Effect of the Brugada syndrome mutation A39V on calmodulin regulation of Cav1.2 channels.
BACKGROUND
The L-type calcium channel Cav1.2 is important for brain and heart function. The ubiquitous calcium sensing protein calmodulin (CaM) regulates calcium dependent gating of Cav1.2 channels by reducing calcium influx, a process known as calcium-dependent inactivation (CDI). Dissecting the calcium-dependence of CaM in this process has benefited greatly from the use of mutant CaM molecules which are unable to bind calcium to their low affinity (N-lobe) and high affinity (C-lobe) binding sites. Unlike CDI, it is unknown whether CaM can modulate the activation gating of Cav1.2 channels.


RESULTS
We examined a Cav1.2 point mutant in the N-terminus region of the channel (A39V) that has been previously linked to Brugada syndrome. Using mutant CaM constructs in which the N- and/or C-lobe calcium binding sites were ablated, we were able to show that this Brugada syndrome mutation disrupts N-lobe CDI of the channel. In the course of these experiments, we discovered that all mutant CaM molecules were able to alter the kinetics of channel activation even in the absence of calcium for WT-Cav1.2, but not A39V-Cav1.2 channels. Moreover, CaM mutants differentially shifted the voltage-dependence of activation for WT and A39V-Cav1.2 channels to hyperpolarized potentials. Our data therefore suggest that structural changes in CaM that arise directly from site directed mutagenesis of calcium binding domains alter activation gating of Cav1.2 channels independently of their effects on calcium binding, and that the N-terminus of the channel contributes to this CaM dependent process.


CONCLUSIONS
Our data indicate that caution must be exercised when interpreting the effects of CaM mutants on ion channel gating.
Brett A
BA
Simms
Ivana Assis
IA
Souza
Department of Physiology and Pharmacology, Hotchkiss Brain Institute, University of Calgary, 3330 Hospital Dr, NW, Calgary T2N 4N1, Canada. Zamponi@ucalgary.ca.
Gerald W
GW
Zamponi
D019943:Amino Acid Substitution; D000818:Animals; D053840:Brugada Syndrome / Q000235:genetics*; D002118:Calcium / Q000494:pharmacology; D020746:Calcium Channels, L-Type / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects; D007700:Kinetics; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D011485:Protein Binding / Q000187:drug effects; D051381:Rats
D020746:Calcium Channels, L-Type; D002147:Calmodulin; C419385:L-type calcium channel alpha(1C); D050505:Mutant Proteins; D002118:Calcium