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Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 Podcast
Structural and functional analysis of the Cav1.4 L-type calcium channel from mouse retina

Structural and functional analysis of the Cav1.4 L-type calcium channel from mouse retina

Fakultät für Chemie und Pharmazie - Digitale Hochschulschriften der LMU - Teil 02/06 vor 20 Jahren

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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.