Résumé :
|
Communication n° 79 Hypokalemic periodic paralysis type 1 (HypoPP1) is associated with mutations within the pore subunit (CaV1.1/a1S) of the skeletal muscle L-type voltage-dependent calcium channel (L-type VDCCs). To date, three missense mutations that introduced amino-acid changes within the voltage sensor of domain II (R528H) and domain IV (R1239H & R1239G) have been described. Previous studies indicated that these mutations have minor impact on the electrophysiological properties of recombinant skeletal muscle L-type voltage-dependent calcium channels. Intriguingly, a significant drop in current density was observed, suggesting that HypoPP1 mutations may affect membrane targeting of the CaV1.1 subunit. More specifically, we have hypothesized that the "loss of function" of HypoPP1 mutations depend on a reduction of membrane expression of the CaV1.1 subunit. We decided to conduct this analysis on human CaV1.1 channels and we describe here, to our knowledge for the first time, the functional expression of recombinant human CaV1.1 channels. Functional expression of recombinant human CaV1.1 channels was obtained in GLT cells, a cell line that derive from muscular dysgenesis mouse muscle. Typical slow inward calcium current were recorded and immunostaining experiments with mab427, a specific antibody against the CaV1.1 subunit, revealed a correct triad expression of the human CaV1.1 protein. We next engineered this human cDNA encoding the CaV1.1 subunit in order to analyze of the HypoPP1 mutations. An extracellular hemagglutin (HA) epitope was introduced in order to quantify the membrane expression of the wild-type and mutated CaV1.1 subunits. Combining electrophysiological experiments and membrane detection of the CaV1.1 subunit should provide a comprehensive view of the functional consequences of HypoPP1 mutations on human skeletal muscle L-type L-type VDCCs.
|