In vivo sodium channel structure/function studies: Consecutive Arg1448 changes to Cys, His, and Pro at the extracellular surface of IVS4

Structure/function relationships in ion channels have been intensively studied through expression of cloned channel subunits in heterologous cellular environments. Considerable information has been gleaned via this approach. However, it is difficult to know if the residues and regions identified as important retain this prominent role in vivo: there are many differences between heterologous systems and functioning nerves and muscle in vivo, any one of which is likely to affect channel function. Examples of such variables include glycosylation status of the channel protein, association of muscle-specific membrane or cytoskeletal proteins, and fluctuations of intracellular and extracellular fluid milieu as a function of fluctuating cellular physiology. The identification of single amino acid changes in the voltage-sensitive muscle sodium channel α subunit in human and horse genetic disease has permitted a new approach to the study of structure/function relationships in ion channels. Importantly, the interactions between the environment and the abnormal channel can be studied in this in vivo system. Here we report the identification of a novel human sodium channel mutation (R1448P), which causes a severe type of cold-sensitive myotonia and weakness. This patient is compared to a series of other patients have R1448C, and R1448H mutations. We show that the severity of the amino acid change correlates with the severity of clinical symptoms. This data shows that different amino acid replacements in the extracellular surface of domain IV S4 are important for a channel function, despite the paucity of heterologous expression data suggesting functional importance of this region. The extreme cold sensitivity of the proline subsitution of R1443 suggests that cold temperatures may affect the structural integrity of the channel, and that proline may destabilize the normal structure.