DOUBLE-PULSE FACILITATION OF SMOOTH-MUSCLE ALPHA-1-SUBUNIT CA2+ CHANNELS EXPRESSED IN CHO CELLS

Frequent strong depolarizations facilitate Ca2+ channels in various ce ll types by shifting their gating behavior towards mode 2, which is ch aracterized by long openings and high probability of being open. In ca rdiac cells, the same type of gating behavior is potentiated by beta-a drenoceptors presumably acting via phosphorylation of a protein identi cal to or associated with the channel. Voltage-dependent phosphorylati on has also been reported to underlie Ca2+ channel facilitation in chr omaffin adrenal medulla and in skeletal muscle cells. We studied a pos sible voltage-dependent facilitation of the principal channel forming alpha(1)-subunit of the dihydropyridine-sensitive smooth muscle Ca2+ c hannel. Single channel and whole-cell Ca2+ currents were recorded in C hinese hamster ovary cells stably expressing the class C-b Ca2+ channe l alpha(1)-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca2channel activity and induction of mode 2-like gating behavior. Accordi ngly we observed a significant potentiation of the whole-cell current by similar to 50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by p rotein kinases (cAMP-dependent protein kinase, protein kinase C and ot her protein kinases utilizing ATP gamma S) in the control and facilita ted current. The data demonstrate that the L-type Ca2+ channel alpha(1 )-subunit solely expressed in Chinese hamster ovary cells is subject t o a voltage-dependent facilitation but not to phosphorylation. We sugg est that this newly identified type of voltage-dependent facilitation of Ca2+ channels is due to a direct voltage-dependent conformational c hange inducing the same type of gating behavior as otherwise induced b y phosphorylation.