Abstract 19413: Regulation of I Ks Current Amplitude by Differential Trafficking Patterns and Interactions Of KCNQ1 and KCNE Subunits

Abstract

KCNQ1 (Q1, pore-forming subunit) and KCNE1 (E1, modulatory subunit) associate to form the slow delayed rectifier (I Ks ) channel that functions as ‘repolarization reserve’ in human heart. We have shown that in adult guinea pig (GP) cardiomyocytes Q1 & E1 cluster to different subcellular compartments, and the degree of Q1/E1 colocalization affects the I Ks amplitude. We have also shown that KCNE2 (E2) is expressed in human heart, and can associate with Q1 or Q1/E1 to suppress the I Ks amplitude. Objectives: To study the patterns of trafficking and interactions of Q1, E1 and E2. Methods: Q1, E1 and E2 are labeled with eGFP (Q1-G) or mono-dsR (Ex-R, x = 1 or 2) and expressed in COS-7 cells, neonatal rat (NRVM) and adult GP (AGPV) ventricular myocytes; the latter 2 by adenoviral (Adv) gene transfer. Protein trafficking/interactions are monitored by live cell imaging, in conjunction with patch clamping, immunocytochemistry & immunoblotting. Results: Time courses of Q1-G & Ex-R distribution in COS-7 cells suggest that they are translated, packaged and shipped to the cell surface independent of each other. They meet on the cell surface and form Q1-G/Ex-R complexes. Q1-G/Ex-R complexes are endocytosed and colocalized in a cytosolic tubulo-vesicular system. We identify 2 main effects of Ex-R on Q1-G trafficking: (1) Ex-R accelerates Q1-G recovery after photobleaching, (2) 24-48 hr after transfection, E2-R brings Q1-G with it to lysosomes for increased degradation. The distribution patterns of Q1-G and Ex-R in NRVM are similar to those in COS-7 cells. However, under the same Adv incubation conditions, Q1-G and Ex-R are expressed at a much lower level in AGPV than NRVM. In AGPV, Q1-G is distributed mainly as cytosolic striations while Ex-R mainly on cell surface, similar to the native Q1 and Ex immunofluorescence patterns seen in control myocytes. Conclusions: Assembly of Q1 & E1 into I Ks does not occur early during biogenesis. Q1 & E1 meet after they reach the plasma membrane and travel together thereafter. E1 increases the mobility of Q1, while E2 increases Q1 degradation in lysosomes. In adult cardiomyocytes the expression levels and subcellular distribution patterns of Q1 & Ex are tightly regulated, with the sarcomere structure playing a key role in Q1 localization.