GIRK4 confers appropriate processing and cell surface localization to G-protein-gated potassium channels

GIRK1 and GIRK4 subunits combine to form the heterotetrameric acetylcholine -activated potassium current (I-KACh) channel in pacemaker cells of the hea rt. The channel is activated by direct binding of G-protein G(beta gamma) s ubunits, The GIRK1 subunit is atypical in the GIRK family in having a uniqu e (similar to 125-amino acid) domain in its distal C terminus. GIRK1 cannot form functional channels by itself but must combine with another GIRK fami ly member (GIRK2, GIRK3, or GIRK4), which are themselves capable of forming functional homotetramers. Here we show, using an extracellularly Flag-tagg ed GIRK1 subunit, that GIRK1 requires association with GIRK4 for cell surfa ce localization. Furthermore, GIRK1 homomultimers reside in core-glycosylat ed and nonglycosylated states. Coexpression of GIRK4 caused the appearance of the mature glycosylated form of GIRK1. [S-35]Methionine pulse-labeling e xperiments demonstrated that GIRK4 associates with GIRK1 either during or s hortly after subunit synthesis. Mutant and chimeric channel subunits were u tilized to identify domains responsible for GIRK1 localization. Truncation of the unique C-terminal domain of Delta 374-501 resulted in an intracellul ar GIRK1 subunit that produced normal I-KACh-like channels when coexpressed with GIRK4. Chimeras containing the C-terminal domain of GIRK1 from amino acid 194 to 501 were intracellularly localized, whereas chimeras containing the C terminus of GIRK4 localized to the cell surface. Deletion analysis o f the GIRK4 C terminus identified a 25-amino acid region required for cell surface targeting of GIRK1/GIRK4 heterotetramers and a 25-amino acid region required for cell surface localization of GIRK4 homotetramers. GIRK1 appea red intracellular in atrial myocytes isolated from GIRK4 knockout mice and was not maturely glycosylated, supporting an essential role for GIRK4 in th e processing and cell surface localization of I,,, in vivo.