For an excitable cell to function properly, a precise number of ion channel proteins need to be trafficked to distinct locations on the cell surface membrane, through a network and anchoring activity of cytoskeletal proteins. Not surprisingly, mutations in anchoring proteins have profound effects on membrane excitability. Ca 2+ -activated K + channels (K Ca 2 or SK) have been shown to play critical roles in shaping the cardiac atrial action potential profile. Here, we demonstrate that filamin A, a cytoskeletal protein, augments the trafficking of SK2 channels in cardiac myocytes. The trafficking of SK2 channel is Ca 2+ -dependent. Further, the Ca 2+ dependence relies on another channel-interacting protein, α-actinin2, revealing a tight, yet intriguing, assembly of cytoskeletal proteins that orchestrate membrane expression of SK2 channels in cardiac myocytes. We assert that changes in SK channel trafficking would significantly alter atrial action potential and consequently atrial excitability. Identification of therapeutic targets to manipulate the subcellular localization of SK channels is likely to be clinically efficacious. The findings here may transcend the area of SK2 channel studies and may have implications not only in cardiac myocytes but in other types of excitable cells.ion channel trafficking | atrial myocytes | atrial fibrillation S mall-conductance Ca 2+ -activated K + (SK or K Ca 2) channels are highly unique in that they are gated solely by changes in intracellular Ca 2+ (Ca 2+ i ) concentration. Hence, the channels function to integrate changes in Ca 2+ concentration with changes in membrane potentials. SK channels have been shown to be expressed in a wide variety of cells (1-3) and mediate afterhyperpolarizations following action potentials in neurons (1, 4, 5). We have previously documented the expression of several isoforms of SK channels in human and mouse atrial myocytes that mediate the repolarization phase of the atrial action potentials (6, 7). We further demonstrated that SK2 (K Ca 2.2) channel knockout mice are prone to the development of atrial arrhythmias and atrial fibrillation (AF) (8). Conversely, a recent study by Diness et al. suggests that inhibition of SK channels may prevent AF (9). Together, these studies underpin the importance of the precise control for the expression of these ion channels in atria and their potential to serve as a future therapeutic target for AF.Current antiarrhythmic agents target the permeation and gating properties of ion channel proteins; however, increasing evidence suggests that membrane localization of ion channels may also be pharmacologically altered (10). Furthermore, a number of disorders have been associated with mistrafficking of ion channel proteins (11,12). We have previously demonstrated the critical role of α-actinin2, a cytoskeletal protein, in the surface membrane localization of cardiac SK2 channels (13,14). Specifically, we demonstrated that cardiac SK2 channel interacts with α-actinin2 cytoskeletal protein via the EF hand motifs in α...

Rationale Adenylyl cyclase (AC) represents one of the principal molecules in the β-adrenergic receptor (βAR) signaling pathway, responsible for the conversion of ATP to the second messenger, cAMP. AC type 5 (ACV) and 6 (ACVI) are the two main isoforms in the heart. While highly homologous in sequence, these two proteins nevertheless play different roles during the development of heart failure. Caveolin-3 is a scaffolding protein, integrating many intracellular signaling molecules in specialized areas called caveolae. In cardiomyocytes, caveolin is predominantly located along invaginations of the cell membrane known as t-tubules. Objective We take advantage of ACV and ACVI knockout mouse models to test the hypothesis that there is distinct compartmentalization of these two isoforms in ventricular myocytes. Methods and Results We demonstrate that ACV and ACVI isoforms exhibit distinct subcellular localization. ACVI isoform is localized in the plasma membrane outside of the t-tubular region, and is responsible for β1AR signaling-mediated enhancement of the L-type Ca2+ current (ICa,L) in ventricular myocytes. In contrast, ACV isoform is localized mainly in the t-tubular region where its influence on ICa,L is restricted by phosphodiesterase (PDE). We further demonstrate that the interaction between caveolin-3 with ACV and PDE is responsible for the compartmentalization of ACV signaling. Conclusions Our results provide new insights into the compartmentalization of the two AC isoforms in the regulation of ICa,L in ventricular myocytes. Since caveolae are found in most mammalian cells, the mechanism of βAR and AC compartmentalization may also be important for βAR signaling in other cell types.

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Calmodulin (CaM), a Ca2؉ -sensing protein, is constitutively bound to IQ domains of the C termini of human Kv7 (hKv7, KCNQ) channels to mediate Ca 2؉ -dependent reduction of Kv7 currents. However, the mechanism remains unclear. We report that CaM binds to two isoforms of the hKv7.4 channel in a Ca 2؉ -independent manner but that only the long isoform (hKv7.4a) is regulated by Ca 2؉ /CaM. Ca 2؉ /CaM mediate reduction of the hKv7.4a channel by decreasing the channel open probability and altering activation kinetics. We took advantage of a known missense mutation (G321S) that has been linked to progressive hearing loss to further examine the inhibitory effects of Ca 2؉ / CaM on the Kv7.4 channel. Using multidisciplinary techniques, we demonstrate that the G321S mutation may destabilize CaM binding, leading to a decrease in the inhibitory effects of Ca 2؉ on the channels. Our study utilizes an expression system to dissect the biophysical properties of the WT and mutant Kv7.4 channels. This report provides mechanistic insights into the critical roles of Ca 2؉ /CaM regulation of the Kv7.4 channel under physiological and pathological conditions.

Influenza A viruses cause widespread human respiratory disease. The viral multifunctional NS1 protein inhibits host antiviral responses. This inhibition results from the binding of specific cellular antiviral proteins at various positions on the NS1 protein. Remarkably, binding of several proteins also requires the two amino-acid residues in the NS1 N-terminal RNA-binding domain (RBD) that are required for binding double-stranded RNA (dsRNA). Here we focus on the host restriction factor DHX30 helicase that is countered by the NS1 protein, and establish why the dsRNA-binding activity of NS1 is required for its binding to DHX30. We show that the N-terminal 152 amino-acid residue segment of DHX30, denoted DHX30N, possesses all the antiviral activity of DHX30 and contains a dsRNA-binding domain, and that the NS1-DHX30 interaction in vivo requires the dsRNA-binding activity of both DHX30N and the NS1 RBD. We demonstrate why this is the case using bacteria-expressed proteins: the DHX30N-NS1 RBD interaction in vitro requires the presence of a dsRNA platform that binds both NS1 RBD and DHX30N. We propose that a similar dsRNA platform functions in interactions of the NS1 protein with other proteins that requires these same two amino-acid residues required for NS1 RBD dsRNA-binding activity.