Regulation of CaV1.2 Channels by Sphingolipids and Cholesterol. Specific Role of the Gamma Subunit

Abstract

Previoulsy (Biophys. Meetings 2010 and 2011), we showed that inactivation of L-type Ca2+ channels containing the γ1 subunit changes during cell cycle. The half-inactivation voltage is very negative (in the −70 - −50 mV range) in cells at the G1 phase, but also after serum starvation, or after the ER shock.Here we report that the novel regulatory pathway is membrane-delimited because it depends on cholesterol and sphingolipids. Alterations of the membrane lipid fluidity (e.g., addition of Triton X-100) affected inactivation regardless of the γ1 subunit. However, various manipulations with membrane cholesterol, ceramide, and/or shpingosine were efficient only when the γ1 subunit was present. Although tested positively in our experimental conditions, phosphoinositides, C-8-ceramide-1-phosphate, sphingosine-1-phosphate, and glycosphingolipid GM1 did not act specifically on inactivation with the γ1 subunit.When cholesterol was depleted from the membrane, or upon application of sphingosine, ceramide-C2, or ceramide-C8, inactivation shifted to the −70 - −50 mV range. Isomers epi-cholesterol and dihydro-ceramide-C2 did not act. Because ceramide and sphingosine strongly activate phosphatases PP1 and PP2A, phosphatase inhibitors calyculin A, or tautomycetin, were applied in order to prevent a rapid current run-down. Thus, a role of phosphorylation-dependent pathways can be excluded as well.We compared the membrane cholesterol content in cells at different cell-cycle stages, but did not find any significant difference. However, the membrane contents of sphingosine and ceramide are well known to change during cell cycle.Therefore, we propose that the enhancement of inactivation of L-type channels by the γ1 subunit is dependent on sphingosine and ceramide. Possibly, the sphingolipids interact directly with the subunit. The large scale of changes of inactivation properties due to these interactions allows efficient spatiotemporal tuning of functional availability of L-type Ca2+ channels.Supported by R01MH079406.