Separation of Q beta and Q gamma charge components in frog cut twitch fibers with tetracaine. Critical comparison with other methods

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. 25 microM tetracaine had very little effect on the maximum amounts of Q beta and Q gamma but slowed the kinetics of the I gamma humps in the ON segments of TEST-minus-CONTROL current traces, giving rise to biphasic transients in the difference traces. This concentration of tetracaine also shifted V gamma 3.7 (SEM 0.7) mV in the depolarizing direction, resulting in a difference Q-V plot that was bell-shaped with a peak at approximately -50 mV. 0.5-1.0 mM tetracaine suppressed the total amount of charge. The suppressed component had a sigmoidal voltage distribution with V = -56.6 (SEM 1.1) mV, k = 2.5 (SEM 0.5) mV, and qmax/cm = 9.2 (SEM 1.5) nC/microF, suggesting that the tetracaine-sensitive charge had a steep voltage dependence, a characteristic of the Q gamma component. An intermediate concentration (0.1-0.5 mM) of tetracaine shifted V gamma and partially suppressed the tetracaine-sensitive charge, resulting in a difference Q-V plot that rose to a peak and then decayed to a plateau level. Following a TEST pulse to greater than -60 mV, the slow inward current component during a post-pulse to approximately -60 mV was also tetracaine sensitive. The voltage distribution of the charge separated by tetracaine (method 1) was compared with those separated by three other existing methods: (a) the charge associated with the hump component separated by a sum of two kinetic functions from the ON segment of a TEST-minus-CONTROL current trace (method 2), (b) the steeply voltage-dependent component separated from a Q-V plot of the total charge by fitting with a sum of two Boltzmann distribution functions (method 3), and (c) the sigmoidal component separated from the Q-V plot of the final OFF charge obtained with a two-pulse protocol (method 4). The steeply voltage-dependent components separated by all four methods are consistent with each other, and are therefore concluded to be equivalent to the same Q gamma component. The shortcomings of each separation method are critically discussed. Since each method has its own advantages and disadvantages, it is recommended that, as much as possible, Q gamma should be separated by more than one method to obtain more reliable results.     

Q beta and Q gamma components of intramembranous charge movement in frog cut twitch fibers

Intramembranous charge movement was measured in frog cut twitch fibers mounted in a double Vaseline-gap chamber with a TEA.Cl solution at 13-14 degrees C in the central pool. When a fiber was depolarized from a holding potential of -90 mV to a potential near -60 mV, the current from intramembranous charge movement was outward in direction and had an early, rapid component and a late, more slowly developing component, referred to as I beta and I gamma, respectively (1979. J. Physiol. [Lond.]. 289:83-97). When the pulse to -60 mV was preceded by a 100-600-ms pulse to -40 mV, early I beta and late I gamma components were also observed, but in the inward direction. The shape of the Q gamma vs. voltage curve can be estimated with this two-pulse protocol. The first pulse to voltage V allows the amounts of Q beta and Q gamma charge in the active state to change from their respective resting levels, Q beta (-90) and Q gamma (-90), to new steady levels, Q beta (V) and Q gamma (V). A second 100-120-ms pulse, usually to -60 mV, allows the amount of Q beta charge in the active state to change from Q beta (V) to Q beta (-60) but is not sufficiently long for the amount of Q gamma charge to change completely from Q gamma (V) to Q gamma (-60). The difference between the amount of Q gamma charge at the end of the second pulse and Q gamma (-60) is estimated from the OFF charge that is observed on repolarization to -90 mV. The OFF charge vs. voltage data were fitted, with gap corrections, with a Boltzmann distribution function plus a constant. The mean values of V (the potential at which, in the steady state, charge is distributed equally between the resting and active states) and k (the voltage dependence factor) were -59.2 mV (SEM, 1.1 mV) and 1.2 mV (SEM, 0.6 mV), respectively. The one-pulse charge vs. voltage data from the same fibers were fitted with a sum of two Boltzmann functions (1990. J. Gen. Physiol. 96:257-297). The mean values of V and k for the steeply voltage-dependent Boltzmann function, which is likely to be associated with the Q gamma component of charge, were -55.3 mV (SEM, 1.3 mV) and 3.3 mV (SEM, 0.6 mV), respectively, similar to the corresponding values obtained with the two-pulse protocol.(ABSTRACT TRUNCATED AT 400 WORDS)     

Distribution and kinetics of membrane dielectric polarization. 1. Long- term inactivation of gating currents

Gating currents were measured by subtracting the linear component of the capacitative current recorded at very positive or very negative potentials. When the membrane is depolarized for a few minutes, repolarized to the usual holding potential (HP) of --70 mV for 1 ms, and then pulsed to 0 mV, the charge transferred in 2--4 ms is approximately 50% of that which was transferred during the same pulse holding at --70 mV. This charge decrease, called slow inactivation of the gating current, was found to be consistent with a shift of the charge vs. potential (Q-V) curve to more hyperpolarized potentials. When the HP is 0 mV, the total charge available to move is the same as the total charge available when the HP is --70 mV. The time constants of the fast component of the ON gating current are smaller at depolarized holding potentials than at --70 mV. When the HP is --70 mV and a prepulse of 50 ms duration is given to 0 mV, the Q-V curve is also shifted to more hyperpolarized potentials (charge immobilization), but the effect is not as pronounced as the one obtained by holding at 0 mV. When the HP is 0 mV, a prepulse to --70 mV for 50 ms partially shifts back the Q-V curve, indicating that fast inactivation of the gating charge may be recovered in the presence of slow inactivation. A physical model consisting of a gating particle that interacts with a fast inactivating particle, and a slow inactivating particle, reproduces most of the experimental results.     

The steady-state distribution of gating charge in crayfish giant axons.

Progressive shifts of holding potential (Vh) in crayfish giant axons, from -140 to -70 mV, reduce gating currents seen in depolarizing steps (to 0 mV test potential) while proportionately increasing gating currents in hyperpolarizing steps (to -240 mV). The resulting sigmoid equilibrium charge distribution (Q-Vh curve) shows an effective valence of 1.9e and a midpoint of -100 mV. By contrast, Q-V curves obtained using hyperpolarizing and/or depolarizing steps from a single holding potential, change their "shape" depending on the chosen holding potential. For holding potentials at the negative end of the Q-Vh distribution (e.g., -140 mV), negligible charge moves in hyperpolarizing pulses and the Q-V curve can be characterized entirely from depolarizing voltage steps. The slope of the resulting simple sigmoid Q-V curve also indicates an effective valence of 1.9e. When the axon is held at less negative potentials significant charge moves in hyperpolarizing voltage steps. The component of the Q-V curve collected using hyperpolarizing pulses shows a significantly reduced slope (approximately 0.75e) by comparison with the 1.9e slope found using depolarizing pulses or from the Q-Vh curve. As holding potential is shifted in the depolarizing direction along the Q-Vh curve, an increasing fraction of total charge movement must be assessed in hyperpolarizing voltage steps. Thus charge moving in the low slope component of the Q-V curve increases as holding potential is depolarized, while charge moving with high apparent valence decreases proportionately. Additional results, together with simulations based on a simple kinetic model, suggest that the reduced apparent valence of the low slope component of the Q-V curve results from gating charge immobilization occurring at holding potential. Immobilization selectively retards that fraction of total charge moving in hyperpolarizing pulses. Misleading conclusions, as to the number and valence of the gating particles, may therefore be derived from Q-V curves obtained by other than depolarizing pulses from negative saturated holding potentials.     

Kinetic isoforms of intramembrane charge in intact amphibian striated muscle

The effects of the ryanodine receptor (RyR) antagonists ryanodine and daunorubicin on the kinetic and steady-state properties of intramembrane charge were investigated in intact voltage-clamped frog skeletal muscle fibers under conditions that minimized time-dependent ionic currents. A hypothesis that RyR gating is allosterically coupled to configurational changes in dihydropyridine receptors (DHPRs) would predict that such interactions are reciprocal and that RyR modification should influence intramembrane charge. Both agents indeed modified the time course of charging transients at 100-200-microM concentrations. They independently abolished the delayed charging phases shown by q gamma currents, even in fibers held at fully polarized, -90-mV holding potentials; such waveforms are especially prominent in extracellular solutions containing gluconate. Charge movements consistently became exponential decays to stable baselines in the absence of intervening inward or other time-dependent currents. The steady-state charge transfers nevertheless remained equal through the ON and the OFF parts of test voltage steps. The charge-voltage function, Q(VT), shifted by approximately +10 mV, particularly through those test potentials at which delayed q gamma currents normally took place but retained steepness factors (k approximately 8.0 to 10.6 mV) that indicated persistent, steeply voltage-dependent q gamma contributions. Furthermore, both RyR antagonists preserved the total charge, and its variation with holding potential, Qmax (VH), which also retained similarly high voltage sensitivities (k approximately 7.0 to 9.0 mV). RyR antagonists also preserved the separate identities of q gamma and q beta species, whether defined by their steady-state voltage dependence or inactivation or pharmacological properties. Thus, tetracaine (2 mM) reduced the available steady-state charge movement and gave shallow Q(VT) (k approximately 14 to 16 mV) and Qmax (VH) (k approximately 14 to 17 mV) curves characteristic of q beta charge. These features persisted with exposure to test agent. Finally, q gamma charge movements showed steep voltage dependences with both activation (k approximately 4.0 to 6.5 mV) and inactivation characteristics (k approximately 4.3 to 6.6 mV) distinct from those shown by the remaining q beta charge, whether isolated through differential tetracaine sensitivities, or the full approximation of charge-voltage data to the sum of two Boltzmann distributions. RyR modification thus specifically alters q gamma kinetics while preserving the separate identities of steady-state q beta and q gamma charge. These findings permit a mechanism by which transverse tubular voltage provides the primary driving force for configurational changes in DHPRs, which might produce q gamma charge movement. However, they attribute its kinetic complexities to the reciprocal allosteric coupling by which DHPR voltage sensors and RyR-Ca2+ release channels might interact even though these receptors reside in electrically distinct membranes. RyR modification then would still permit tubular voltage change to drive net q gamma charge transfer but would transform its complex waveforms into simple exponential decays.     

Existence of Q gamma in frog cut twitch fibers with little Q beta.

Charge movements were measured in frog cut twitch fibers with the double Vaseline-gap voltage-clamp technique. In most fibers, when a depolarizing pulse to -60 to -40 mV was applied at 13-14 degrees C, the ON segment of a charge movement trace showed an early I beta component and a late I gamma hump component. An ongoing controversy is whether the I gamma hump component triggers calcium release from the sarcoplasmic reticulum or arises as a consequence of the release. Interestingly, a number of cut fibers showed normal I gamma components but greatly diminished, or unresolvable, I beta components. When the amount of charge associated with the current transient was plotted against the membrane potential, the steeply voltage-dependent Q gamma component appeared normal whereas the less steeply voltage-dependent Q beta component was also greatly diminished or unresolvable. These results suggest that I gamma can flow in the absence of I beta, thereby ruling out the possibility that Q beta triggers calcium release which, in turn, causes Q gamma to move. The results, however, do not rule out the positive feedback of calcium release to activate Q gamma, if calcium release is not triggered by Q beta but by Q gamma itself or by some other signal.     

Comparison of charge movement components in intact and cut twitch fibers of the frog. Effects of stretch and temperature

Charge movements were measured in frog intact fibers with the three-microelectrode technique and in cut fibers with the double Vaseline gap technique. At 13-14 degrees C, the ON segments of charge movement records from both preparations showed an early I beta component and a late I gamma hump component. When an intact fiber was cooled to 4-7 degrees C, the time-to-peak of I gamma (tp,gamma) was prolonged, but I gamma still appeared as a hump. Q-V plots from intact fibers at 4-7 degrees C were fitted with a sum of two Boltzmann distribution functions (method 1). The more steeply voltage-dependent component, identified with Q gamma, accounted for 32.1% (SEM 2.2%) of the total charge. This fraction was larger than the 22.6% (SEM 1.5%) obtained by separating the ON currents with a sum of two kinetic functions (method 2). The total charge in cut fibers stretched to a sarcomere length of 3.5 microns at 13-14 degrees C was separated into Q beta and Q gamma by methods 1 and 2. The fraction of Q gamma in the total charge was 51.3% (SEM 1.7%) and 53.7% (SEM 1.8%), respectively, suggesting that cut fibers have a larger proportion of Q gamma:Q beta than intact fibers. When cut fibers were stretched to a sarcomere length of 4 microns, the proportion of Q gamma:Q beta was unchanged. Between 4 and 13 degrees C, the Q10 of l/tp,gamma in intact fibers was 2.33 (SEM 0.33) and that of 1/tau beta was less than 1.44 (SEM 0.04), implying that the kinetics of I gamma has a steeper temperature dependence than the kinetics of I beta. When cut fibers were cooled from 14 to 6 degrees C, I gamma in the ON segment generally became too broad to be manifested as a hump. In a cut fiber in which I gamma was manifested as a hump, the Q10 of l/tp,gamma was 2.08 and that of l/tau beta was less than 1.47. Separating the Q-V plots from cut fibers at different temperatures by method 1 showed that the proportion of Q gamma:Q beta was unaffected by temperature change. The appearance of I gamma humps at low temperatures in intact fibers but generally not in cut fibers suggests an intrinsic difference between the two fiber preparations.     

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.     

Steady-state availability of sodium channels. Interactions between activation and slow inactivation.

Changes in holding potential (Vh), affect both gating charge (the Q(Vh) curve) and peak ionic current (the F(Vh) curve) seen at positive test potentials. Careful comparison of the Q(Vh) and F(Vh) distributions indicates that these curves are similar, having two slopes (approximately 2.5e for Vh from -115 to -90 mV and approximately 4e for Vh from -90 to -65 mV) and very negative midpoints (approximately -86 mV). Thus, gating charge movement and channel availability appear closely coupled under fully-equilibrated conditions. The time course by which channels approach equilibration was explored using depolarizing prepulses of increasing duration. The high slope component seen in the F(Vh) and Q(Vh) curves is not evident following short depolarizing prepulses in which the prepulse duration approximately corresponds to the settling time for fast inactivation. Increasing the prepulse duration to 10 ms or longer reveals the high slope, and left-shifts the midpoint to more negative voltages, towards the F(Vh) and Q(Vh) distributions. These results indicate that a separate slow-moving voltage sensor affects the channels at prepulse durations greater than 10 ms. Charge movement and channel availability remain closely coupled as equilibrium is approached using depolarizing pulses of increasing durations. Both measures are 50% complete by 50 ms at a prepulse potential of -70 mV, with proportionately faster onset rates when the prepulse potential is more depolarized. By contrast, charge movement and channel availability dissociate during recovery from prolonged depolarizations. Recovery of gating charge is considerably faster than recovery of sodium ionic current after equilibration at depolarized potentials. Recovery of gating charge at -140 mV, is 65% complete within approximately 100 ms, whereas less than 30% of ionic current has recovered by this time. Thus, charge movement and channel availability appear to be uncoupled during recovery, although both rates remain voltage sensitive. These data suggest that channels remain inactivated due to a separate process operating in parallel with the fast gating charge. We demonstrate that this behavior can be simulated by a model in which the fast charge movement associated with channel activation is electrostatically-coupled to a separate slow voltage sensor responsible for the slow inactivation of channel conductance.     

Holding potential affects the apparent voltage-sensitivity of sodium channel activation in crayfish giant axons.

Sodium channel activations, measured as the fraction of channels open to peak conductance for different test potentials (F[V]), shows two statistically different slopes from holding potential more positive than -90 mV. A high valence of 4-6e is indicated a test potentials within 35 mV of the apparent threshold potential (circa -65 mV at -85 mV holding potential). However, for test potentials positive to -30 mV, the F(V) curve shows a 2e valence. The F(V) curve for crayfish axon sodium channels at these "depolarized" holding potentials thus closely resembles classic data obtained from other preparations at holding potentials between -80 and -60 mV. In contrast, at holding potentials more negative than -100 mV, the high slope essentially disappears and the F(V) curve follows a single Boltzmann distribution with a valence of approximately 2e at all potentials. Neither the slope of this simple distribution nor its midpoint (-20 mV) was significantly affected by removal of fast inactivation with pronase. The change in F(V) slope, when holding potential is increased from -85 to -120 mV, does not appear to be caused by the contribution of a second channel type. The simple voltage dependence of sodium current found at Vh -120 mV be used by to discriminate between models of sodium channel activation, and rules out models with three particles of equal valence.     

Effects of the Enantiomers of BayK 8644 on the Charge Movement of L-type Ca Channels in Guinea-pig Ventricular Myocytes

The effects of the agonist enantiomer S(-)Bay K 8644 on gating charge of L-type Ca channels were studied in single ventricular myocytes. From a holding potential (Vh) of -40 mV, saturating (250 nm) S(-)Bay K shifted the half-distribution voltage of the activation charge (Q1) vs. V curve -7.5 +/- 0.8 mV, almost identical to the shift produced in the Ba conductance vs. V curve (-7.7 +/- 2 mV). The maximum Q1 was reduced by 1.7 +/- 0.2 nC/microF, whereas Q2 (charge moved in inactivated channels) was increased in a similar amount (1.4 +/- 0.4 nC/microF). The steady-state availability curves for Q1, Q2, and Ba current showed almost identical negative shifts of -14.8 +/- 1.7 mV, -18.6 +/- 5.8 mV, and -15.2 +/- 2.7 mV, respectively. The effects of the antagonist enantiomer R(+)BayK 8644 were also studied, the Q1 vs. V curve was not significantly shifted, but Q1max (Vh = -40 mV) was reduced and the Q1 availability curve shifted by -24.6 +/- 1.2 mV. We concluded that: a) the left shift in the Q1 vs. V activation curve produced by S(-)BayK is a purely agonistic effect; b) S(-)BayK induced a significantly larger negative shift in the availability curve than in the Q1 vs. V relation, consistent with a direct promotion of inactivation; c) as expected for a more potent antagonist, R(+)Bay K induced a significantly larger negative shift in the availability curve than did S(-)Bay K.     

Sodium channel gating currents in frog skeletal muscle

Charge movements similar to those attributed to the sodium channel gating mechanism in nerve have been measured in frog skeletal muscle using the vaseline-gap voltage-clamp technique. The time course of gating currents elicited by moderate to strong depolarizations could be well fitted by the sum of two exponentials. The gating charge exhibits immobilization: at a holding potential of -90 mV the proportion of charge that returns after a depolarizing prepulse (OFF charge) decreases with the duration of the prepulse with a time course similar to inactivation of sodium currents measured in the same fiber at the same potential. OFF charge movements elicited by a return to more negative holding potentials of -120 or -150 mV show distinct fast and slow phases. At these holding potentials the total charge moved during both phases of the gating current is equal to the ON charge moved during the preceding prepulse. It is suggested that the slow component of OFF charge movement represents the slower return of charge "immobilized" during the prepulse. A slow mechanism of charge immobilization is also evident: the maximum charge moved for a strong depolarization is approximately doubled by changing the holding potential from -90 to -150 mV. Although they are larger in magnitude for a -150-mV holding potential, the gating currents elicited by steps to a given potential have similar kinetics whether the holding potential is -90 or -150 mV.     

Gating of the bacterial sodium channel, NaChBac: voltage-dependent charge movement and gating currents

The bacterial sodium channel, NaChBac, from Bacillus halodurans provides an excellent model to study structure-function relationships of voltage-gated ion channels. It can be expressed in mammalian cells for functional studies as well as in bacterial cultures as starting material for protein purification for fine biochemical and biophysical studies. Macroscopic functional properties of NaChBac have been described previously (Ren, D., B. Navarro, H. Xu, L. Yue, Q. Shi, and D.E. Clapham. 2001. Science. 294:2372-2375). In this study, we report gating current properties of NaChBac expressed in COS-1 cells. Upon depolarization of the membrane, gating currents appeared as upward inflections preceding the ionic currents. Gating currents were detectable at -90 mV while holding at -150 mV. Charge-voltage (Q-V) curves showed sigmoidal dependence on voltage with gating charge saturating at -10 mV. Charge movement was shifted by -22 mV relative to the conductance-voltage curve, indicating the presence of more than one closed state. Consistent with this was the Cole-Moore shift of 533 micros observed for a change in preconditioning voltage from -160 to -80 mV. The total gating charge was estimated to be 16 elementary charges per channel. Charge immobilization caused by prolonged depolarization was also observed; Q-V curves were shifted by approximately -60 mV to hyperpolarized potentials when cells were held at 0 mV. The kinetic properties of NaChBac were simulated by simultaneous fit of sodium currents at various voltages to a sequential kinetic model. Gating current kinetics predicted from ionic current experiments resembled the experimental data, indicating that gating currents are coupled to activation of NaChBac and confirming the assertion that this channel undergoes several transitions between closed states before channel opening. The results indicate that NaChBac has several closed states with voltage-dependent transitions between them realized by translocation of gating charge that causes activation of the channel.     

Effects of D-600 on intramembrane charge movement of polarized and depolarized frog muscle fibers

Intramembrane charge movement has been measured in frog cut skeletal muscle fibers using the triple vaseline gap voltage-clamp technique. Ionic currents were reduced using an external solution prepared with tetraethylammonium to block potassium currents, and O sodium + tetrodotoxin to abolish sodium currents. The internal solution contained 10 mM EGTA to prevent contractions. Both the internal and external solutions were prepared with impermeant anions. Linear capacitive currents were subtracted using the P-P/4 procedure, with the control pulses being subtracted either at very negative potentials, for the case of polarized fibers, or at positive potentials, for the case of depolarized fibers. In 63 polarized fibers dissected from Rana pipiens or Leptodactylus insularis frogs the following values were obtained for charge movement parameters: Qmax = 39 nC/microF, V = 36 mV, k = 18.5 mV. After depolarization we found that the total amount of movable charge was not appreciably reduced, while the voltage sensitivity was much changed. For 10 fibers, in which charge movement was measured at -100 and at 0 mV, Qmax changed from 46 to 41 nC/microF, while V changed from -41 to -103 mV and k changed from 20.5 to 30 mV. Thus membrane depolarization to 0 mV produces a shift of greater than 50 mV in the Q-V relationship and a decrease of the slope. Membrane depolarization to -20 and -30 mV, caused a smaller shift of the Q-V relationship. In normally polarized fibers addition of D-600 at concentrations of 50-100 microM, does not cause important changes in charge movement parameters. However, the drug appears to have a use-dependent effect after depolarization. Thus in depolarized fibers, total charge is reduced by approximately 20%. D-600 causes no further changes in the voltage sensitivity of charge movement in fibers depolarized to 0 mV, while in fibers depolarized to -20 and -30 mV it causes the same effects as that obtained with depolarization to 0 mV. These results are compatible with the idea that after depolarization charge 1 is transformed into charge 2. D-600 appears to favor the conversion of charge 1 into charge 2. Since D-600 also favors contractile inactivation, charge 2 could represent the state of the voltage sensor for excitation-contraction coupling in the inactivated state.     

Contractile inactivation in frog skeletal muscle fibers. The effects of low calcium, tetracaine, dantrolene, D-600, and nifedipine

Short muscle fibers (approximately 1.5 mm) of Rana pipiens were voltage-clamped with a two-microelectrode technique at a holding potential of -100 mV. Using conditioning depolarizing ramps, with slopes greater than 0.2 mV/s, partially inactivated responses are obtained at threshold values between -55 and -35 mV. With slopes equal to or slower than 0.1 mV/s, one inactivates contraction without ever activating it. When the membrane potential is brought slowly to values more positive than about -40 mV, test pulses, applied on top of the ramps, bringing the membrane potential to values up to +100 mV, are ineffective in eliciting contractile responses, which indicates complete inactivation. After inactivation, contractile threshold is shifted by perhaps 10 mV, to about -40 mV. The sensitivity of fibers to depolarizing ramps is increased by D-600 (50 microM), dantrolene (50 microM), tetracaine (100 microM), and low calcium (10(-8) M). In the presence of these agents, complete inactivation was obtained using ramp slopes of 1, 0.8, 0.4, and 0.2 mV/s, respectively. Nifedipine was less effective. With D-600, once inactivation had been induced, no repriming occurred after repolarization to -100 mV, and partial recovery occurred after washing out the drug. With low calcium, tetracaine, and nifedipine, the tension-voltage relationship was not affected, whereas the steady state inactivation curve (obtained in repriming experiments) was shifted by 10-25 mV toward more negative potentials. With D-600, the activation curve was not modified, whereas the inactivation curve could not be obtained, because of repriming failure. With dantrolene, the inactivation curve was not affected, whereas the activation curve was shifted toward less negative potentials and peak tension diminished, depending on the pulse duration. The results indicate that it is possible to induce complete inactivation without activation, and to differentiate activation and inactivation parameters pharmacologically, which suggests that the two are separate processes.     

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.     

Factors affecting the appearance of the hump charge movement component in frog cut twitch fibers.

Charge movement was measured in frog cut twitch fibers with the double Vaseline gap technique. Five manipulations listed below were applied to investigate their effects on the hump component (I gamma) in the ON segments of TEST minus CONTROL current traces. When external Cl-1 was replaced by MeSO3- to eliminate Cl current, I gamma peaked earlier due to a few millivolts shift of the voltage dependence of I gamma kinetics in the negative direction. The Q-V plots in the TEA.Cl and TEA.MeSO3 solutions were well fitted by a sum of two Boltzmann distribution functions. The more steeply voltage-dependent component (Q gamma) had a V approximately 6 mV more negative in the TEA.MeSO3 solution than in the TEA.Cl solution. These voltage shifts were partially reversible. When creatine phosphate in the end pool solution was removed, the I gamma hump disappeared slowly over the course of 20-30 min, partly due to a suppression of Q gamma. The hump reappeared when creatine phosphate was restored. When 0.2-1.0 mM Cd2+ was added to the center pool solution to block inward Ca current, the I gamma hump became less prominent due to a prolongation in the time course of I gamma but not to a suppression of Q gamma. When the holding potential was changed from -90 to -120 mV, the amplitude of I beta was increased, thereby obscuring the I gamma hump. Finally, when a cut fiber was stimulated repetitively, I gamma lost its hump appearance because its time course was prolonged. In an extreme case, a 5-min resting interval was insufficient for a complete recovery of the waveform. In general, a stimulation rate of once per minute had a negligible effect on the shape of I gamma. Of the five manipulations, MeSO3- has the least perturbation on the appearance of I gamma and is potentially a better substitute for Cl- than SO2-(4) in eliminating Cl current if the appearance of the I gamma hump is to be preserved.     

Calcium currents in embryonic and neonatal mammalian skeletal muscle

The whole-cell patch-clamp technique was used to study the properties of inward ionic currents found in primary cultures of rat and mouse skeletal myotubes and in freshly dissociated fibers of the flexor digitorum brevis muscle of rats. In each of these cell types, test depolarizations from the holding potential (-80 or -90 mV) elicited three distinct inward currents: a sodium current (INa) and two calcium currents. INa was the dominant inward current: under physiological conditions, the maximum inward INa was estimated to be at least 30-fold larger than either of the calcium currents. The two calcium currents have been termed Ifast and Islow, corresponding to their relative rates of activation. Ifast was activated by test depolarizations to around -40 mV and above, peaked in 10-20 ms, and decayed to baseline in 50-100 ms. Islow was activated by depolarizations to approximately 0 mV and above, peaked in 50-150 ms, and decayed little during a 200-ms test pulse. Ifast was inactivated by brief, moderate depolarizations; for a 1-s change in holding potential, half-inactivation occurred at -55 to -45 mV and complete inactivation occurred at -40 to -30 mV. Similar changes in holding potential had no effect on Islow. Islow was, however, inactivated by brief, strong depolarizations (e.g., 0 mV for 2 s) or maintained, moderate depolarizations (e.g., -40 mV for 60 s). Substitution of barium for calcium had little effect on the magnitude or time course of either Ifast or Islow. The same substitution shifted the activation curve for Islow approximately 10 mV in the hyperpolarizing direction without affecting the activation of Ifast. At low concentrations (50 microM), cadmium preferentially blocked Islow compared with Ifast, while at high concentrations (1 mM), it blocked both Ifast and Islow completely. The dihydropyridine calcium channel antagonist (+)-PN 200-110 (1 microM) caused a nearly complete block of Islow without affecting Ifast. At a holding potential of -80 mV, the half-maximal blocking concentration (K0.5) for the block of Islow by (+)-PN 200-110 was 182 nM. At depolarized holding potentials that inactivated Islow by 35-65%, K0.5 decreased to 5.5 nM.     

Saxitoxin and tetrodotoxin. Electrostatic effects on sodium channel gating current in crayfish axons.

The effects of extracellular saxitoxin (STX) and tetrodotoxin (TTX) on gating current (IgON) were studied in voltage clamped crayfish giant axons. At a holding potential (VH) of -90 mV, integrated gating charge (QON) was found to be 56% suppressed when 200 nM STX was added to the external solution, and 75% suppressed following the addition of 200 nM TTX. These concentrations of toxin are sufficiently high to block greater than 99% of sodium channels. A smaller suppression of IgON was observed when 1 nM STX was used (KD = 1-2 nM STX). The suppression of IgON by external toxin was found to be hold potential dependent, with only minimal suppression observed at the most hyperpolarized hold potentials, -140 to -120 mV. The maximal effect of these toxins on IgON was observed at hold potentials where the QON vs. VH plot was found to be steepest, -100 to -80 mV. The suppression of IgON induced by TTX is partially relieved following the removal of fast inactivation by intracellular treatment with N-bromoacetamide (NBA). The effect of STX and TTX on IgON is equivalent to a hyperpolarizing shift in the steady state inactivation curve, with 200 nM STX and 200 nM TTX inducing shifts of 4.9 +/- 1.7 mV and 10.0 +/- 2.1 mV, respectively. Our results are consistent with a model where the binding of toxin displaces a divalent cation from a negatively charged site near the external opening of the sodium channel, thereby producing a voltage offset sensed by the channel gating apparatus.     

Presteady-state and steady-state kinetics and turnover rate of the mouse gamma-aminobutyric acid transporter (mGAT3)

We expressed mouse gamma-aminobutyric acid (GABA) transporter (mGAT3) in Xenopus laevis oocytes and examined its steady-state and presteady-state kinetics and turnover rate by using tracer flux and electrophysiological methods. In oocytes expressing mGAT3, GABA evoked a Na+-dependent and Cl(-)-facilitated inward current. The dependence on Na+ was absolute, whereas that for Cl(-) was not. At a membrane potential of -50 mV, the half-maximal concentrations for Na+, Cl(-), and GABA were 14 mM, 5 mM, and 3 microM. The Hill coefficient for GABA activation and Cl(-) enhancement of the inward current was 1, and that for Na+ activation was > or =2. The GABA-evoked inward current was directly proportional to GABA influx (2.2 +/- 0.1 charges/GABA) into cells, indicating that under these conditions, there is tight ion/GABA coupling in the transport cycle. In response to step changes in the membrane voltage and in the absence of GABA, mGAT3 exhibited presteady-state current transients (charge movements). The charge-voltage (Q-V) relation was fitted with a single Boltzmann function. The voltage at half-maximal charge (V(0.5)) was +25 mV, and the effective valence of the moveable charge (zdelta) was 1.6. In contrast to the ON transients, which relaxed with a time constant of < or =30 msec, the OFF transients had a time constant of 1.1 sec. Reduction in external Na+ ([Na+]o) and Cl(-) ([Cl(-)]o) concentrations shifted the Q-V relationship to negative membrane potentials. At zero [Na+]o (106 mM Cl(-)), no mGAT3-mediated transients were observed, and at zero [Cl(-)]o (100 mM Na+), the charge movements decreased to approximately 30% of the maximal charge (Q(max)). GABA led to the elimination of charge movements. The half-maximal concentrations for Na+ activation, Cl(-) enhancement, and GABA elimination of the charge movements were 48 mM, 19 mM, and 5 mM, respectively. Q(max) and I(max) obtained in the same cells yielded the mGAT3 turnover rate, 1.7 sec(-1) at -50 mV. The low turnover rate of mGAT3 may be due to the slow return of the empty transporter from the internal to the external membrane surface.