Epithelial Na+ Channel Regulation by Extracellular and Intracellular Factors

Abstract

Epithelial Na⁺ channels (ENaCs) are members of the ENaC/degenerin family of ion channels that evolved to respond to extracellular factors. In addition to being expressed in the distal aspects of the nephron, where ENaCs couple the absorption of filtered Na⁺ to K⁺ secretion, these channels are found in other epithelia as well as nonepithelial tissues. This review addresses mechanisms by which ENaC activity is regulated by extracellular factors, including proteases, Na⁺, and shear stress. It also addresses other factors, including acidic phospholipids and modification of ENaC cytoplasmic cysteine residues by palmitoylation, which enhance channel activity by altering interactions of the channel with the plasma membrane.

Keywords: ENaC; palmitoylation; palmitoyltransferase; protease; shear stress; sodium self-inhibition.

Figure 1

ASIC1 structure. Left, cartoon of ASIC1 trimer. Domains of one subunit highlighted. Approximate location of the membrane is indicated. Right, schematic of an ASIC1 subunit. Cylinders represent helices, arrows represent β strands. The peripheral finger, knuckle and thumb domains are contiguous α helices arising from the central β-ball or palm domains. Modified from (1).

Figure 2

Na⁺ self-inhibition reduces channel activity by reducing open probability. Top: in the presence of high extracellular Na⁺ concentrations, Na⁺ (green circle) binds to a low affinity site in the extracellular domain and drives channels to strongly favor the closed state, restricting Na⁺ flux. Excised inside-out patch clamp recording shown, with 110 mM Na⁺ in the pipette, and pipette potential clamped to -100 mV to drive Na⁺ from the cell into the pipette. Channel mutant shown is not cleaved by furin, retains the α and γ subunit inhibitory tracts, and exhibits a strong Na⁺ self-inhibition response. Bottom: in the presence of a low Na⁺ concentration, the inhibitory site is unoccupied, and open probability and Na⁺ flux are higher. Patch clamp recording was performed similarly to top recording, with NMDG⁺ replacing Na⁺ in the pipette. Modified from (31).

Figure 3

Location of palmitoylation sites on the β and γ subunit. Rectangles represent predicted α-helices in cytoplasmic domains. Rotation, angle and length of first (TM1) and second (TM2) transmembrane domains are based on crystal structure of a related channel (ASIC1), although inter-subunit distances are exaggerated. Palmitoylation of βC43, γC33 and γC41 may tether these sites to the plasma membrane, and may affect the orientation of a His-Gly (HG) motif (132). βC557 is within an amphipathic α-helix. Extracellular domains are not shown.

Figure 4

ENaC channels with both a degenerin mutation and mutated furin cleavage sites exhibit an intermediate open probability with frequent switching between open and closed states. Top: Channels with the βS518K degenerin site mutation in the pore have a high open probability and short mean closed times. Bottom: Adding in α and γ subunits with mutated furin cleavage sites that prevent subunit processing by furin reduces open probability, and the channel exhibits both short mean open times and short mean closed times. Modified from (27).