Differentiation of voltage-gated potassium current and modulation of excitability in cultured amphibian spinal neurones.

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Gigaohm-seal whole-cell voltage-clamp techniques were used to study the development of ionic currents in the membrane of embryonic amphibian (Ambystoma) spinal neurones during in vitro differentiation. Dissociated neural plate cells, some of which are neuronal precursor cells, were placed into culture. Cells became excitable at the time of neurite outgrowth, 2-3 days later, and over the next 2-10 days the duration of the action potential shortened from about 100 ms to about 1 ms. Voltage-clamp recordings demonstrated that at the time of appearance of neurites, activatable Na, Ca and voltage-gated K channels were present in the membrane (Ca-dependent K channels were not studied). Over succeeding days in culture, records of total membrane current indicated that the amplitudes of peak inward and steady-state outward currents both increased. As a result of these increases, the pattern of total membrane current came to be increasingly dominated by outward currents. With inward Na and Ca currents blocked, a voltage-gated K current (IK(V] could be studied in isolation. The reversal potential of this current varied in good agreement with the equilibrium potential for K ions predicted by the Nernst relation. The wave form of IK(V) activation was sigmoidal. Activation was more rapid at more positive voltages (relative to the usual holding potential of -70 mV), and deactivation was more rapid at more negative voltages. The amplitude of IK(V) increased during neural development, while cell size remained approximately constant. Increases in rates of activation and deactivation were observed in parallel with the increase in current density. When measured at 0 mV, cells studied on day 4 of culture or earlier showed steady-state chord conductances (gK(V] of less than 20 nS, and one-half activation times (t1/2) of 2 X 5-10 ms. Older cells showed gK(V)s of 10-80 nS, and t1/2s of 0 X 8-2 X 5 ms. As Na, and to a lesser extent Ca, current amplitudes were also increasing during differentiation, these observations concerning IK(V) suggested that its amplitude and kinetic changes might in part be responsible for the observed decrease in action potential duration during development. This hypothesis was tested by modelling Na, Ca and voltage-gated K currents and testing the effects of changes in amplitude and kinetics of IK(V) on the duration and ionic dependence of reconstructed action potentials. The results obtained using this model suggested that the increase in IK(V) amplitude and activation rate was sufficient to change action potential duration and apparent ionic sensitivity.

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