Voltage-dependent block of charge movement components by nifedipine in frog skeletal muscle

Potential-dependent inhibition of charge movement components by nifedipine was studied in intact, voltage-clamped, frog skeletal muscle fibers. Available charge was reduced by small shifts in holding potential (from -100 mV to -70 mV) in 2 microM nifedipine, without changes in the capacitance deduced from control (-120 mV to -100 mV) voltage steps made at a fully polarized (-100 mV) holding potential. These voltage-dependent effects did not occur in lower (0-0.5 microM) nifedipine concentrations. The voltage dependence of membrane capacitance at higher (10 microM) nifedipine concentrations was reduced even in fully polarized fibers, but shifting the holding voltage produced no further block. Voltage-dependent inhibition by nifedipine was associated with a fall in available charge, and a reduction in the charge and capacitance-voltage relationships and of late (q gamma) charging transients. It thus separated a membrane-capacitance with a distinct and steep steady-state voltage dependence. Tetracaine (2 mM) reduced voltage-dependent membrane capacitance and nonlinear charge more than did nifedipine. However, nifedipine did not exert voltage-dependent effects on charging currents, membrane capacitance, or inactivation of tetracaine-resistant (q beta) charge. This excludes participation of q beta, or the membrane charge as a whole, from the voltage-dependent effects of nifedipine. Rather, the findings suggest that the charge susceptible to potential-dependent block by nifedipine falls within the tetracaine-sensitive (q gamma) category of intramembrane charge.     

Membrane tension directly shifts voltage dependence of outer hair cell motility and associated gating charge.

The unique electromotility of the outer hair cell (OHC) is believed to promote sharpening of the passive mechanical vibration of the mammalian basilar membrane. The cell also presents a voltage-dependent capacitance, or equivalently, a nonlinear gating current, which correlates well with its mechanical activity, suggesting that membrane-bound voltage sensor-motor elements control OHC length. We report that the voltage dependence of the gating charge and motility are directly related to membrane stress induced by intracellular pressure. A tracking procedure was devised to continuously monitor the voltage at peak capacitance (VpkCm) after obtaining whole cell voltage clamp configuration. In addition, nonlinear capacitance was more fully evaluated with a stair step voltage protocol. Upon whole cell configuration, VpkCm was typically near -20 mV. Negative patch pipette pressure caused a negative shift in VpkCm, which obtained a limiting value near the normal resting potential of the OHC (approximately -70 mV) at the point of cell collapse. Positive pressure in the pipette caused a positive shift that could reach values greater than 0 mV. Measures of the mechanical activity of the OHC mirrored those of charge movement. Similar membrane-tension dependent peak shifts were observed after the cortical cytoskeletal network was disrupted by intracellular dialysis of trypsin from the patch pipette. We conclude that unlike stretch receptors, which may sense tension through elastic cytoskeletal elements, the OHC motor senses tension directly. Furthermore, since the voltage dependence of the OHC nonlinear capacitance and motility is directly regulated by intracellular turgor pressure, we speculate that modification of intracellular pressure in vivo provides a mechanism for controlling the gain of the mammalian "cochlear amplifier".     

Probing conformational changes of prestin with thiol-reactive optical switches

Thiol-reactive optical switch probes were used to examine conformational changes of prestin-based membrane motor. Because this motor is based on mechanoelectric coupling similar to piezoelectricity, the motile activity can be monitored by charge movements across the plasma membrane, which appears as nonlinear capacitance. When the plasma membrane is conjugated with the probes, optically induced spiro-merocyanine transition positively shifted nonlinear capacitance of outer hair cells and prestin-transfected cells by ∼10 mV. These shifts were reversible and were eliminated by pretreatment with iodoacetamide. However, they were little affected by pretreatment with biotin maleimide, which cannot reach the cytoplasmic surface. Our results showed that merocyanine states, with a larger dipole moment, interact with the motor's extended conformation stronger than with the compact conformation by 1.6 × 10⁻²¹ J/molecule. The interaction sites are near the cytoplasmic side of the motor protein.     

Distribution and kinetics of membrane dielectric polarization. II. Frequency domain studies of gating currents

We have studied the admittance of the membrane of squid giant axon under voltage clamp in the absence of ionic conductances in the range of 0-12 kHz for membrane potentials (V) between --130 and 70 mV. The admittance was measured at various holding potentials (HP) or 155 ms after pulsing from a given holding potential. Standard P/4 procedure was used to study gating currents in the same axons. We found that the membrane capacity Cm (omega) is voltage as well as frequency dependent. For any given V, the voltage-dependent part of the membrane capacitance has a maximum as the frequency approaches zero and requires at least a two-time constant equivalent circuit to be described. When the holding potential is varied, the voltage-dependent capacitance follows a bell- shaped curve with a maximum change of 0.15 muF/cm2 at about --60 mV. With the pulse method, the maximum is at --40 mV for HP = --70 and it shifts to --70 mV for HP = 0. The shift in the maximum of the voltage- dependent capacitance is consistent with the shift in the charge (Q) vs. V curve observed in our experiments with regular P/4 procedure when the HP is varied. Our data can be explained qualitatively by a four- state model for the sodium channel gating, where a charged particle can move within the field and interact with another particle not affected by the field.     

Sensitivity of Prestin-Based Membrane Motor to Membrane Thickness

Prestin is the membrane protein in outer hair cells that harnesses electrical energy by changing its membrane area in response to changes in the membrane potential. To examine the effect of membrane thickness on this protein, phosphatidylcholine (PC) with various acyl-chain lengths were incorporated into the plasma membrane by using γ-cyclodextrin. Incorporation of short chain PCs increased the linear capacitance and positively shifted the voltage dependence of prestin, up to 120 mV, in cultured cells. PCs with long acyl chains had the opposite effects. Because the linear capacitance is inversely related to the membrane thickness, these voltage shifts are attributable to membrane thickness. The corresponding voltage shifts of electromotility were observed in outer hair cells. These results demonstrate that electromotility is extremely sensitive to the thickness of the plasma membrane, presumably involving hydrophobic mismatch. These observations indicate that the extended state of the motor molecule, which is associated with the elongation of outer hair cells, has a conformation with a shorter hydrophobic height in the lipid bilayer.     

Amphipath-Induced Nanoscale Changes in Outer Hair Cell Plasma Membrane Curvature

Outer hair cell (OHC) electromotility enables frequency selectivity and sensitivity in mammalian audition. Electromotility is generated by the transmembrane protein prestin and is sensitive to amphipathic compounds including salicylate, chlorpromazine (CPZ), and trinitrophenol (TNP). Although these compounds induce observable membrane curvature changes in erythrocytes, their effects on OHC membrane curvature are unknown. In this work, fluorescence polarization microscopy was applied to investigate the effects of salicylate, CPZ, and TNP on di-8-ANEPPS orientation in the OHC plasma membrane. Our results demonstrate the ability of fluorescence polarization microscopy to measure amphipath-induced changes in di-8-ANEPPS orientation, consistent with nanoscale changes in membrane curvature between regularly spaced proteins connecting the OHC plasma membrane and cytoskeleton. Simultaneous application of oppositely charged amphipaths generally results in no net membrane bending, consistent with predictions of the bilayer couple hypothesis; however, the application of salicylate (10 mM), which inhibits electromotility, is not reversed by the addition of CPZ. This result supports other findings that suggest salicylate primarily influences electromotiliy and OHC nonlinear capacitance via a direct interaction with prestin. In contrast, we find that CPZ and TNP influence the voltage sensitivity of prestin via membrane bending, demonstrating the mechanosensitivity of this unique membrane motor protein.     

Protein- and Lipid-Reactive Agents Alter Outer Hair Cell Lateral Membrane Motor Charge Movement

Outer hair cells from the mamma*lian cochlea are mechanically active cells that rely on charged voltage sensors within their lateral plasma membrane to gate the integral membrane motor protein, prestin, into one of two area states. Here we use protein and lipid reactive reagents to probe the influence of these bilayer components on motor-induced nonlinear membrane capacitance. Of the protein-reactive reagents tested, cross-linking and sulfhydryl reagents were most effective in altering steady state and time-varying motor activity. Of the lipid-altering agents, chloroform and HePC were most effective. Chloroform, in particular, drastically modified the susceptibility of the motor to prior voltage (initial conditions). Our data suggest that outer hair cell motor activity derives substantially from interactions with its lipid environment.This revised version was published online in June 2005 with a corrected cover date.     

Further evidence that membrane thickness influences voltage-gated sodium channels.

The short-chain phospholipid, diheptanoyl phosphatidylcholine, at 520 microM, reduced the maximum inward sodium current in voltage-clamped squid giant axons by greater than 50%. Analysis of these currents by means of the Hodgkin-Huxley equations showed this reduction to be mainly the result of a large depolarizing shift in the voltage dependence of the steady state activation parameter, m infinity. The voltage dependence of the steady state inactivation parameter, h infinity, was also moved in the depolarizing direction and the axonal membrane capacitance per unit area measured at 100 kHz was increased. A longer chain length derivative, didecanoyl phosphatidylcholine, had no significant effect on the axonal sodium current at concentrations of 3.7 and 18.5 microM. Dioctanoyl phosphatidylcholine was intermediate in its effects, 200 microM producing approximately the same current suppression as 520 microM diheptanoyl phosphatidylcholine, together with depolarizing shifts in m infinity and h infinity. These effects may be contrasted with those of the normal and cyclic alkanes (1-3), which tend to move both m infinity and h infinity in the hyperpolarizing direction and to reduce the capacitance per unit area at 100 kHz. The above results are all consistent with the hypothesis that small hydrocarbons thicken, while short-chain phospholipids thin, the axonal membrane. Thus membrane thickness changes may be of considerable importance in determining the behavior of the voltage-gated sodium channel.     

Chloride and Salicylate Influence Prestin-dependent Specific Membrane Capacitance: SUPPORT FOR THE AREA MOTOR MODEL*

The outer hair cell is electromotile, its membrane motor identified as the protein SLC26a5 (prestin). An area motor model, based on two-state Boltzmann statistics, was developed about two decades ago and derives from the observation that outer hair cell surface area is voltage-dependent. Indeed, aside from the nonlinear capacitance imparted by the voltage sensor charge movement of prestin, linear capacitance (C_lin) also displays voltage dependence as motors move between expanded and compact states. Naturally, motor surface area changes alter membrane capacitance. Unit linear motor capacitance fluctuation (δC_sa) is on the order of 140 zeptofarads. A recent three-state model of prestin provides an alternative view, suggesting that voltage-dependent linear capacitance changes are not real but only apparent because the two component Boltzmann functions shift their midpoint voltages (V_h) in opposite directions during treatment with salicylate, a known competitor of required chloride binding. We show here using manipulations of nonlinear capacitance with both salicylate and chloride that an enhanced area motor model, including augmented δC_sa by salicylate, can accurately account for our novel findings. We also show that although the three-state model implicitly avoids measuring voltage-dependent motor capacitance, it registers δC_sa effects as a byproduct of its assessment of C_lin, which increases during salicylate treatment as motors are locked in the expanded state. The area motor model, in contrast, captures the characteristics of the voltage dependence of δC_sa, leading to a better understanding of prestin.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin’s voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein’s ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin’s unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells’ role as a cochlear amplifier.     

Exploratory studies of some drug charge transfer interactions

Conductivity and capacitance titrations yield minima for the chlorpromazine hydrochloride-heparin interaction, confirming clinical suspicions of its occurrence. The effective dosage of heparin thus is reduced if administered in conjunction with chlorpromazine. The interaction is interpreted as charge transfer complex formation, occurring as an (electrode) surface reaction. It is suggested that the charge transfer complexing capability of heparin preparations, as evidenced by conductance and/or capacitance changes, evaluated against a well defined donor such as chlorpromazine hydrochloride, may be adapted as a more precise method of measuring heparin activity than coagulation time determinations. Phenytoin and chlorpromazine likewise yield conductance and capacitance minima; voltammetry indicates new peaks at +250mV and −300mV vers.SCE supporting the suggestions that an uncharged 1∶1 complex is being formed, again in a type of surface reaction. Phenytoin and lignocain form a precipitate at 0.002 equimolar; in conductance and capacitance titrations phenytoin behaves as a weak electron donor against iodine though as a weak acceptor against lignocain. Lignocain and chlorpromazine conductance and capacitance titrations using gold electrodes fail to show any evidence for their previously reported interaction on Pt/Pt electrodes. Voltammetry on Pt/Pt electrodes indicates 2 new peaks at zero and at −750mV vers.SCE. It is thought that these two compounds interact only on catalytically highly active surfaces, where they form a weak surface charge transfer complex. Adrenalin, in conductance and capacitance titrations, behaves amphoteric, i.e. as an electron acceptor against the strong donor chlorpromazine and as a donor against the strong acceptor tetramethyl-p-phenylenediamine. Voltammograms of the above listed interactions are interpreted as of the ECE type exhibiting mainly irreversible behaviour.     

IR Laser-Induced Perturbations of the Voltage-Dependent Solute Carrier Protein SLC26a5

Alterations in membrane capacitance can arise from linear and nonlinear sources. For example, changes in membrane surface area or dielectric properties can modify capacitance linearly, whereas sensor residues of voltage-dependent proteins can modify capacitance nonlinearly. Here, we examined the effects of fast temperature jumps induced by an infrared (IR) laser in control and prestin (SLC26a5)-transfected human embryonic kidney (HEK) cells under whole-cell voltage clamp. Prestin’s voltage sensor imparts a characteristic bell-shaped, voltage-dependent nonlinear capacitance (NLC). Temperature jumps in control HEK cells cause a monophasic increase in membrane capacitance (C_m) regardless of holding voltage due to double-layer effects. Prestin-transfected HEK cells, however, additionally show a biphasic increase/decrease in C_m with a reversal potential corresponding to the voltage at peak NLC of prestin (V_h), attributable to a rapid temperature-following shift in V_h, with shift rates up to 14 V/s over the course of a 5 ms IR pulse. Treatment with salicylate, a known inhibitor of NLC, reestablishes control cell behavior. A simple kinetic model recapitulates our biophysical observations. These results verify a voltage-dependent protein’s ability to respond to fast temperature perturbations on a par with double-layer susceptibility. This likely arises from prestin’s unique ability to move sensor charge at kilohertz rates, which is required for the outer hair cells’ role as a cochlear amplifier.     

Rapid changes in flagellar rotation induced by external electric pulses.

The bacterial flagellar motor is the only molecular rotary machine found in living organisms, converting the protonmotive force, i.e., the membrane voltage and proton gradients across the cell membrane, into the mechanical force of rotation (torque). We have developed a method for holding a bacterial cell at the tip of a glass micropipette and applying electric pulses through the micropipette. This method has enabled us to observe the dynamical responses of flagellar rotation to electric pulses that change the membrane voltage transiently and repeatedly. We have observed that acceleration and deceleration of motor rotation are induced by application of these electric pulses. The change in the rotation rate occurred within 5 ms after pulse application.     

Asymmetry currents and admittance in squid axons.

The complex admittance of squid (Loligo pealei) axon was measured rapidly (within 1 s) with pseudo-random small signals and discrete Fourier transform techniques under guarded, "space-clamp" conditions and during suppression of ion conduction. Asymmetry currents were measured by paired step clam pulses of +/-70 mV from a holding potential of -97 mV and gave an apparent capacitance of 0.36 muF/cm2. However, the admittance data showed no change in capacitance at holding potentials from -97 to -67 mV and gave a decrease of 0.07 of 0.15 muF/cm2 at -37 mV. The failure to observe a capacitance increase at low membrane potentials suggests the following possibilities: (a) the asymmetry current is a displacement current that inactivates completely with time, and (b) the asymmetry current is not a displacement current and arises from large signal effects (i.e., delayed nonlinearity in ionic current) on the membrane.     

Membrane currents in retinal bipolar cells of the axolotl

By whole-cell patch-clamping bipolar cells isolated from enzymatically dissociated retinae, we have studied the nonsynaptic ionic currents that may play a role in shaping the bipolar cell light response and in determining the level of voltage noise in these cells. Between -30 and -70 mV, the membrane current of isolated bipolar cells is time independent, and the input resistance is 1-2 G omega. Depolarization past -30 mV activates an outward current (in less than 100 ms), which then inactivates slowly (approximately 1 s). Inactivation of this current is removed by hyperpolarization over the range -20 to -80 mV. This current is carried largely by K ions. It is not activated by internal Ca2+. The membrane current of isolated bipolar cells is noisy, and the variance of this noise has a minimum between -40 and -60 mV. At its minimum, the standard deviation of the voltage noise produced by nonsynaptic membrane currents is at least 100 microV. The membrane currents of depolarizing bipolar cells in slices of retina were investigated by whole-cell patch-clamping. Their membrane properties were similar to those of isolated bipolar cells, but with a larger membrane capacitance and a smaller input resistance. Their membrane current noise also showed a minimum near -40 to -60 mV. The time-dependent potassium current in axolotl bipolar cells is not significantly activated in the physiological potential range and can therefore play little role in shaping the bipolar cells' voltage response to light. Differences in the waveform of the light response of bipolar cells and photoreceptors must be ascribed to shaping by the synapses between these cells. The noise minimum in the bipolar membrane current is near the dark potential of these cells, and this may be advantageous for the detection of weak signals by the bipolar cells.     

Current noise parameters derived from voltage noise and impedance in embryonic heart cell aggregates.

We have recorded membrane impedance and voltage noise in the pacemaker range of potentials (-70 to -59 mV) from spheroidal aggregates of 7-d embryonic chick ventricle cells made quiescent by exposure to tetrodotoxin in medium containing 4.5 mM K+. The input capacitance is proportional to aggregate volume and therefore to total membrane area. The specific membrane capacitance is 1.24 microF/cm2. The input resistance at constant potential is inversely proportional to aggregate volume and therefore to total membrane area. The specific membrane resistance in 18 k omega . cm2 at -70 mV and increases to 81 k omega . cm2 at -59 mV. The RC time constant is 22 ms at -70 mV and increases to 146 ms at -59 mV. The aggregate transmembrane small-signal impedance can be represented by a parallel RC circuit itself in parallel with an inductive branch consisting of a resistor (rL) and an inductor (L) in series. The time constant of the inductive branch (L/rL) is 340 ms, and is only weakly dependent on potential. Correlation functions of aggregate voltage noise and the impedance data were modeled by a population of channels with simple open-close kinetics. The time constant of a channel (tau s) derived from the noise analysis is 300 ms. The low frequency limit of the pacemaker current noise (SI[0]), derived from the voltage noise and impedance, increases from 10(-20) A2/Hz . cm2 at -67 mV to 10(-19) A2/Hz . cm2 at -61 mV.     

Modulation of rat brain cytosolic phosphatidate phosphohydrolase: effect of cationic amphiphilic drugs and divalent cations

The effects of three cationic amphiphilic drugs on rat brain cytosolic phosphatidate phosphohydrolase and their mechanisms of action were studied utilizing membrane-bound, emulsified, and emulsified sonicated phosphatidate as substrates. With the membrane-bound substrate, chlorpromazine, desmethylimipramine, and propranolol inhibited the activity in a dose-dependent fashion with an IC50 of 30-50 microM. In the presence of the emulsified substrate, chlorpromazine was a more potent inhibitor than desmethylimipramine or propranolol but 200 microM was needed for 50% inhibition of activity. Addition of heat-inactivated microsomes to the emulsified substrate, to simulate the conditions with the membrane-bound substrate, did not alter this value. Both Mg2+ and Ca2+ stimulated the enzyme activity but only Ca2+ counteracted the effect of chlorpromazine. Kinetic studies indicate that chlorpromazine acts as a noncompetitive inhibitor of the enzyme. Emulsified sonicated phosphatidate was a good substrate at low (less than 10 microM) concentrations. It was a poor substrate at 1 mM, but at this concentration chlorpromazine stimulated the activity instead of inhibiting. This drug altered the integrity of phosphatidate vesicle membranes as visualized by electron microscopy. The different results obtained with the three types of substrate indicate the importance of the configuration of phosphatidate for the expression of enzyme activity and for its susceptibility to the action of cationic amphiphilic drugs.     

Developmental Expression of the Outer Hair Cell Motor Prestin in the Mouse

The development of motor protein activity in the lateral membrane of the mouse outer hair cell (OHC) from postnatal day 5 (P5) to P18 was investigated under whole-cell voltage clamp. Voltage-dependent, nonlinear capacitance (C _v), which represents the conformational fluctuations of the motor molecule, progressively increased during development. At P12, the onset of hearing in the mouse, C _v was about 70% of the mature level. C _v saturated at P18 when hearing shows full maturation. On the other hand, C _lin, which represents the membrane area of the OHC, showed a relatively small increase with development, reaching steady state at P10. This early maturation of linear capacitance is further supported by morphological estimates of surface area during development. These results, in light of recent prestin knockout experiments and our results with quantitative polymerase chain reaction, suggest that, rather than the incorporation of new motors into the lateral membrane after P10, molecular motors mature to augment nonlinear capacitance. Thus, current estimates of motor protein density based on charge movement may be exaggerated. A corresponding indicator of motor maturation, the motor’s operating voltage midpoint, V _pkcm, tended to shift to depolarized potentials during postnatal development, although it was unstable prior to P10. However, after P14, V _pkcm reached a steady-state level near −67 mV, suggesting that intrinsic membrane tension or intracellular chloride, each of which can modulate V _pkcm, may mature at P14. These developmental data significantly alter our understanding of the cellular mechanisms that control cochlear amplification and provide a foundation for future analysis of genetic modifications of mouse auditory development.     

Kinetic properties of intramembrane charge movement under depolarized conditions in frog skeletal muscle fibers

Intramembrane charge movement was measured on skeletal muscle fibers of the frog in a single Vaseline-gap voltage clamp. Charge movements determined both under polarized conditions (holding potential, VH = -100 mV; Qmax = 30.4 +/- 4.7 nC/micro(F), V = -44.4 mV, k = 14.1 mV; charge 1) and in depolarized states (VH = 0 mV; Qmax = 50.0 +/- 6.7 nC/micro(F), V = -109.1 mV, k = 26.6 mV; charge 2) had properties as reported earlier. Linear capacitance (LC) of the polarized fibers was increased by 8.8 +/- 4.0% compared with that of the depolarized fibers. Using control pulses measured under depolarized conditions to calculate charge 1, a minor change in the voltage dependence (to V = -44.6 mV and k = 14.5 mV) and a small increase in the maximal charge (to Qmax = 31.4 +/- 5.5 nC/micro(F] were observed. While in most cases charge 1 transients seemed to decay with a single exponential time course, charge 2 currents showed a characteristic biexponential behavior at membrane potentials between -90 and -180 mV. The voltage dependence of the rate constant of the slower component was fitted with a simple constant field diffusion model (alpha m = 28.7 s-1, V = -124.0 mV, and k = 15.6 mV). The midpoint voltage (V) was similar to that obtained from the Q-V fit of charge 2, while the steepness factor (k) resembled that of charge 1. This slow component could also be isolated using a stepped OFF protocol; that is, by hyperpolarizing the membrane to -190 mV for 200 ms and then coming back to 0 mV in two steps. The faster component was identified as an ionic current insensitive to 20 mM Co2+ but blocked by large hyperpolarizing pulses. These findings are consistent with the model implying that charge 1 and the slower component of charge 2 interconvert when the holding potential is changed. They also explain the difference previously found when comparing the steepness factors of the voltage dependence of charge 1 and charge 2.     

Nonlinear charge movement in mammalian cardiac ventricular cells. Components from Na and Ca channel gating [published erratum appears in J Gen Physiol 1989 Aug;94(2):401]

Intramembrane charge movement was recorded in rat and rabbit ventricular cells using the whole-cell voltage clamp technique. Na and K currents were eliminated by using tetraethylammonium as the main cation internally and externally, and Ca channel current was blocked by Cd and La. With steps in the range of -110 to -150 used to define linear capacitance, extra charge moves during steps positive to approximately -70 mV. With holding potentials near -100 mV, the extra charge moving outward on depolarization (ON charge) is roughly equal to the extra charge moving inward on repolarization (OFF charge) after 50-100 ms. Both ON and OFF charge saturate above approximately +20 mV; saturating charge movement is approximately 1,100 fC (approximately 11 nC/muF of linear capacitance). When the holding potential is depolarized to -50 mV, ON charge is reduced by approximately 40%, with little change in OFF charge. The reduction of ON charge by holding potential in this range matches inactivation of Na current measured in the same cells, suggesting that this component might arise from Na channel gating. The ON charge remaining at a holding potential of -50 mV has properties expected of Ca channel gating current: it is greatly reduced by application of 10 muM D600 when accompanied by long depolarizations and it is reduced at more positive holding potentials with a voltage dependence similar to that of Ca channel inactivation. However, the D600-sensitive charge movement is much larger than the Ca channel gating current that would be expected if the movement of channel gating charge were always accompanied by complete opening of the channel.