A Novel Animal Model to Study the In Vivo Role of a C-Terminal Regulatory Domain in Cav1.3 L-Type Calcium Channels

Abstract

Cav1.3 channels-mediated calcium-signals are crucial for hearing, cardiac pacemaking, and for shaping activity patterns in neurons and endocrine cells. We previously found that Cav1.3 activity is strongly modulated by alternative splicing of their pore-forming α1-subunit.α1-variants with a long C-terminus can form a C-terminal modulatory domain (CTM) that reduces open probability, slows inactivation and decreases sensitivity to activation voltage. These modulatory properties are absent in short splice variants, which results in different dynamics of calcium inward current. Long and short splice variants are expressed together in brain and other tissues. However, the (patho-)physiological role of this CTM is unknown.We therefore generated a mutant mouse strain in which CTM function is disrupted by an HA-tag in one of the putative a-helices (DCRD) forming the CTM. Homozygous mutants (Cav1.3-DCRD-HA/HA mice) are viable and reproduce normally. Heterozygous mice show no overt differences in locomotive activity. As predicted, HA-immunoreactivity in Western blots of mutant mouse brains was only associated with the long Cav1.3 splice variant (230 kDa), and the mutation did not interfere with its protein expression level. Anti-Cav1.3 alpha1-antibodies recognizing all C-terminal splice variants revealed also the presence of short variants (180 kDa). These may arise from alternative splicing and/or from C-terminal post-translational proteolytic processing as described for Cav1.1 and Cav1.2 channels. Proteolytic processing would generate an HA-tagged low molecular mass fragment in Cav1.3-DCRD-HA/HA tissues, a possibility which we currently evaluate. using these animals we will also study the physiological role of CTM function in vivo. Furthermore, the HA-tagged α1-subunit will present an excellent target for specific detection with anti-HA antibodies in mouse tissues.Support: Austrian Science Fund (SFB F4402), EC-project MRTN-CT-2006-35367 (CavNet), University of Innsbruck