Single-channel characteristics and regulation of endogenously expressed cation channels in human cardiac microvascular endothelial cells and human aortic endothelial cells.

BACKGROUND: Previous studies showed that endothelial sodium channels (EnNaC) endogenously expressed in human aortic endothelial cells (hAoEC) are positively regulated by extracellular vesicles enriched in the lipid raft protein MARCKS-like protein 1 (MLP-1) and bioactive lipids, and negatively regulated by C-type natriuretic peptide receptor (NPRC) ligand binding. Here, we hypothesize that human cardiac microvascular endothelial cells (hCMEC) express an EnNaC/ENaC-like channel that is sensitive to the NPR-C agonist cANF, MLP-1 protein expression, and nystatin mediated lipid raft modulation.

METHODS AND RESULTS: Single-channel patch clamp studies revealed human cardiac microvascular endothelial cells express at least two types of cation channels that have a conductance of 43.5pS and 8.6pS, respectively. Like in hAoEC, the EnNaC/ENaC-like channel activity was attenuated after treating hCMEC with cANF. Protein expression of the alpha subunit of the channel was augmented after treating hCMEC with nystatin. A proteomic analysis identified 228 differentially expressed proteins in the nystatin group compared to the vehicle group. hCMEC and hAoEC that were transiently transfected with MLP-1 siRNA compared to non-targeting siRNA showed rearrangement of the actin cytoskeleton indicated by disorganization of actin filaments assessed by fluorescence microscopy.

CONCLUSIONS: Taken together, hCMEC and hAoEC have similar single characteristics. NPR-C activation downregulates the function of an EnNaC/ENaC-like channel while nystatin enhances expression of the alpha subunit of the channel. Transient knockdown of MLP-1 in both hCMEC and hAoEC caused rearrangement of the actin cytoskeleton, which is known to regulate channel function.