Interaction between capacitative Ca²⁺ influx and Ca²⁺-dependent Cl⁻ currents inXenopus oocytes

Abstract

The relationship between capacitative Ca²⁺ influx and activation of Ca²⁺-dependent Cl⁻ channels was monitored in intactXenopus oocytes following stimulation of 5-hydroxytryptamine (5-HT) receptors, through the activity of Ca²⁺-dependent Cl⁻ channels using the double-electrode voltage-clamp technique. Under voltage-clamp conditions, 5-HT evoked a rapid transient inward current followed by a slowly developing secondary 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 evoked hump-like currents which inactivated rapidly when applied during the secondary inward current. Hump currents arose from Ca²⁺ entering through the depletion-activated pathway. The hump currents inactivated with hyperpolarising pulses at <5-s intervals, and recovered monoexponentially with a time constant of around 8 s. Currents in response to hyperpolarising pulses during the transient current did not inactivate, suggesting that inactivation was associated with Ca²⁺ entry. When Ca²⁺ release evoked by inositol 1,4,5-triphosphate [Ins(1,4,5)P ₃] was prevented by heparin injection, hyperpolarising pulses during Ca²⁺ ionophore application also generated hump currents that were dependent on external 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 (Ca²⁺ATPase) inhibitor thapsigargin reduced the rate of rise of the hump current, increased the time-to-peak of the current and slowed the rate of decay. Pharmacological interventions 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⁻ channels in a way that might reflect both Ca²⁺ inhibition of Ca²⁺ entry and clustering of Cl⁻ channels in the plasma membrane.