INTERACTION BETWEEN CAPACITATIVE CA2-DEPENDENT CL- CURRENTS IN XENOPUS OOCYTES( INFLUX AND CA2+)

The relationship between capacitative Ca2+ influx and activation of Ca 2+-dependent Cl- channels was monitored in intact Xenopus oocytes foll owing stimulation of 5-hydroxytryptamine (5-HT) receptors, through the activity of Ca2+-dependent Cl- channels using the double-electrode vo ltage-clamp technique. Under voltage-clamp conditions, 5-HT evoked a r apid transient inward current. followed by a slowly developing seconda ry inward current. The secondary current reflected depletion-activated Ca2+ entry. Hyperpolarising pulses evoked sustained Ca2+-dependent Cl - currents when applied during the transient inward current, but evoke d hump-like currents which inactivated rapidly when applied during the secondary inward current. Hump currents arose from Ca2+ entering thro ugh the depletion-activated pathway. The hump currents inactivated wit h hyperpolarising pulses at < 5-s intervals, and recovered monoexponen tially with a time constant of around 8 s. Currents in response to hyp erpolarising pulses during the transient current did not inactivate, s uggesting that inactivation was associated with Ca2+ entry. When Ca2release evoked by inositol 1,4,5-triphosphate [Ins(1,4,5)P-3] was prev ented by heparin injection, hyperpolarising pulses during Ca2+ ionopho re application also generated hump currents that were dependent on ext ernal Ca2+, inactivated and recovered from inactivation with a similar time course as the humps following 5-HT treatment. Pretreatment with the Ca2+ adenosine 5'-triphosphatase (Ca(2+)ATPase) inhibitor thapsiga rgin reduced the rate of rise of the hump current, increased the time- to-peak of the current and slowed the rate of decay. Pharmacological i nterventions to disrupt the cytoskeleton reduced the amplitude of the hump current. It is suggested that, following hyperpolarisation in the presence of Ca2+ entry, the ensuing Ca2+ influx interacts with Cl- ch annels in a way that might reflect both Ca2+ inhibition of Ca2+ entry and clustering of Cl- channels in the plasma membrane.