The trafficking protein, EHD2, positively regulates cardiac sarcolemmal K _ATP channel surface expression: role in cardioprotection

Abstract

ATP-sensitive K (K) channels uniquely link cellular energy metabolism to membrane excitability and are expressed in diverse cell types that range from the endocrine pancreas to neurons and smooth, skeletal, and cardiac muscle. A decrease in the surface expression of K channels has been linked to various disorders, including dysregulated insulin secretion, abnormal blood pressure, and impaired resistance to cardiac injury. In contrast, up-regulation of K channel surface expression may be protective, for example, by mediating the beneficial effect of ischemic preconditioning. Molecular mechanisms that regulate K channel trafficking are poorly understood. Here, we used cellular assays with immunofluorescence, surface biotinylation, and patch clamping to demonstrate that Eps15 homology domain-containing protein 2 (EHD2) is a novel positive regulator of K channel trafficking to increase surface K channel density. EHD2 had no effect on cardiac Na channels (Nav1.5). The effect is specific to EHD2 as other members of the EHD family-EHD1, EHD3, and EHD4-had no effect on K channel surface expression. EHD2 did not directly affect K channel properties as unitary conductance and ATP sensitivity were unchanged. Instead, we observed that the mechanism by which EHD2 increases surface expression is by stabilizing K channel-containing caveolar structures, which results in a reduced rate of endocytosis. EHD2 also regulated K channel trafficking in isolated cardiomyocytes, which validated the physiologic relevance of these observations. Pathophysiologically, EHD2 may be cardioprotective as a dominant-negative EHD2 mutant sensitized cardiomyocytes to ischemic damage. Our findings highlight EHD2 as a potential pharmacologic target in the treatment of diseases with K channel trafficking defects.-Yang, H. Q., Jana, K., Rindler, M. J., Coetzee, W. A. The trafficking protein, EHD2, positively regulates cardiac sarcolemmal K channel surface expression: role in cardioprotection.

Cardiomyocyte damage control in heart failure and the role of the sarcolemma

“…Caveolae have also been reported in the heart to function as sarcolemma ‘reservoirs’ to buffer membrane tension (Sinha et al 2011). A critical link between EHD2 and caveolae stability has also recently been demonstrated by Coetzee and colleagues showing EHD2 is necessary for stabilising caveolae, and for co-localisation of a sub-population of K ATP channels leading to a proposed cardioprotective role of EHD2 against ischaemic damage (Yang et al 2018). Other reports have characterised EHD2 in COS-7 cells as a transcriptional supressor, regulating gene expression via a nuclear localisation signal (NLS), a conserved nuclear export signal (NES) and SUMOylation site within the helical linker between the N-terminal and C-terminal domains (Pekar et al 2012).…”

Palmitoylation of the K ATP channel Kir6.2 subunit promotes channel opening by regulating PIP 2 sensitivity

“…Immunocytochemistry. As previously described (24), isolated rat cardiomyocytes were plated on glass coverslips and fixed with 4% paraformaldehyde. Cells were permeabilized with 0.1% Triton X-100 and blocked with 5% donkey serum in PBS.…”

“…To investigate whether the increased mean patch K ATP channel current is due to enhanced membrane targeting, we biochemically assessed the surface expression of K ATP channels. As in our previous study (24), we performed surface biotinylation of Kir6.2 with an extracellular Avi tag (Avi-Kir6.2), which functions and traffics similar to wildtype Kir6.2. The Avi-Kir6.2/SUR1 channels were expressed in HEK-293 cells and we confirmed that they are palmitoylated similarly to the wild-type channel (SI Appendix, Fig.…”

Cellular functions and intrinsic attributes of the ATP‐binding Eps15 homology domain‐containing proteins

“…The significance of EHDs in this process however remains unclear. In addition, EHD paralogs, more specifically EHD3, appear to be crucial for the structural and functional specialization of membrane tubules of cardiomyocytes, a process attributed to endocytic recycling of critical proteins required for normal excitability of cardiac myocyotes 54–58 …”