Multiple Regions Inhibit Expression of Cav1.1 Ca2+ Channels in Non-Muscle Cells

Abstract

Compared to the cardiac CaV1.2, the skeletal CaV1.1 Ca2+ channel has been very difficult to express in non-muscle cells. To identify domains of CaV1.1 that inhibit such expression, we have co-transfected tsA201 cells with β1a and chimeric constructs of CaV1.1 and CaV1.2 tagged with YFP. A tandem construct of CaV1.1 linked to CaV1.2 expressed robustly in tsA201 cells, suggesting that either CaV1.2 masks a sequence of CaV1.1 which causes ER retention in non-muscle cells, or that CaV1.1 lacks a signal important for ER export. Replacement of individual transmembrane repeats of CaV1.1 (in non-tandem constructs) with the corresponding repeat of CaV1.2 was ineffective at increasing functional expression as indicated by slowly activating, L-type Ca2+ current and/or membrane-bound charge movement. However, simultaneous replacement of all four CaV1.1 repeats with those of CaV1.2, while retaining CaV1.1 sequence for the cytoplasmic domains (construct “CSk9”), resulted in increased expression, although not to the level of CaV1.2. By contrast, expression levels of CSk9 and CaV1.2 were equivalent in dysgenic myotubes. Thus, both the transmembrane and cytoplasmic domains appear to play a role in suppressing expression of CaV1.1 in non-muscle cells. To investigate which cytoplasmic domains might be particularly important at suppressing expression, we tested CaV1.2 constructs in which the I-II loop, II-III loop, I-II and II-III loops, or C-terminus were replaced with CaV1.1 sequence and found expression not to differ for any of these constructs from that of the entirely CaV1.2 construct. We are currently testing constructs of CaV1.2 with other combinations of cytoplasmic domains replaced by CaV1.1.