

1995 ◽  
Vol 106 (4) ◽  
pp. 617-640 ◽  
Author(s):  
M F Sheets ◽  
D A Hanck
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Na Channel ◽  
Total Charge ◽  
Na Channels ◽  
Gating Currents ◽  
Gating Charge ◽  
Cardiac Purkinje Cells ◽  
Voltage Dependent ◽  
Cardiac Sodium Channels

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.

scholarly journals Kinetic effects of quaternary lidocaine block of cardiac sodium channels: a gating current study.

1994 ◽  
Vol 103 (1) ◽  
pp. 19-43 ◽  
Author(s):  
D A Hanck ◽  
J C Makielski ◽  
M F Sheets
Keyword(s):  
Antiarrhythmic Drugs ◽  
Na Channel ◽  
Na Channels ◽  
Gating Currents ◽  
Cardiac Purkinje Cells ◽  
Affinity Receptor ◽  
New Interpretation ◽  
Kinetic Effects ◽  
Cardiac Sodium Channels ◽  
Modulated Receptor

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.

scholarly journals Gating charge displacement in a monomeric voltage-gated proton (Hv1) channel

2018 ◽  
Vol 115 (37) ◽  
pp. 9240-9245 ◽  
Author(s):  
Emerson M. Carmona ◽  
H. Peter Larsson ◽  
Alan Neely ◽  
Osvaldo Alvarez ◽  
Ramon Latorre ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Time Course ◽  
Voltage Sensor ◽  
Total Charge ◽  
Gating Currents ◽  
Voltage Gated ◽  
Central Transition ◽  
Physiological Processes ◽  
Gating Charge ◽  
Voltage Dependent

The voltage-gated proton (Hv1) channel, a voltage sensor and a conductive pore contained in one structural module, plays important roles in many physiological processes. Voltage sensor movements can be directly detected by measuring gating currents, and a detailed characterization of Hv1 charge displacements during channel activation can help to understand the function of this channel. We succeeded in detecting gating currents in the monomeric form of the Ciona-Hv1 channel. To decrease proton currents and better separate gating currents from ion currents, we used the low-conducting Hv1 mutant N264R. Isolated ON-gating currents decayed at increasing rates with increasing membrane depolarization, and the amount of gating charges displaced saturates at high voltages. These are two hallmarks of currents arising from the movement of charged elements within the boundaries of the cell membrane. The kinetic analysis of gating currents revealed a complex time course of the ON-gating current characterized by two peaks and a marked Cole–Moore effect. Both features argue that the voltage sensor undergoes several voltage-dependent conformational changes during activation. However, most of the charge is displaced in a single central transition. Upon voltage sensor activation, the charge is trapped, and only a fast component that carries a small percentage of the total charge is observed in the OFF. We hypothesize that trapping is due to the presence of the arginine side chain in position 264, which acts as a blocking ion. We conclude that the movement of the voltage sensor must proceed through at least five states to account for our experimental data satisfactorily.

scholarly journals Augmentation of Recovery from Inactivation by Site-3 Na Channel Toxins

1999 ◽  
Vol 113 (2) ◽  
pp. 333-346 ◽  
Author(s):  
G. Richard Benzinger ◽  
Gayle S. Tonkovich ◽  
Dorothy A. Hanck
Keyword(s):  
Time Course ◽  
Genetic Diseases ◽  
Low Frequency ◽  
Periodic Paralysis ◽  
Na Channel ◽  
Na Channels ◽  
Cell Recovery ◽  
Reverse Transcription Pcr ◽  
Recovery From Inactivation ◽  
Voltage Dependent

Site-3 toxins isolated from several species of scorpion and sea anemone bind to voltage-gated Na channels and prolong the time course of INa by interfering with inactivation with little or no effect on activation, effects that have similarities to those produced by genetic diseases in skeletal muscle (myotonias and periodic paralysis) and heart (long QT syndrome). Some published reports have also reported the presence of a noninactivating persistent current in site-3 toxin-treated cells. We have used the high affinity site-3 toxin Anthopleurin B to study the kinetics of this current and to evaluate kinetic differences between cardiac (in RT4-B8 cells) and neuronal (in N1E-115 cells) Na channels. By reverse transcription–PCR from N1E-115 cell RNA multiple Na channel transcripts were detected; most often isolated were sequences homologous to rBrII, although at low frequency sequences homologous to rPN1 and rBrIII were also detected. Toxin treatment induced a voltage-dependent plateau current in both isoforms for which the relative amplitude (plateau current/peak current) approached a constant value with depolarization, although the magnitude was much greater for neuronal (17%) than cardiac (5%) INa. Cell-attached patch recordings revealed distinct quantitative differences in open times and burst durations between isoforms, but for both isoforms the plateau current comprised discrete bursts separated by quiescent periods, consistent with toxin induction of an increase in the rate of recovery from inactivation rather than a modal failure of inactivation. In accord with this hypothesis, toxin increased the rate of whole-cell recovery at all tested voltages. Moreover, experimental data support a model whereby recovery at negative voltages is augmented through closed states rather than through the open state. We conclude that site-3 toxins produce qualitatively similar effects in cardiac and neuronal channels and discuss implications for channel kinetics.

scholarly journals Kinetics of 9-aminoacridine block of single Na channels.

1984 ◽  
Vol 84 (3) ◽  
pp. 361-377 ◽  
Author(s):  
D Yamamoto ◽  
J Z Yeh
Keyword(s):  
Rate Constant ◽  
Voltage Dependence ◽  
Single Channel ◽  
Na Channel ◽  
Na Channels ◽  
Patch Clamp Technique ◽  
Open Time ◽  
Voltage Dependent ◽  
The Mean ◽  
Kinetics Of

The kinetics of 9-aminoacridine (9-AA) block of single Na channels in neuroblastoma N1E-115 cells were studied using the gigohm seal, patch clamp technique, under the condition in which the Na current inactivation had been eliminated by treatment with N-bromoacetamide (NBA). Following NBA treatment, the current flowing through individual Na channels was manifested by square-wave open events lasting from several to tens of milliseconds. When 9-AA was applied to the cytoplasmic face of Na channels at concentrations ranging from 30 to 100 microM, it caused repetitive rapid transitions (flickering) between open and blocked states within single openings of Na channels, without affecting the amplitude of the single channel current. The histograms for the duration of blocked states and the histograms for the duration of open states could be fitted with a single-exponential function. The mean open time (tau o) became shorter as the drug concentration was increased, while the mean blocked time (tau b) was concentration independent. The association (blocking) rate constant, kappa, calculated from the slope of the curve relating the reciprocal mean open time to 9-AA concentration, showed little voltage dependence, the rate constant being on the order of 1 X 10(7) M-1s-1. The dissociation (unblocking) rate constant, l, calculated from the mean blocked time, was strongly voltage dependent, the mean rate constant being 214 s-1 at 0 mV and becoming larger as the membrane being hyperpolarized. The voltage dependence suggests that a first-order blocking site is located at least 63% of the way through the membrane field from the cytoplasmic surface. The equilibrium dissociation constant for 9-AA to block the Na channel, defined by the relation of l/kappa, was calculated to be 21 microM at 0 mV. Both tau -1o and tau -1b had a Q10 of 1.3, which suggests that binding reaction was diffusion controlled. The burst time in the presence of 9-AA, which is the sum of open times and blocked times, was longer than the lifetime of open channels in the absence of drug. All of the features of 9-AA block of single Na channels are compatible with the sequential model in which 9-AA molecules block open Na channels, and the blocked channels could not close until 9-AA molecules had left the blocking site in the channels.

scholarly journals Inactivation of cloned Na channels expressed in Xenopus oocytes.

1990 ◽  
Vol 96 (4) ◽  
pp. 689-706 ◽  
Author(s):  
D S Krafte ◽  
A L Goldin ◽  
V J Auld ◽  
R J Dunn ◽  
N Davidson ◽  
...  
Keyword(s):  
Time Course ◽  
Xenopus Oocytes ◽  
Voltage Dependence ◽  
Single Channel ◽  
Single Amino Acid ◽  
Na Channel ◽  
Low Molecular Weight ◽  
Cdna Clones ◽  
Na Channels ◽  
Single Amino Acid Residue

This study investigates the inactivation properties of Na channels expressed in Xenopus oocytes from two rat IIA Na channel cDNA clones differing by a single amino acid residue. Although the two cDNAs encode Na channels with substantially different activation properties (Auld, V. J., A. L. Goldin, D. S. Krafte, J. Marshall, J. M. Dunn, W. A. Catterall, H. A. Lester, N. Davidson, and R. J. Dunn. 1988. Neuron. 1:449-461), their inactivation properties resemble each other strongly but differ markedly from channels induced by poly(A+) rat brain RNA. Rat IIA currents inactivate more slowly, recover from inactivation more slowly, and display a steady-state voltage dependence that is shifted to more positive potentials. The macroscopic inactivation process for poly(A+) Na channels is defined by a single exponential time course; that for rat IIA channels displays two exponential components. At the single-channel level these differences in inactivation occur because rat IIA channels reopen several times during a depolarizing pulse; poly(A+) channels do not. Repetitive stimulation (greater than 1 Hz) produces a marked decrement in the rat IIA peak current and changes the waveform of the currents. When low molecular weight RNA is coinjected with rat IIA RNA, these inactivation properties are restored to those that characterize poly(A+) channels. Slow inactivation is similar for rat IIA and poly(A+) channels, however. The data suggest that activation and inactivation involve at least partially distinct regions of the channel protein.

scholarly journals Extracellular Mg2+ Modulates Slow Gating Transitions and the Opening of Drosophila Ether-à-Go-Go Potassium Channels

2000 ◽  
Vol 115 (3) ◽  
pp. 319-338 ◽  
Author(s):  
Chih-Yung Tang ◽  
Francisco Bezanilla ◽  
Diane M. Papazian
Keyword(s):  
Time Course ◽  
Ionic Current ◽  
Ionic Currents ◽  
K Channel ◽  
Gating Currents ◽  
Gating Charge ◽  
Channel Opening ◽  
Voltage Dependent ◽  
Pore Opening ◽  
Sequential Activation

We have characterized the effects of prepulse hyperpolarization and extracellular Mg2+ on the ionic and gating currents of the Drosophila ether-à-go-go K+ channel (eag). Hyperpolarizing prepulses significantly slowed channel opening elicited by a subsequent depolarization, revealing rate-limiting transitions for activation of the ionic currents. Extracellular Mg2+ dramatically slowed activation of eag ionic currents evoked with or without prepulse hyperpolarization and regulated the kinetics of channel opening from a nearby closed state(s). These results suggest that Mg2+ modulates voltage-dependent gating and pore opening in eag channels. To investigate the mechanism of this modulation, eag gating currents were recorded using the cut-open oocyte voltage clamp. Prepulse hyperpolarization and extracellular Mg2+ slowed the time course of ON gating currents. These kinetic changes resembled the results at the ionic current level, but were much smaller in magnitude, suggesting that prepulse hyperpolarization and Mg2+ modulate gating transitions that occur slowly and/or move relatively little gating charge. To determine whether quantitatively different effects on ionic and gating currents could be obtained from a sequential activation pathway, computer simulations were performed. Simulations using a sequential model for activation reproduced the key features of eag ionic and gating currents and their modulation by prepulse hyperpolarization and extracellular Mg2+. We have also identified mutations in the S3–S4 loop that modify or eliminate the regulation of eag gating by prepulse hyperpolarization and Mg2+, indicating an important role for this region in the voltage-dependent activation of eag.

Reduced voltage dependence of inactivation in the SCN5A sodium channel mutation delF1617

2005 ◽  
Vol 288 (6) ◽  
pp. H2666-H2676 ◽  
Author(s):  
Tiehua Chen ◽  
Masashi Inoue ◽  
Michael F. Sheets
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Single Channel ◽  
Expression System ◽  
Single Amino Acid ◽  
Wild Type ◽  
Gating Charge ◽  
Voltage Curve ◽  
Voltage Dependent ◽  
Single Channel Currents

Deletion of a phenylalanine at position 1617 (delF1617) in the extracellular linker between segments S3 and S4 in domain IV of the human heart Na+ channel (hH1a) has been tentatively associated with long QT syndrome type 3 (LQT3). In a mammalian cell expression system, we compared whole cell, gating, and single-channel currents of delF1617 with those of wild-type hH1a. The half points of the peak activation-voltage curve for the two channels were similar, as were the deactivation time constants at hyperpolarized test potentials. However, delF1617 demonstrated a significant negative shift of −7 mV in the half point of the voltage-dependent Na+ channel availability curve compared with wild type. In addition, both the time course of decay of Na+ current ( INa) and two-pulse development of inactivation of delF1617 were faster at negative test potentials, whereas they tended to be slower at positive potentials compared with wild type. Mean channel open times for delF1617 were shorter at potentials <0 mV, whereas they were longer at potentials >0 mV compared with wild type. Using anthopleurin-A, a site-3 toxin that inhibits movement of segment S4 in domain IV (S4-DIV), we found that gating charge contributed by the S4-DIV in delF1617 was reduced 37% compared with wild type. We conclude that deletion of a single amino acid in the S3-S4 linker of domain IV alters the voltage dependence of fast inactivation via a reduction in the gating charge contributed by S4-DIV and can cause either a gain or loss of INa, depending on membrane potential.

scholarly journals Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels.

1995 ◽  
Vol 106 (6) ◽  
pp. 1193-1209 ◽  
Author(s):  
H B Nuss ◽  
G F Tomaselli ◽  
E Marbán
Keyword(s):  
Skeletal Muscle ◽  
Local Anesthetics ◽  
Time Course ◽  
Large Fraction ◽  
Expression System ◽  
Na Channel ◽  
Na Channels ◽  
Na Current ◽  
Unexpected Findings ◽  
Cardiac Sodium Channels

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use-dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1-beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.

scholarly journals Gating currents associated with Na channels in canine cardiac Purkinje cells.

1990 ◽  
Vol 95 (3) ◽  
pp. 439-457 ◽  
Author(s):  
D A Hanck ◽  
M F Sheets ◽  
H A Fozzard
Keyword(s):  
Purkinje Cells ◽  
Single Channel ◽  
State Transitions ◽  
Na Channel ◽  
Channel Measurements ◽  
Na Channels ◽  
Gating Currents ◽  
Closed State ◽  
Conductance Curve ◽  
Cardiac Purkinje Cells

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.

Time-dependent changes in kinetics of Na+ current in single canine cardiac Purkinje cells

1992 ◽  
Vol 262 (4) ◽  
pp. H1197-H1207 ◽  
Author(s):  
D. A. Hanck ◽  
M. F. Sheets
Keyword(s):  
Purkinje Cells ◽  
Time Constant ◽  
Decay Time ◽  
Voltage Dependence ◽  
Na Channel ◽  
Time Constants ◽  
Voltage Range ◽  
Time To Peak ◽  
Cardiac Purkinje Cells ◽  
Voltage Dependent

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.

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