Calcium entry in response to maintained depolarization of squid axons

1. Intracellular aequorin was used to monitor changes in Ca entry in response to maintained depolarization either produced electrically or by exposure to K-rich solutions.2. External K concentrations greater than 50 mM produce a phasic light response. The light rises to a peak in a few sec and then falls in 0.5-5 min to a new steady level that is always greater than the level in the absence of K.3. The phasic light response does not result from depletion of available aequorin at the periphery of the axon, but rather seems to reflect a phasic entry of Ca in response to depolarization.4. Similar phasic responses are produced by prolonged electrical depolarization. These results are consistent with depolarization serving both to activate and also to inactivate Ca entry.5. Following inactivation and after return to normal sea-water, there is an appreciable relative refractory period during which the response both to K-rich sea-water and electrical depolarization is reduced in size. Complete recovery takes 10-15 min.6. The response to 410 mM-KCl is dependent on the previous treatment of the preparation. Pre-treatment with 100 or 200 mM-KCl reduced the response to 410 mM-KCl. The potential for half inactivation was about -25 mV in 112 mM-Ca and -40 mV in 20 mM-Ca.7. The rate of onset of inactivation is potential dependent and is faster for depolarizations to zero potential than for smaller ones.8. The phasic Ca entry produced by K-rich solutions is insensitive to external tetrodotoxin and internal tetraethylammonium ions, but is blocked by external Mn(2+), Co(2+) and Ni(2+) ions and by the drugs D-600 and iproveratril. This suggests that the phasic Ca entry involves the late Ca channel.9. Recovery of the outward K current after a long depolarization is much faster than recovery of the late Ca entry system. This provides further support for the view that the late Ca channel and the K channel are distinct.