Effects of protein phosphorylation on the regulation of capacitative calcium influx in xenopus oocytes

Abstract

The regulation of capacitative Ca²⁺ influx in Xenopus oocytes was investigated using both the two electrode voltage-clamp (where Ca²⁺ is monitored through the Ca²⁺-dependent Cl^- current) and patchclamp techniques. Following stimulation of expressed 5-hydroxytryptamine (5-HT) receptors, capacitative Ca²⁺ influx deactivated in around 15 min. Following injection of [adenosine 5′-O-(3-Thiotriphosphate)] (ATP [γ-S]), an ATP analogue that is readily used by protein kinases, capacitative Ca²⁺ influx activated by 5-HT application either did not deactivate or was prolonged around twofold. However, injection of adenylyl 5′-(β,γ-methylene)-diphosphonate (AMP-PCP), another ATP analogue that is not utilised by kinases, did not affect the time-course of Ca²⁺ influx. When capacitative Ca²⁺ influx was activated by readmission of Ca²⁺ to oocytes incubated in thapsigargin/0 Ca²⁺ solution for several hours, Ca²⁺ influx occurred and a weakly saturating relationship between external Ca²⁺ and Ca²⁺ influx was found. Ca²⁺ influx in thapsigargintreated cells was unaffected by ATP [γ-S]. ATP [γ-s] and several kinases had no effect on the Ca²⁺-dependent Cl^- current when the latter was activated by elevation of Ca²⁺ independent of capacitative Ca²⁺ influx. Protein kinase C slowly and partially inhibited the Cl^- current. Outside-out patches taken from thapsigargin-treated cells failed to demonstrated any Ca²⁺ current or Ca²⁺-dependent Cl^- current on reapplying high Ca²⁺ to the patch, despite the oocyte showing a large capacitative Ca²⁺ influx. The results suggest that a kinase, activated on receptor stimulation, prolongs the activation time-course of capacitative Ca²⁺ influx.