The fourth wheel: Structural studies of heterotrimeric delta beta gamma ENaC.

Abstract

In addition to voltage-gated sodium channels, there are a multitude of non-voltage gated sodium channels in the human brain. A lesser studied sodium channels in the nervous system is the Epithelial Sodium Channel (ENaC). The ENaC family of proteins are characterized by their sensitivity to amiloride and their high selectivity of sodium ions. ENaC is a uniquely proteolytically activated channel that is canonically composed of three subunits, alpha, beta, and gamma, to form a heterotrimer. Though shortly after alpha-beta-gamma-ENaC was cloned, a fourth subunit was found, delta, which is most closely related to the α subunit and is predicted to form a heterotrimer with the beta and gamma subunit. There are several differences between alpha and delta containing ENaC though, such as expression pattern, protease activation, and channel selectivity. Unlike alpha-ENaC, delta-ENaC is expressed in the brain similar to another member of the Degenerin/Epithelial Sodium (Deg/ENaC) Superfamily, Acid Sensing Ion Channels (ASICs). Like ASIC, some literature suggests delta-ENaC is activated by protons instead of proteolysis. Unfortunately, there is not much known about delta-ENaC or its role in the human brain. Discerning delta-ENaC's function is critical to our understanding of sodium regulation in neurons. To elucidate delta-ENaC's function I have heterologously expressed human delta-beta-gamma-ENaC in HEK293S GnTI- (Ric15) cells. Whole cell amiloride sensitive currents recorded from Ric15 cells expressing transgenic delta-beta-gamma-ENaC have shown that the channel is functional and matches the selectivity pattern previously suggested for delta-beta-gamma-ENaC in the literature. I then utilized single particle Cryo-EM to study the structure of delta-beta-gamma-ENaC. A structure of 3.45 Å has been developed thus far, but further work needs to be done to resolve the transmembrane domains and have a better understanding of the functional differences between alpha and delta containing channels.