Double-pulse facilitation of smooth muscle α₁ -subunit Ca²⁺ channels expressed in CHO cells

Frequent strong depolarizations facilitate Ca²⁺ channels in various cell types by shifting their gating behavior towards mode 2, which is characterized by long openings and high probability of being open. In cardiac cells, the same type of gating behavior is potentiated by β-adrenoceptors presumably acting via phosphorylation of a protein identical to or associated with the channel. Voltage-dependent phosphorylation has also been reported to underlie Ca²⁺ channel facilitation in chromaffin adrenal medulla and in skeletal muscle cells. We studied a possible voltage-dependent facilitation of the principal channel forming α₁-subunit of the dihydropyridine-sensitive smooth muscle Ca²⁺ channel. Single channel and whole-cell Ca²⁺ currents were recorded in Chinese hamster ovary cells stably expressing the class C_b Ca²⁺ channel α₁,-subunit. Strong depolarizing voltage-clamp steps preceding the test pulse resulted in a 2- to 3-fold increase of the single Ca²⁺ channel activity and induction of mode 2-like gating behavior. Accordingly we observed a significant potentiation of the whole-cell current by ∼50%. In contrast to the previous suggestions we found no experimental evidence for involvement of channel phosphorylation by protein kinases (cAMP-dependent protein kinase, protein kinase C and other protein kinases utilizing ATPγS) in the control and facilitated current. The data demonstrate that the L-type Ca²⁺ channel α-submit solely expressed in Chinese hamster ovary cells is subject to a voltage-dependent facilitation but not to phosphorylation. We suggest that this newly identified type of voltage-dependent facilitation of Ca²⁺ channels is due to a direct voltage-dependent conformational change inducing the same type of gating behavior as otherwise induced by phosphorylation.