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Baumann, Ludwig (2006): Structural and functional analysis of the Cav1.4 L-type calcium channel from mouse retina. Dissertation, LMU München: Faculty of Chemistry and Pharmacy
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Abstract

This study provides novel insights to the function and regulation of Cav1.4 LTCCs. In the first part of the sudy the basic biophysical and pharmacological properties of Cav1.4 have been characterized. To this end Cav1.4 was cloned from murine retinal cDNA. The full-length cDNA comprises 6111bp and contains an open reading frame encoding for a protein of 1984 amino acids. Cav1.4 was functionally expressed in HEK 293 cells. Like in the case of other LTCCs the coexpression of alpha2delta and beta subunits was necessary to get measurable currents. The electrophysiological properties of Cav1.4 found in patch clamp experiments distinguish these channels from other LTCCs. Activation kinetics were very fast, the activation threshold was relatively low and the time course of inactivation was extremely slow. Also the pharmacological properties were different from those of classical LTCCs. Cav1.4 channels show a much lower sensitivity for LTCC blockers compared to Cav1.2b channels. The most important findings of this study are the novel insights on the regulation of CDI. Surprisingly, no CDI was observed in Cav1.4 LTCCs in electrophysiological experiments. CDI is a negative feedback mechanism by which Ca2+ limits its own influx into the cell. This feedback inhibition is essential for many cell types to prevent excessive and potentially toxic Ca2+ levels and is widespread among HVA calcium channels. The sequences conferring CDI are conserved throughout the whole HVA calcium channel family and also in Cav1.4 raising the question of how this channel manages to switch off CDI. We identified an autoinhibitory domain in the distal C- terminus of Cav1.4 that serves to abolish CDI. This domain (ICDI, inhibitor of CDI) uncouples the molecular machinery conferring CDI from the inactivation gate by binding to the EF hand motif in the proximal C-terminus. Deletion of ICDI completely restores Ca2+-calmodulin mediated CDI in Cav1.4. CDI can be switched off again in the truncated Cav1.4 channel by coexpression of ICDI indicating that it works as an autonomous unit. Furthermore, replacement of the distal C-terminus in the Cav1.2b LTCC by the corresponding sequence of Cav1.4 is sufficient to block CDI. This finding suggests that autoinhibition of CDI can be principally introduced into other Ca2+ channel types. The novel mechanism described is also of great physiological impact. In vivo, Cav1.4 is expressed in photoreceptors and bipolar cells of the retina. In these cells the lack of CDI is of great physiological importance since it is required to generate a sustained Ca2+ influx and, hence, to mediate tonic glutamate release from synaptic terminals. Mutations in the gene coding for the Cav1.4alpha1 subunit in humans are linked to a disease called congenital stationary nightblindness type 2 (CSNB2). Some of these mutations lead to truncated channels nearly identical to channel mutants analyzed in this study that show CDI. Thus, the phenotype of these mutations can be explained by the recovery of CDI.