Search

The scaffolding protein Yotiao is a member of a large family of protein A-kinase anchoring proteins with important roles in the organization of spatial and temporal signaling. In heart, Yotiao directly associates with the slow outward potassium ion current (IKs) and recruits both PKA and PP1 to regulate IKs phosphorylation and gating. Human mutations that disrupt IKs-Yotiao interaction result in reduced PKA-dependent phosphorylation of the IKs subunit KCNQ1 and inhibition of sympathetic stimulation of IKs, which can give rise to long-QT syndrome. We have previously identified a subset of adenylyl cyclase (AC) isoforms that interact with Yotiao, including AC1-3 and AC9, but surprisingly, this group did not include the major cardiac isoforms AC5 and AC6. We now show that either AC2 or AC9 can associate with KCNQ1 in a complex mediated by Yotiao. In transgenic mouse heart expressing KCNQ1-KCNE1, AC activity was specifically associated with the IKs-Yotiao complex and could be disrupted by addition of the AC9 N terminus. A survey of all AC isoforms by RT-PCR indicated expression of AC4-6 and AC9 in adult mouse cardiac myocytes. Of these, the only Yotiao-interacting isoform was AC9. Furthermore, the endogenous IKs-Yotiao complex from guinea pig also contained AC9. Finally, AC9 association with the KCNQ1-Yotiao complex sensitized PKA phosphorylation of KCNQ1 to β-adrenergic stimulation. Thus, in heart, Yotiao brings together PKA, PP1, PDE4D3, AC9, and the IKs channel to achieve localized temporal regulation of β-adrenergic stimulation. [ABSTRACT FROM AUTHOR]

Regulation of the heart by the sympathetic nervous system, fundamental to the physiological response to stress and exercise, requires coordinated phosphorylation of multiple downstream molecular targets, including the IKs (slowly activating potassium current) channel. Sympathetic nervous system stimulation increases intracellular cAMP for which targeted regulation is directed in large part by distinct scaffold or anchoring proteins. Yotiao is an A-kinase-anchoring protein (AKAP) that recruits the cyclic AMP-dependent protein kinase (protein kinase A (PKA)) and protein phosphatase 1 to the carboxyl terminus of the IKs channel to form a molecular complex and control its phosphorylation state, crucial to the cardiac cellular response to sympathetic nervous system stimulation. Here we report that Yotiao itself is a substrate for PKA phosphorylation, and we identify a Yotiao amino-terminal (N-T) residue (Ser-43) that is PKA-phosphorylated in response to β-adrenergic receptor stimulation. The replacement of Ser-43 by Ala ablates the PKA phosphorylation of N-T Yotiao and markedly diminishes the functional response of the wild type and pseudo-phosphorylated IKs channel to cAMP but neither prevents the PKA phosphorylation of KCNQ1 nor its binding to Yotiao. These results suggest, for the first time, a critical role for the PKA phosphorylation of an AKAP in the functional regulation of an ion channel protein and postphosphorylation allosteric modulation of the IKs channel by Yotiao. [ABSTRACT FROM AUTHOR]

Ca2+ has been proposed to regulate Na+ channels through the action of calmodulin (CaM) bound to an IQ motif or through direct binding to a paired EF hand motif in the Nav1 C terminus. Mutations within these sites cause cardiac arrhythmias or autism, but details about how Ca2+ confers sensitivity are poorly understood. Studies on the homologous Cav1.2 channel revealed non-canonical CaM interactions, providing a framework for exploring Na+ channels. In contrast to previous reports, we found that Ca2+ does not bind directly to Na+ channel C termini. Rather, Ca2+ sensitivity appears to be mediated by CaM bound to the C termini in a manner that differs significantly from CAM regulation of Cav1.2. In Nav1.2 or Nav1.5, CaM bound to a localized region containing the IQ motif and did not support the large Ca2+-dependent conformational change seen in the Cav1.2·CaM complex. Furthermore, CaM binding to Nail C termini lowered Ca2+ binding affinity and cooperativity among the CaM-bindlng sites compared with CaM alone. Nonetheless, we found suggestive evidence for Ca2+/CaM-dependent effects upon Nail channels. The R1902C autism mutation conferred a Ca2+-dependent conformational change in Nav1.2 C terminus·CaM complex that was absent in the wild-type complex. In Nav1.5, CaM modulates the Cterminal interaction with the III-IV linker, which has been suggested as necessary to stabilize the inactivation gate, to minimize sustained channel activity during depolarization, and to prevent cardiac arrhythmias that lead to sudden death. Together, these data offer new biochemical evidence for Ca2+/CaM modulation of Na+ channel function. [ABSTRACT FROM AUTHOR]

The cardiac slow delayed rectifier potassium channel (IKs), comprised of α (KCNQ1) and β (KCNE1) subunits, is regulated by sympathetic nervous stimulation, with activation of β-adrenergic receptors PKA phosphorylating IKs channels. We examined the effects of β2-adrenergic receptors (β2-AR) on IKs in cardiac ventricular myocytes from transgenic mice expressing fusion proteins of IKs subunits and hβ2-ARs. KCNQ1 and β2-ARs were localized to the same subcellular regions, sharing intimate localization within nanometers of each other. In IKs/B2-AR myocytes, IKs density was increased, and activation shifted in the hyperpolarizing direction; IKs was not further modulated by exposure to isoproterenol, and KCNQ1 was found to be PKA-phosphorylated. Conversely, β2-AR overexpression did not affect L-type calcium channel current (ICaL) under basal conditions with ICaL remaining responsive to cAMP. These data indicate intimate association of KCNQ1 and β2-ARs and that β2-AR signaling can modulate the function of IKs channels under conditions of increased β2-AR expression, even in the absence of exogenous β-AR agonist. [ABSTRACT FROM AUTHOR]