Potassium conductance and internal calcium accumulation in a molluscan neurone

Abstract

1. The Aplysia neurone R-15 was injected with the Ca²⁺ sensitive dye arsenazo III. Changes in dye absorbance were measured with a differential spectrophotometer to monitor changes in the free internal Ca²⁺ concentration, [Ca]_i, during membrane depolarization and during intracellular Ca²⁺ ion injection under voltage clamp conditions.

2. The absorbance change, and thus [Ca]_i, increases linearly with Ca²⁺ injection intensity at constant duration. The absorbance change produced by a constant intensity Ca²⁺ injection also increases with injection duration, but this increase is asymptotic.

3. The Ca²⁺ activated K⁺ current, I_{K, Ca}, increases linearly with the increase in [Ca]_i and its rise and decay follows closely the time course of the absorbance change produced by internal Ca²⁺ injection.

4. The Ca²⁺ activated K⁺ conductance increases exponentially with membrane depolarization. The increase in K⁺ conductance activated by a constant intensity and duration Ca²⁺ injection is on average e-fold for a 25.3 mV change in membrane potential.

5. The difference in net outward K⁺ current measured during depolarizing pulses to different membrane potentials in normal and in Ca²⁺ free ASW was used as an index of I_{K, Ca}. Its time course was approximately linear for the first 50-100 msec of depolarization, but for longer times the relation approached a maximum. Simultaneous measurements of the arsenazo III absorbance changes were broadly consistent with the activation of I_{K, Ca} being brought about by the rise in [Ca]_i during a pulse.

6. The relation between Ca²⁺ activated K⁺ conductance and membrane potential is bell shaped and resembles the absorbance vs. potential curve, but its maximum is displaced to more positive membrane potentials. The shift in the two curves on the voltage axis can be explained by the potential dependence of G_{K, Ca}.

7. The net outward K⁺ current measured with depolarizing voltage pulses in normal and in Ca²⁺ free ASW is increased when [Ca]_i is elevated by internal Ca²⁺ injection. With large and prolonged Ca²⁺ injections the net outward current is depressed following the decline of [Ca]_i.

8. The time and frequency dependent depression of the net outward K⁺ current which occurs during repetitive stimulation is shown to have no obvious temporal relation to the increase in [Ca]_i. The depression is relieved by an increase in [Ca]_i caused by internal Ca²⁺ injection.

9. The net outward K⁺ current measured with brief depolarizing pulses which approach the estimated Ca²⁺ equilibrium potential and therefore do not cause Ca²⁺ influx and accumulation is facilitated by a previous depolarizing pulse which causes a rise in [Ca]_i..

10. The facilitation experiments also suggest that the activation of I_{K, Ca} by [Ca]_i has a significant time constant. During a depolarizing pulse, the rise in [Ca]_i next to the membrane, and hence I_{K, Ca} is expected to follow the square root of time, but a delay in the activation of I_{K, Ca} by [Ca]_i could explain why the observed time course of I_{K, Ca} is initially almost linear.

11. The potential dependence of the Ca²⁺ activated K⁺ conductance can be explained if the internal Ca²⁺ binding site is about half way through the membrane.

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