Chloride conductance in xenopus laevis skeletal muscle membrane
Creators: name: Loo, Donald Doo Fuey
Publisher: name: University of British Columbia
Year: 2010

description: The chloride current-voltage characteristics of the membrane of sartorius fibers from Xenopus laevis were studied using a three microelectrode voltage clamp system. In fibers with normal resting potentials (-70 to -90 mV) and in fibers depolarized in 115 mM KC1 (resting potential -20 mV) the direction and degree of steady state rectification depended on extracellular pH. In alkaline solutions (pH 8.4) the current rectified outwards; with large hyperpolarizations the current recorded in normally polarized fibers was sometimes seen to diminish as the voltage was made extremely negative (the current-voltage relation exhibited a negative slope). In the depolarizing region (in depolarized fibers) the slope of the I-V relation became constant (limiting conductance) in alkaline solutions. In acid solutions (pH 5.4) the current rectified inwards with hyperpolarization and reached a limiting value with depolarization. Chloride currents decay ('inactivate') following changes of membrane potential from the resting potential (for both polarized and depolarized fibers). The kinetics of current relaxation exhibited voltage-dependent time constants depending on the size of the voltage step with a sensitivity of about -1.5 msec/mV but were independent of absolute membrane potential and external pH. Inactivation of chloride conductance was studied in two-pulse (conditioning (v₁) and test (V₂), pulses) voltage clamp experiments. In variable experiments the dependence of the initial current at the onset of was sigmoidally related to (inactivation relation). The slope of the inactivation relation was twice as steep in acid as in alkaline solutions, but was independent of the resting potential. In variable V₂ experiments, the current-voltage relation was linear over a wide voltage range and for different values of V₁, the instantaneous I-V relations converged in the outward current region; they also had zero-current potentials that became increasingly negative with respect to the holding potential as V₁ was made negative. Instantaneous chloride currents and the kinetics of current relaxation were found to depend on initial conditions when the membrane potential was changed under non-stationary conditions. The inactivation and recovery of initial current had similar timecourses as did the prolongation and recovery of the time constants. Initial currents recovered from conditioning with an exponential or sigmoid timecourse. Relaxation time constants exhibited a similar recovery pattern. The decline of initial current was initially exponentially dependent on the duration of conditioning. The time constant increased sigmoidally, or exponentially as the duration of conditioning increased. Using the data from variable conditioning step and variable test step experiments a manifold (or state space representation) was constructed that enables much of the current-voltage behavior of the chloride permeation system to be predicted. Currents recorded in voltage clamp experiments can be visualized as time-dependent flows along trajectories that are determined by the voltage. The rectification of the steady state and instantaneous current-voltage relations are related to the dispersion of the trajectories. The dependence of time constants of current transients can also be accounted for by the manifold. The results are examined in light of models for channel behavior. The instantaneous I-V characteristics exhibit some properties of channels of the electrodiffusion type. The steady state current-voltage relations are qualitatively similar to those of a model incorporating a particle within the chloride channel that either blocks or unblocks it depending on the extracellular pH. The dependence of relaxation kinetics on the size of the voltage step and on initial conditions suggest the participation of a molecule acting in a catalytic role controlling the relaxation of current transients.