Modified Ca(v)1.4 expression in the Cacna1f(nob2) mouse due to alternative splicing of an ETn inserted in exon 2.

Abstract

The Cacna1f(nob2) mouse is reported to be a naturally occurring null mutation for the Ca(v)1.4 calcium channel gene and the phenotype of this mouse is not identical to that of the targeted gene knockout model. We found two mRNA species in the Cacna1f(nob2) mouse: approximately 90% of the mRNA represents a transcript with an in-frame stop codon within exon 2 of CACNA1F, while approximately 10% of the mRNA represents a transcript in which alternative splicing within the ETn element has removed the stop codon. This latter mRNA codes for full length Ca(v)1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Ca(v)1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Ca(v)1.4(wt) or Ca(v)1.4(nob2) cDNA revealed that the alternatively spliced protein does not differ from wild type with respect to activation and inactivation characteristics; however, while the wild type N-terminus interacted with filamin proteins in a biochemical pull-down experiment, the alternatively spliced N-terminus did not. The Cacna1f(nob2) mouse electroretinogram displayed reduced b-wave and oscillatory potential amplitudes, and the retina was morphologically disorganized, with substantial reduction in thickness of the outer plexiform layer and sprouting of bipolar cell dendrites ectopically into the outer nuclear layer. Nevertheless, the spatial contrast sensitivity (optokinetic response) of Cacna1f(nob2) mice was generally similar to that of wild type mice. These results suggest the Cacna1f(nob2) mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Ca(v)1.4 protein, albeit at reduced levels, and the functional Ca(v)1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1f(nob2) mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts.