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.
Subject(s)
Alternative Splicing , Calcium Channels, L-Type/genetics , Calcium Channels/genetics , Exons , Amino Acid Sequence , Animals , Base Sequence , Calcium Channels/metabolism , Calcium Channels, L-Type/metabolism , Mice , Molecular Sequence Data , RNA, Messenger/metabolism , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Ca(v)1.4 channels are the latest calcium channels to be described in the literature. Originally identified in 1997 from the human genome project, several reports have since been published describing mutations in the CACNA1F gene encoding Ca(v)1.4 channels, and implicated these mutations in human disorders such as X-linked cone rod dystrophy (CORDX3) and incomplete X-linked congenital stationary night blindness type 2 (CSNB2). The gene was subsequently cloned and expressed in heterologous expression systems beginning in 2003, and many of the mutations linked to CSNB2 have been tested. Here, we review literature describing the discovery of the CACNA1F gene, its tissue expression profile, alternative splicing events, and biophysical and pharmacological characteristics of the channel in various expression systems. Channel biophysics are also compared to those obtained from recordings made from vertebrate photoreceptors, suggesting that these studies may have been describing Ca(v)1.4 channels in native cells.
Subject(s)
Calcium Channels, L-Type/metabolism , Eye Proteins/metabolism , Genetic Diseases, X-Linked/metabolism , Night Blindness/metabolism , Photoreceptor Cells, Vertebrate/metabolism , Retinitis Pigmentosa/metabolism , Animals , Calcium Channels, L-Type/genetics , Eye Proteins/genetics , Gene Expression Regulation/genetics , Genetic Diseases, X-Linked/genetics , Human Genome Project , Humans , Mutation , Night Blindness/genetics , Organ Specificity/genetics , Retinitis Pigmentosa/geneticsABSTRACT
Direct interactions between the presynaptic N-type calcium channel and the beta subunit of the heterotrimeric G-protein complex cause voltage-dependent inhibition of N-type channel activity, crucially influencing neurotransmitter release and contributing to analgesia caused by opioid drugs. Previous work using chimeras of the G-protein beta subtypes Gbeta1 and Gbeta5 identified two 20-amino acid stretches of structurally contiguous residues on the Gbeta1 subunit as critical for inhibition of the N-type channel. To identify key modulation determinants within these two structural regions, we performed scanning mutagenesis in which individual residues of the Gbeta1 subunit were replaced by corresponding Gbeta5 residues. Our results show that Gbeta1 residue Ser189 is critical for N-type calcium channel modulation, whereas none of the other Gbeta1 mutations caused statistically significant effects on the ability of Gbeta1 to inhibit N-type channels. Structural modeling shows residue 189 is surface exposed, consistent with the idea that it may form a direct contact with the N-type calcium channel alpha1 subunit during binding interactions.
Subject(s)
Calcium Channels, N-Type/physiology , GTP-Binding Protein beta Subunits/chemistry , GTP-Binding Protein beta Subunits/physiology , Serine/analysis , Serine/physiology , Cell Line , DNA, Complementary/analysis , DNA, Complementary/genetics , Electrophysiology , G Protein-Coupled Inwardly-Rectifying Potassium Channels/physiology , GTP-Binding Protein beta Subunits/genetics , Humans , Mutagenesis , Patch-Clamp Techniques , Protein Structure, TertiaryABSTRACT
PURPOSE: Childhood absence epilepsy (CAE) is an idiopathic form of seizure disorder that is believed to have a genetic basis. METHODS: We examined the biophysical consequences of seven mutations in the Ca(v)3.2 T-type calcium channel gene linked to CAE. RESULTS: Of the channel variants examined, one of the mutants, a replacement of glycine 848 in the domain II-S2 region with serine, resulted in significant slowing of the time courses of both activation and inactivation across a wide range of membrane potentials. These changes are consistent with increased channel activity in response to prolonged membrane depolarizations. CONCLUSIONS: Taken together, these findings suggest that such little changes in channel gating may contribute to the etiology of CAE.
Subject(s)
Calcium Channels, T-Type/genetics , Epilepsy, Absence/genetics , Mutation/genetics , Animals , Calcium Channels, T-Type/physiology , Cells, Cultured , Child , Epilepsy, Absence/physiopathology , Glycine/genetics , Glycine/physiology , Humans , In Vitro Techniques , Ion Channel Gating/genetics , Ion Channel Gating/physiology , Membrane Potentials/genetics , Mutation/physiology , Mutation, Missense/genetics , Mutation, Missense/physiology , Rats , Serine/geneticsABSTRACT
The inhibition of N-type calcium channels by opioid receptor like receptor 1 (ORL1) is a key mechanism for controlling the transmission of nociceptive signals. We recently reported that signaling complexes consisting of ORL1 receptors and N-type channels mediate a tonic inhibition of calcium entry. Here we show that prolonged ( approximately 30 min) exposure of ORL1 receptors to their agonist nociceptin triggers an internalization of these signaling complexes into vesicular compartments. This effect is dependent on protein kinase C activation, occurs selectively for N-type channels and cannot be observed with mu-opioid or angiotensin receptors. In expression systems and in rat dorsal root ganglion neurons, the nociceptin-mediated internalization of the channels is accompanied by a significant downregulation of calcium entry, which parallels the selective removal of N-type calcium channels from the plasma membrane. This may provide a new means for long-term regulation of calcium entry in the pain pathway.
