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.