Regulation of L-type calcium channels by protein processing and phosphorylation

It is well known that a cyclic AMP and protein kinase A-dependent pathway regulates L-type calcium channels. However, the molecular events underlying this regulation are not completely defined. In heterologous expression systems two modes of regulation have been observed. One mode requires the presence of a full-length alpha (1C) subunit, whereas the second made has only been observed in the presence of a C-terminally truncated alpha (1C) subunit. The alpha (1C) subunit is proteolytically processed in intact cardiac myocytes, but we hypothesize that the C-terminus remains functionally associated with the channels. As first proof of this hypothesis, we have developed an in vitro model system to test whether C-terminal fragments of the expected size can be produced by proteolysis and remain associated with the channel. Chymotrypsin cleaved the expressed alpha (1C) subunit into a "body" of 190 kDa and C-terminal fragments of 55-30 kDa. The hydrophilic C-terminal fragments remained associated with the membrane after cleavage via a proline rich domain that could bind to SH3 domains. We propose that full effect of protein kinase A on channel regulation requires proteolytic cleavage and membrane tethering of the C-terminus, and that the role of phosphorylation is to relieve the autoinhibition caused by the C-terminus.