INSERTION MUTATIONS OF THE RIIA NA-KINASE-A MODULATION( CHANNEL REVEAL NOVEL FEATURES OF VOLTAGE GATING AND PROTEIN)

A linker insertion mutagenesis strategy was developed to probe functio nal subdomains of the RIIA Na+ channel alpha-subunit. We describe muta tions within the first two repeat domains that provide new functional information for three segments of the channel structure. 1. The insert ion of two alanine residues within the short peptide segment joining h elices S4 and S5 in domain II had two effects: a depolarizing shift of steady-state activation and reduced single-channel currents. These re sults suggest that the peptide segment following the S4 voltage sensor is involved in the activation process and is facing the ion pore. 2. An insertion immediately N-terminal. to the proposed transmembrane hel ix S1 in domain II shifted the steady-state activation in the depolari zing direction, suggesting a functional role in channel gating. 3. Ins ertions in the large, cytoplasmic loop between domains I and II affect two channel functions: inactivation and protein kinase A modulation. These results demonstrate that the Linker insertion approach can provi de novel insights into the structure-function relationships of large, multi-domain ion channel proteins.