Summary Small conductance Ca2+-activated K+ (SK) channels have recently been documented in human and mouse cardiac myocytes that contribute importantly towards cardiac action potential profiles. Three isoforms of SK channel subunits (SK1, 2 and 3) have been demonstrated in the heart. The channels are more prominently expressed in atrial and pacemaking tissues compared to the ventricles. Significance of the channels is underscored by the findings that SK2 channels may play a role in atrial fibrillation. The present study demonstrates the heteromultimerization of different SK channel subunits in human and mouse atrial myocytes. Moreover, the study provides evidence for the direct interaction between the coiled-coil domains in the C-termini of the different SK subunits. Disruption of the coiled-coil domain interaction results in a significant decrease in the Ca2+-activated K+ current in atrial myocytes which is important for cardiac repolarization. Formation of heteromeric channels provides an increase in functional diversity for K+ channels. Moreover, different isoforms of SK channels may represent therapeutic targets to directly modify atrial cells without interfering with ventricular myocytes. Thus, new knowledge into the structure and function of SK channels is important not only from a fundamental viewpoint, but might also have important therapeutic implications in cardiac arrhythmias. Rationale Ca2+-activated K+ channels are present in a wide variety of cells. We have previously reported the presence of small conductance Ca2+-activated K+ (SK or KCa) channels in human and mouse cardiac myocytes that contribute functionally towards the shape and duration of cardiac action potentials. Three isoforms of SK channel subunits (SK1, 2 and 3) are found to be expressed. Moreover, there is differential expression with more abundant SK channels in the atria and pacemaking tissues compared to the ventricles. SK channels are proposed to be assembled as tetramers similar to other K+ channels, but the molecular determinants driving their subunit interaction and assembly are not defined in cardiac tissues. Objective The goal of the study is to investigate the heteromultimeric formation and the domain necessary for the assembly of three SK channel subunits (SK1-3) into complexes in human and mouse hearts. Methods and Results Here, we provide evidence to support the formation of heteromultimeric complexes among different SK channel subunits in native cardiac tissues. SK1, 2 and 3 subunits contain coiled-coil domains (CCDs) in the C-termini. In vitro interaction assay supports the direct interaction between CCDs of the channel subunits. Moreover, specific inhibitory peptides derived from CCDs block the Ca2+-activated K+ current in atrial myocytes which is important for cardiac repolarization. Conclusions The data provide evidence for the formation of heteromultimeric complexes among different SK channel subunits in atrial myocytes. Since SK channels are predominantly expressed in atrial myocytes, specific ligands of th...

The importance of proper ion channel trafficking is underpinned by a number of channel-linked genetic diseases whose defect is associated with failure to reach the cell surface. Conceptually, it is reasonable to suggest that the function of ion channels depends critically on the precise subcellular localization and the number of channel proteins on the cell surface membrane, which is determined jointly by the secretory and endocytic pathways. Yet the precise mechanisms of the entire ion channel trafficking pathway remain unknown. Here, we directly demonstrate that proper membrane localization of a smallconductance Ca 2ϩ -activated K ؉ channel (SK2 or KCa2.2) is dependent on its interacting protein, ␣-actinin2, a major F-actin crosslinking protein. SK2 channel localization on the cell-surface membrane is dynamically regulated, and one of the critical steps includes the process of cytoskeletal anchoring of SK2 channel by its interacting protein, ␣-actinin2, as well as endocytic recycling via early endosome back to the cell membrane. Consequently, alteration of these components of SK2 channel recycling results in profound changes in channel surface expression. The importance of our findings may transcend the area of K ؉ channels, given that similar cytoskeletal interaction and anchoring may be critical for the membrane localization of other ion channels in neurons and other excitable cells.ion channel trafficking ͉ early endosome ͉ cardiac myocytes ͉ small conductance Ca 2ϩ -activated K ϩ channel ͉ calmodulin binding domain T he function of ion channels depends critically on the precise number and subcellular localization of the channel proteins on the cell-surface membrane (1, 2). The steady-state cell-surface expression of ion channels is intricately and dynamically governed by the anterograde (forward) and retrograde trafficking (2, 3). Ion channel molecules are first synthesized in the endoplasmic reticulum (ER), assembled and processed, then trafficked to the membrane where they function. Trafficking of ion channel proteins to the surface membrane involves a series of tightly regulated events coordinated by ER resident proteins, microtubules, transport vesicle and Golgi apparatus, the actin cytoskeleton, myosins, and anchoring proteins (2). The importance of correct ion channel trafficking is highlighted by a number of channel-linked genetic diseases whose defect is associated with failure to reach the cell surface (4-8).Small-conductance Ca 2ϩ -activated K ϩ (SK or K Ca 2) channels belong to a family of Ca 2ϩ -activated K ϩ channels (K Ca ) that have been reported from a wide variety of cells (9-11). SK channels represent a highly unique family of K ϩ channels, in that they are directly gated by changes in intracellular Ca 2ϩ concentration and hence function to integrate changes in Ca 2ϩ concentration with changes in K ϩ conductance and membrane potentials. SK channels have been shown to mediate afterhyperpolarizations in neurons (9, 12, 13) and action potential repolarization in cardiac tissues (14,15). Prev...

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...

Patients who were treated with IMRT had fewer instances of dental disease, more salivary flow, and fewer requisite posttreatment extractions compared with those treated with RT. The number of posttreatment extractions has been reduced with the advent of IMRT and more so with a complete dental evaluation prior to treatment.