Marquardt, Michelle L.

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.

A-kinase anchoring proteins (AKAPs) recruit signaling molecules and present them to downstream targets to achieve efficient spatial and temporal control of their phosphorylation state. In the heart, sympathetic nervous system (SNS) regulation of cardiac action potential duration (APD), mediated by β-adrenergic receptor (βAR) activation, requires assembly of AKAP9 (Yotiao) with the I Ks potassium channel α subunit (KCNQ1). KCNQ1 mutations that disrupt this complex cause type 1 long-QT syndrome (LQT1), one of the potentially lethal heritable arrhythmia syndromes. Here, we report identification of ( i ) regions on Yotiao critical to its binding to KCNQ1 and ( ii ) a single putative LQTS-causing mutation (S1570L) in AKAP9 (Yotiao) localized to the KCNQ1 binding domain in 1/50 (2%) subjects with a clinically robust phenotype for LQTS but absent in 1,320 reference alleles. The inherited S1570L mutation reduces the interaction between KCNQ1 and Yotiao, reduces the cAMP-induced phosphorylation of the channel, eliminates the functional response of the I Ks channel to cAMP, and prolongs the action potential in a computational model of the ventricular cardiocyte. 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 a link between genetic perturbations in AKAP and human disease in general and AKAP9 and LQTS in particular.