Abstract
The Cacna1fnob2 mouse is reported to be a naturally occurring null mutation for the Cav1.4 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 Cacna1fnob2 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 Cav1.4 protein, detectable by Western blot analysis that is predicted to differ from wild type Cav1.4 protein in a region of approximately 22 amino acids in the N-terminal portion of the protein. Electrophysiological analysis with either mouse Cav1.4wt or Cav1.4nob2 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 Cacna1fnob2 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 Cacna1fnob2 mice was generally similar to that of wild type mice. These results suggest the Cacna1fnob2 mouse is not a CACNA1F knockout model. Rather, alternative splicing within the ETn element can lead to full-length Cav1.4 protein, albeit at reduced levels, and the functional Cav1.4 mutant may be incapable of interacting with cytoskeletal filamin proteins. These changes, do not alter the ability of the Cacna1fnob2 mouse to detect and follow moving sine-wave gratings compared to their wild type counterparts.
Highlights
Influx of calcium through voltage-gated calcium channels (VGCCs) leads to excitation-contraction coupling, excitationtranscription coupling, neurotransmitter release, and programmed cell death
Two mRNA species are detected in the Cacna1fnob2 mouse Total RNA from the eyes of Cacna1fwt and Cacna1fnob2 mice was analyzed by RT-PCR
The cDNA bands were subsequently isolated and sequenced; the larger and more intense band corresponded to the CACNA1F-encoding sequence containing the ETn transposable element with an in-frame stop codon (Figure 2A), whereas the smaller and less intense band corresponded to the CACNA1F-encoding sequence containing the shorter ETn element that lacks the in-frame stop codon (Figure 2B)
Summary
Influx of calcium through voltage-gated calcium channels (VGCCs) leads to excitation-contraction coupling, excitationtranscription coupling, neurotransmitter release, and programmed cell death. Knockout of CACNA1F protein in mice following insertion of a self-excising Cre-lox-neo cassette into exon 7 results in an in-frame premature stop codon (G305X) in the Cav1.4 protein [21]. These Cacna1fG305X mice are characterized by complete loss of the bwave and oscillatory potentials of the electroretinogram (ERG), absence of cone-driven visually-evoked activity in the superior colliculus, .90% reduction in calcium influx in photoreceptors, and disrupted retinal morphology with loss of photoreceptor synapses and sprouting of horizontal and bipolar cell dendrites into the outer nuclear layer [21]. While the Cacna1fnob mouse displays disrupted retina morphology similar to that of the Cacna1fG305X mouse, the ERG of the Cacna1fG305X mouse is more similar to that of CSNB2 patients, being characterized by a reduced b-wave and oscillatory potentials while cone-driven responses are maintained
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