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.     

Effects of conditioning depolarization and repetitive stimulation on Q beta and Q gamma charge components in frog cut twitch fibers

Charge movement was measured in frog cut twitch fibers with the double Vaseline-gap technique. Steady-state inactivation of charge movement was studied by changing the holding potential from -90 mV to a level ranging from -70 to -30 mV. Q beta and Q gamma at each holding potential were separated by fitting the Q-V plot with a sum of two Boltzmann distribution functions. At -70 mV Q beta and Q gamma were inactivated to 54.0% (SEM 2.2) and 82.7% (SEM 3.0) of the amounts at -90 mV. At holding potentials greater than or equal to -60 mV, more Q gamma was inactivated than Q beta, and at -30 mV Q gamma was completely inactivated but Q beta was not. There was no holding potential at which Q beta was unaffected and Q gamma was completely inactivated. The differences between the residual fractions of Q beta and Q gamma are significant at all holding potentials (P less than 0.001-0.05). The plot of the residual fraction of Q beta or Q gamma versus holding potential can be fitted well by an inverted sigmoidal curve that is a mirror image of the activation curve of the respective charge component. The pair of curves for Q gamma correlates well with those for tension generation or Ca release obtained by other investigators. The time courses of the inactivation of Q beta and Q gamma were studied by obtaining several Q-V plots with conditioning depolarizations lasting 1-20 s and separating each Q-V plot into Q beta and Q gamma components by fitting with a sum of two Boltzmann distribution functions. The inactivation time constant of Q beta was found to be 5-10 times as large as that of Q gamma. During repetitive stimulation, prominent I gamma humps could be observed in TEST-minus-CONTROL current traces and normal Q gamma components could be separated from the Q-V plots, whether 20 or 50 mM EGTA was present in the internal solution, whether 2 or 10 stimulations were used, and whether the stimuli were separated by 400 ms or 6 s. Repetitive stimulation slowed the kinetics of the I gamma hump and could shift the Q-V curve slightly in the depolarizing direction in some cases, resulting in an apparent suppression of charge at the potentials that fall on the steep part of the Q-V curve.     

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.     

Two classes of gating current from L-type Ca channels in guinea pig ventricular myocytes.

Intramembrane charge movement was recorded in guinea pig ventricular myocytes at 19-22 degrees C using the whole-cell patch clamp technique. From a holding potential of -110 mV, the dependence of intramembrane charge moved on test voltage (Q(V)) followed the sum of two Boltzmann components. One component had a transition voltage (V) of -48 mV and a total charge (Qmax) of congruent to 3 nC/microF. The other had a V of -18 mV and a Qmax of 11 nC/microF. Ba2+ currents through Ca channels began to activate at -45 mV and peaked at congruent to -15 mV. Na+ current peaked at -35 to -30 mV. Availability of charge (in pulses from -70 to +10 mV) depended on the voltage of conditioning depolarizations as two Boltzmann terms plus a constant. One term had a V of -88 mV and a Qmax of 2.5 nC/microF; the other had a V of -29 mV and a Qmax of 6.3 nC/microF. From the Q(V) dependence, the voltage dependence of the ionic currents, and the voltage dependence of the availability of charge, the low voltage term of Q(V) and availability was identified as Na gating charge, at a total of 3.5 nC/microF. The remainder, 11 nC/microF, was attributed to Ca channels. After pulses to -40 mV and above, the OFF charge movement had a slow exponentially decaying component. Its time constant had a bell-shaped dependence on OFF voltage peaking at 11 ms near -100 mV. Conditioning depolarizations above -40 mV increased the slow component exponentially with the conditioning duration (tau approximately equal to 480 ms). Its magnitude was reduced as the separation between conditioning and test pulses increased (tau approximately equal to 160 ms). The voltage distribution of the slow component of charge was measured after long (5 s) depolarizations. Its V was -100 mV, a shift of -80 mV from the value in normally polarized cells. This voltage was the same at which the time constant of the slow component peaked. Qmax and the steepness of the voltage distribution were unchanged by depolarization. This indicates that the same molecules that produce the charge movement in normally polarized cells also produce the slow component in depolarized cells. 100 microns D600 increased by 77% the slow charge movement after a 500-ms conditioning pulse. These results demonstrate two classes of charge movement associated with L-type Ca channels, with kinetics and voltage dependence similar to charge 1 and charge 2 of skeletal muscle.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

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.     

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.     

Gating of the expressed Cav3.1 calcium channel

Intramembrane charge movement originating from Cav3.1 (T-type) channel expressed in HEK 293 cells was investigated. Ion current was blocked by 1 mM La3+. Charge movement was detectable for depolarizations above approximately -70 mV and saturated above +60 mV. The voltage dependence of charge movement followed a single Boltzmann function with half-maximal activation voltage +12.9 mV and +12.3 mV and with slopes of 22.4 mV and 18.1 mV for the ON- and OFF-charge movement, respectively. Inactivation of I(Ca) by prolonged depolarization pulse did not immobilize intramembrane charge movement in the Cav3.1 channel.     

Intramembranous charge movement in frog cut twitch fibers mounted in a double vaseline-gap chamber

Intramembranous charge movement was measured in cut twitch fibers mounted in a double Vaseline-gap chamber with either a tetraethylammonium chloride (TEA.Cl) or a TEA2.SO4 solution (13-14 degrees C) in the central pool. Charge vs. voltage data were fitted by a single two-state Boltzmann distribution function. The average values of V (the voltage at which steady-state charge is equally distributed between the two Boltzmann states), k (the voltage dependence factor), and qmax/cm (the maximum charge divided by the linear capacitance, both per unit length of fiber) were V = -53.3 mV (SEM, 1.1 mV), k = 6.3 mV (SEM, 0.3 mV), qmax/cm = 18.0 nC/microF (SEM, 1.1 nC/microF) in the TEA.Cl solution; and V = -35.1 mV (SEM, 1.8 mV), k = 10.5 mV (SEM, 0.9 mV), qmax/cm = 36.3 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. These values of k are smaller than those previously reported for cut twitch fibers and are as small as those reported for intact fibers. If a correction is made for the contributions of currents from under the Vaseline seals, V = -51.2 mV (SEM, 1.1 mV), k = 7.2 mV (SEM, 0.4 mV), qmax/cm = 22.9 nC/microF (SEM, 1.4 nC/microF) in the TEA.Cl solution; and V = -34.0 mV (SEM, 1.9 mV), k = 10.1 mV (SEM, 1.1 mV), qmax/cm = 38.8 nC/microF (SEM, 3.2 nC/microF) in the TEA2.SO4 solution. With this correction, however, the fit of the theoretical curve to the data is poor. A good fit with this correction can be obtained with a sum of two Boltzmann distribution functions. The first has average values V = -33.0 mV (SEM, 2.8 mV), k = 11.0 mV (SEM, 0.5 mV), qmax/cm = 10.6 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -20.0 mV (SEM, 3.3 mV), k = 17.0 mV (SEM, 2.0 mV), qmax/cm = 36.4 nC/microF (SEM, 2.3 nC/microF) in the TEA2.SO4 solution. The second has average values V = -56.5 mV (SEM, 1.3 mV), k = 2.9 mV (SEM, 0.4 mV), qmax/cm = 13.2 nC/microF (SEM, 1.0 nC/microF) in the TEA.Cl solution; and V = -41.6 mV (SEM, 1.4 mV), k = 2.5 mV (SEM, 0.8 mV), qmax/cm = 11.8 nC/microF (SEM, 1.7 nC/microF) in the TEA2.SO4 solution. When a fiber is depolarized to near V of the second Boltzmann function, a slowly developing "hump" appears in the ON-segment of the current record.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effect of sarcoplasmic reticulum calcium depletion on intramembranous charge movement in frog cut muscle fibers

Cut muscle fibers from Rana temporaria (sarcomere length, 3.3-3.5 microns; temperature, 13-16 degrees C) were mounted in a double Vaseline-gap chamber and equilibrated for at least an hour with an internal solution that contained 20 mM EGTA and phenol red and an external solution that contained predominantly TEA-gluconate; both solutions were nominally Ca-free. The increase in total myoplasmic concentration of Ca (delta[CaT]) produced by sarcoplasmic reticulum (SR) Ca release was estimated from the change in pH produced when the released Ca was complexed by EGTA (Pape, P.C., D.-S. Jong, and W.K. Chandler. 1995. Journal of General Physiology. 106:259-336). The resting value of SR Ca content, [CaSR]R (expressed as myoplasmic concentration), was taken to be equal to the value of delta[CaT] obtained during a step depolarization (usually to -50 to -40 mV) that was sufficiently long (200-750 ms) to release all of the readily releasable Ca from the SR. In ten fibers, the first depolarization gave [CaSR]R = 839-1,698 microM. Progressively smaller values were obtained with subsequent depolarizations until, after 30-40 depolarizations, the value of [CaSR]R had usually been reduced to < 10 microM. Measurements of intramembranous charge movement, Icm, showed that, as the value of [CaSR]R decreased, ON-OFF charge equality held and the amount of charge moved remained constant. ON Icm showed brief initial I beta components and prominent I gamma "humps", even after the value of [CaSR]R was < 10 microM. Although the amplitude of the hump component decreased during depletion, its duration increased in a manner that preserved the constancy of ON charge. In the depleted state, charge movement was steeply voltage dependent, with a mean value of 7.2 mV for the Boltzmann factor k. These and other results are not consistent with the idea that there is one type of charge, Q beta, and that I gamma is a movement of Q beta caused by SR Ca release, as proposed by Pizarro, Csernoch, Uribe, Rodriguez, and Rios (1991. Journal of General Physiology. 97:913-947). Rather, our results imply that Q beta and Q gamma represent either two distinct species of charge or two transitions with different properties of a single species of charge, and that SR Ca content or release or some related event alters the kinetics, but not the amount of Q gamma. Many of the properties of Q gamma, as well as the voltage dependence of the rate of SR Ca release for small depolarizations, are consistent with predictions from a simple model in which the voltage sensor for SR Ca release consists of four interacting charge movement particles.     

The relationship between Q gamma and Ca release from the sarcoplasmic reticulum in skeletal muscle

Asymmetric membrane currents and fluxes of Ca2+ release were determined in skeletal muscle fibers voltage clamped in a Vaseline-gap chamber. The conditioning pulse protocol 1 for suppressing Ca2+ release and the "hump" component of charge movement current (I gamma), described in the first paper of this series, was applied at different test pulse voltages. The amplitude of the current suppressed during the ON transient reached a maximum at slightly suprathreshold test voltages (-50 to -40 mV) and decayed at higher voltages. The component of charge movement current suppressed by 20 microM tetracaine also went through a maximum at low pulse voltages. This anomalous voltage dependence is thus a property of I gamma, defined by either the conditioning protocol or the tetracaine effect. A negative (inward-going) phase was often observed in the asymmetric current during the ON of depolarizing pulses. This inward phase was shown to be an intramembranous charge movement based on (a) its presence in the records of total membrane current, (b) its voltage dependence, with a maximum at slightly suprathreshold voltages, (c) its association with a "hump" in the asymmetric current, (d) its inhibition by interventions that reduce the "hump", (e) equality of ON and OFF areas in the records of asymmetric current presenting this inward phase, and (f) its kinetic relationship with the time derivative of Ca release flux. The nonmonotonic voltage dependence of the amplitude of the hump and the possibility of an inward phase of intramembranous charge movement are used as the main criteria in the quantitative testing of a specific model. According to this model, released Ca2+ binds to negatively charged sites on the myoplasmic face of the voltage sensor and increases the local transmembrane potential, thus driving additional charge movement (the hump). This model successfully predicts the anomalous voltage dependence and all the kinetic properties of I gamma described in the previous papers. It also accounts for the inward phase in total asymmetric current and in the current suppressed by protocol 1. According to this model, I gamma accompanies activating transitions at the same set of voltage sensors as I beta. Therefore it should open additional release channels, which in turn should cause more I gamma, providing a positive feedback mechanism in the regulation of calcium release.     

Patch-clamp recording of charge movement, Ca(2+) current, and Ca(2+) transients in adult skeletal muscle fibers

Intramembrane charge movement (Q), Ca(2+) conductance (G(m)) through the dihydropyridine-sensitive L-type Ca(2+) channel (DHPR) and intracellular Ca(2+) fluorescence (F) have been recorded simultaneously in flexor digitorum brevis muscle fibers of adult mice, using the whole-cell configuration of the patch-clamp technique. The voltage distribution of Q was fitted to a Boltzmann equation; the Q(max), V(1/2Q), and effective valence (z(Q)) values were 41 +/- 3.1 nC/&mgr;F, -17.6 +/- 0.7 mV, and 2.0 +/- 0.12, respectively. V(1/2G) and z(G) values were -0.3 +/- 0.06 mV and 5.6 +/- 0.34, respectively. Peak Ca(2+) transients did not change significantly after 30 min of recording. F was fit to a Boltzmann equation, and the values for V(F1/2) and z(F) were 6.2 +/- 0.04 mV and 2.4, respectively. F was adequately fit to the fourth power of Q. These results demonstrate that the patch-clamp technique is appropriate for recording Q, G(m), and intracellular [Ca(2+)] simultaneously in mature skeletal muscle fibers and that the voltage distribution of the changes in intracellular Ca(2+) can be predicted by a Hodgkin-Huxley model.     

Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca(2+).

Large-conductance Ca(2+)-activated K(+) channels can be activated by membrane voltage in the absence of Ca(2+) binding, indicating that these channels contain an intrinsic voltage sensor. The properties of this voltage sensor and its relationship to channel activation were examined by studying gating charge movement from mSlo Ca(2+)-activated K(+) channels in the virtual absence of Ca(2+) (<1 nM). Charge movement was measured in response to voltage steps or sinusoidal voltage commands. The charge-voltage relationship (Q-V) is shallower and shifted to more negative voltages than the voltage-dependent open probability (G-V). Both ON and OFF gating currents evoked by brief (0.5-ms) voltage pulses appear to decay rapidly (tau(ON) = 60 microseconds at +200 mV, tau(OFF) = 16 microseconds at -80 mV). However, Q(OFF) increases slowly with pulse duration, indicating that a large fraction of ON charge develops with a time course comparable to that of I(K) activation. The slow onset of this gating charge prevents its detection as a component of I(gON), although it represents approximately 40% of the total charge moved at +140 mV. The decay of I(gOFF) is slowed after depolarizations that open mSlo channels. Yet, the majority of open channel charge relaxation is too rapid to be limited by channel closing. These results can be understood in terms of the allosteric voltage-gating scheme developed in the preceding paper (Horrigan, F.T., J. Cui, and R.W. Aldrich. 1999. J. Gen. Physiol. 114:277-304). The model contains five open (O) and five closed (C) states arranged in parallel, and the kinetic and steady-state properties of mSlo gating currents exhibit multiple components associated with C-C, O-O, and C-O transitions.     

Effect of the calcium buffer EGTA on the "hump" component of charge movement in skeletal muscle.

Three manifestations of excitation-contraction (E-C) coupling were measured in cut skeletal muscle fibers of the frog, voltage clamped in a double Vaseline gap: intramembrane charge movements, myoplasmic Ca2+ transients, and changes in optical transparency. Pulsing patterns in the presence of high [EGTA] intracellularly, shown by García et al. (1989. J. Gen. Physiol. 94:973-986) to deplete Ca2+ in the sarcoplasmic reticulum, were found to change the above manifestations. With an intracellular solution containing 15 mM EGTA and 0 Ca, 10-15 pulses (100 ms) to -20 mV at a frequency of 2 min-1 reduced the "hump" component of charge movement current. This effect was reversible by 5 min of rest. The same effect was obtained in 62.5 mM EGTA and 0 Ca by pulsing at 0.2 min-1. This effect was reversible by adding calcium to the EGTA solution, for a nominal [Ca2+]i of 200 nM, and was prevented by adding calcium to the EGTA solution before pulsing. The suppression of the hump was accompanied by elimination of the optical manifestations of E-C coupling. The current suppressed was found by subtraction and had the following properties: delayed onset, a peak at a variable interval (10-20 ms) into the pulse, a negative phase (inward current) after the peak, and a variable OFF transient that could be multi-phasic and carried less charge than the ON transient. In the previous paper (Csernoch et al., 1991. J. Gen. Physiol. 97:845-884) it was shown that several interventions suppress a similar component of charge movement current, identified with the "hump" or Q gamma current (I gamma). Based on the similarity to that component, the charge movement suppressed by the depletion protocols can also be identified with I gamma. The fact that I gamma is suppressed by Ca2+ depletion and the kinetic properties of the charge suppressed is inconsistent with the existence of separate sets of voltage sensors underlying the two components of charge movement, Q beta and Q gamma. This is explicable if Q gamma is a consequence of calcium release from the sarcoplasmic reticulum.     

Kinetics of intramembrane charge movement and sodium current in frog node of Ranvier

Intramembrane charge movement (Q) and sodium current (INa) were monitored in isolated voltage-clamped frog nodes of Ranvier, ON charge movements (QON) for pulses from the holding potential (-100 mV) to potentials V less than or equal to 0 mV followed single exponential time courses, whereas two exponentials were found for pulses to V greater than or equal to 20 mV. The voltage dependence of both QON and its time constant tauON indicated that the two ON components resolved at V greater than or equal to 20 mV were also present, though not resolvable, for pulses to V less than or equal to 0 mV. OFF charge movements (QOFF) monitored at various potentials were well described by single exponentials. When QOFF was monitored at -30 or -40 mV after a 200-microsecond pulse to +20 mV and QON was monitored at the same potential using pulses directly from -100 mV, tauON/tauOFF = 2.5 +/- 0.3. At a set OFF potential (-90 to -70 mV), tauOFF first increased with increasing duration tON of the preceding pulse to a given potential (0 to +30 mV) and then decreased with further increases in tON. The declining phase of tauOFF followed a time course similar to that of the decline in QOFF with tON. For the same pulse protocol, the OFF time constant tauNa for INA also first increased with tON but then remained constant over the tON interval during which tauOFF and QOFF were declining. After 200- or 300-microsecond pulses to +20, +20, or +50 mV, tauOFF/tauNa at -70 to -90 mV was 1.2 +/- 0.1. Similar tauOFF/tauNa ratios were predicted by channel models having three identical charged gating particles that can rapidly and reversibly form an immobile dimer or trimer after independently crossing the membrane from their OFF to their ON locations.     

A damped oscillation in the intramembranous charge movement and calcium release flux of frog skeletal muscle fibers

Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of frog skeletal muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Fluorescence studies of ligand-induced conformational changes of the Na(+)/glucose cotransporter.

Conformational changes in the human Na(+)/glucose cotransporter (hSGLT1) were examined using hSGLT1 Q457C expressed in Xenopus laevis oocytes and tagged with tetramethylrhodamine-6-maleimide (TMR6M). Na(+)/glucose cotransport is abolished in the TMR6M-labeled mutant, but the protein binds Na(+) and sugar [Loo et al. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 7789-7794]. Under voltage clamp the fluorescence of labeled Q457C was dependent on external cations. Increasing [Na(+)] increased fluorescence with a Hill coefficient of 2 and half-maximal concentration (K(Na)(0.5)) of 49 mM at -90 mV. Li(+) also increased fluorescence, whereas choline, tetraethylammonium, and N-methyl-D-glucamine did not. Fluorescence was increased by sugars with specificity: methyl alpha-D-glucopyranoside > D-glucose > D-galactose > D-mannitol. Voltage-jump experiments (in 100 mM NaCl buffer in absence of sugar) elicited parallel changes in pre-steady-state charge movement and fluorescence. Charge vs voltage and fluorescence vs voltage curves followed Boltzmann relations with the same median voltage (V(0.5) = -50 mV), but the apparent valence was 1 for charge movement and 0.4 for fluorescence. V(0.5) for fluorescence and charge movement was shifted by -100 mV per 10-fold decrease in [Na(+)]. Under Na(+)-free conditions, there was a voltage-dependent change in fluorescence. Voltage-jump experiments showed that the maximal change in fluorescence increased 20% with sugar. These results indicate that Na(+), sugar, and membrane voltage change the local environment of the fluorophore at Q457C. Our interpretation of these results is (1) the conformational change of the empty transporter is voltage dependent, (2) two Na(+) ions can bind cooperatively to the protein before sugar, and (3) sugar binding induces a conformational change.     

Slow charge movement in mammalian skeletal muscle

Voltage-dependent charge movements were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Contraction was abolished with hypertonic sucrose. The standard (ON-OFF) protocol for eliciting charge movements was to depolarize the fiber from -90 mV to a variable test potential (V) and then repolarize the fiber to -90 mV. The quantity of charge moved saturated at test potentials of approximately 0 mV. The steady state dependence of the amount of charge that moves as a function of test potential could be well fitted by the Boltzmann relation: Q = Qmax/(1 + exp[-(V - V)/k]), where Qmax is the maximum charge that can be moved, V is the potential at which half the charge moves, and k is a constant. At 15 degrees C, these values were Qmax = 28.5 nC/microF, V = -34.2 mV, and k = 8.7 mV. Qmax, k, and V exhibited little temperature dependence over the range 7-25 degrees C. "Stepped OFF" charge movements were elicited by depolarizing the fiber from -90 mV to a fixed conditioning level that moved nearly all the mobile charge (0 mV), and then repolarizing the fiber to varying test potentials. The sum of the charge that moved when the fiber was depolarized directly from -90 mV to a given test potential and the stepped OFF charge that moved when the fiber was repolarized to the same test potential had at all test potentials a value close to Qmax for that fiber. In nearly all cases, the decay phase of ON, OFF, and stepped OFF charge movements could be well fitted with a single exponential. The time constant, tau decay, for an ON charge movement at a given test potential was comparable to tau decay for a stepped OFF charge movement at the same test potential. Tau decay had a bell-shaped dependence on membrane potential: it was slowest at a potential near V (the midpoint of the steady state charge distribution) and became symmetrically faster on either side of this potential. Raising the temperature from 7 to 15 degrees C caused tau decay to become faster by about the same proportion at all potentials, with a Q10 averaging 2.16. Raising the temperature from 15 to 25 degrees C caused tau decay to become faster at potentials near V, but not at potentials farther away.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ca(2+)-dependent inactivation of cardiac L-type Ca2+ channels does not affect their voltage sensor

Inactivation of currents carried by Ba2+ and Ca2+, as well as intramembrane charge movement from L-type Ca2+ channels were studied in guinea pig ventricular myocytes using the whole-cell patch clamp technique. Prolonged (2 s) conditioning depolarization caused substantial reduction of charge movement between -70 and 10 mV (charge 1, or charge from noninactivated channels). In parallel, the charge mobile between -70 and -150 mV (charge 2, or charge from inactivated channels) was increased. The availability of charge 2 depended on the conditioning pulse voltage as the sum of two Boltzmann components. One component had a central voltage of -75 mV and a magnitude of 1.7 nC/microF. It presumably is the charge movement (charge 2) from Na+ channels. The other component, with a central voltage of approximately - 30 mV and a magnitude of 3.5 nC/microF, is the charge 2 of L-type Ca2+ channels. The sum of charge 1 and charge 2 was conserved after different conditioning pulses. The difference between the voltage dependence of the activation of L-type Ca2+ channels (half-activation voltage, V, of approximately -20 mV) and that of charge 2 (V of -100 mV) made it possible to record the ionic currents through Ca2+ channels and charge 2 in the same solution. In an external solution with Ba2+ as sole metal the maximum available charge 2 of L-type Ca2+ channels was 10-15% greater than that in a Ca(2+)-containing solution. External Cd2+ caused 20-30% reduction of charge 2 both from Na+ and L-type Ca2+ channels. Voltage- and Ca(2+)-dependent inactivation phenomena were compared with a double pulse protocol in cells perfused with an internal solution of low calcium buffering capacity. As the conditioning pulse voltage increased, inactivation monitored with the second pulse went through a minimum at about 0 mV, the voltage at which conditioning current had its maximum. Charge 2, recorded in parallel, did not show any increase associated with calcium entry. Two alternative interpretations of these observations are: (a) that Ca(2+)- dependent inactivation does not alter the voltage sensor, and (b) that inactivation affects the voltage sensor, but only in the small fraction of channels that open, and the effect goes undetected. A model of channel gating that assumes the first possibility is shown to account fully for the experimental results. Thus, extracellular divalent cations modulate voltage-dependent inactivation of the Ca2+ channel. Intracellular Ca2+ instead, appears to cause inactivation of the channel without affecting its voltage sensor.     

Charge movement by the Na/K pump in Xenopus oocytes

Pre-steady-state transient currents (1986. Nakao, M., and D. C. Gadsby. Nature [Lond.]. 323:628-630) mediated by the Na/K pump were measured under conditions for Na/Na exchange (K-free solution) in voltage- clamped Xenopus oocytes. Signal-averaged (eight times) current records obtained in response to voltage clamp steps over the range -160 to +60 mV after the addition of 100 microM dihydroouabain (DHO) or removal of external Na (control) were subtracted from test records obtained before the solution change. A slow component of DHO- or Na-sensitive difference current was consistently observed and its properties were analyzed. The quantity of charge moved was well described as a Boltzmann function of membrane potential with an apparent valence of 1.0. The relaxation rate of the current was fit by the sum of an exponentially voltage-dependent reverse rate coefficient plus a voltage- independent forward rate constant. The quantity of charge moved at the on and off of each voltage pulse was approximately equal except at extreme negative values of membrane potential where the on charge tended to be less than the off. The midpoint voltage of the charge distribution function (Vq) was shifted by -24.8 +/- 1.7 mV by changing the external [Na] in the test condition from 90 to 45 mM and by +14.7 +/- 1.7 mV by changing the test [Na] from 90 to 120 mM. A pseudo three- state model of charge translocation is discussed in which Na+ is bound and occluded at the internal face of the enzyme and is released into an external-facing high field access channel (ion well). The model predicts a shift of the charge distribution function to more hyperpolarized potentials as extracellular [Na] is lowered; however, several features of the data are not predicted by the model.