Abstract 2925: An inherited mutation of AKAP9 (Yotiao) causes Long QT Syndrome (LQTS)

A-Kinase Anchoring Protein 9 (AKAP9), or Yotiao, binds to the carboxy-terminus (C-T) of the IKs channel alpha subunit KCNQ1 and recruits cAMP-dependent protein kinase A (PKA) and protein phosphatase 1 (PP1) to the channel to control its phosphorylation state. Previously, an inherited LQT1-causing mutation in the KCNQ1 C-T/Yotiao binding domain was shown to disrupt the complex and render the channel functionally insensitive to cAMP-dependent regulation. Despite the critical role of Yotiao in mediating this fundamental physiological response, the large size of the Yotiao protein (over 200 kD) has hindered patient screens and, to date, no disease-linked mutations have been found in this adaptor protein. The objectives of the present study were first to locate reciprocal binding site(s) for KCNQ1 on Yotiao and to test whether mutation(s) in the binding module(s) might cause LQTS. Using GST fusion protein pull down and immunoprecipitation (IP) techniques, we identified two regions in Yotiao necessary for binding to KCNQ1: one within the Yotiao N-terminus (residues 29 – 46), and a second within its C-terminus (1574 –1643). Targeted sequence analysis was then carried out in fifty clinically diagnosed but genotype negative LQTS patients in the coding regions for these two KCNQ1 binding domains in AKAP9-encoded Yotiao. This analysis identified a novel missense mutation, S1570L-Yotiao, present in two siblings diagnosed with LQTS but absent in the normal population. We found that the S1570L mutation, located close to the Yotiao C-T binding motif for KCNQ1, disrupted the interaction between the two proteins and significantly reduced both basal and exogenous cAMP-induced KCNQ1 phosphorylation. Perforated patch experiments in CHO cells revealed that expression of S1570L-Yotiao significantly reduced basal IKS channel activity and completely ablated the response of the IKs channel to cAMP plus the phosphatase inhibitor okadaic acid. These reconstituted cellular consequences of the inherited S1570L-Yotiao mutation are consistent with delayed repolarization of the ventricular action potential observed in the affected siblings. Thus, we have demonstrated for the first time a link between an inherited AKAP mutation and human disease, LQTS.