Gamma subunit second transmembrane domain contributes to epithelial sodium channel gating and amiloride block

Abstract

Shi S, Kleyman TR. Gamma subunit second transmembrane domain contributes to epithelial sodium channel gating and amiloride block. The epithelial sodium channel (ENaC) is comprised of three homologous subunits. Channels composed solely of ␣-and ␤-subunits (␣␤-channels) exhibit a very high open probability (P o) and reduced sensitivity to amiloride, in contrast to channels composed of ␣-and ␥-subunits or of all three subunits (i.e., ␣␥-and ␣␤␥-channels). A mutant channel comprised of ␣-and ␤-subunits, and a chimeric ␥-subunit where the region immediately preceding (␤12 and wrist) and encompassing the second transmembrane domain (TM2) was replaced with the corresponding region of the ␤-subunit (␥-␤TM2), displayed characteristics reminiscent of ␣␤-channels, including a reduced amiloride potency of block and a loss of Na ϩ self-inhibition (reflecting an increased Po). Substitutions at key pore-lining residues of the ␥-␤TM2 chimera enhanced the Na ϩ self-inhibition response, whereas key ␥-subunit substitutions reduced the response. Furthermore, multiple sites within the TM2 domain of the ␥-subunit were required to confer high amiloride potency. In summary, we have identified novel pore-lining residues of the ␥-subunit of ENaC that are important for proper channel gating and its interaction with amiloride.ENaC; amiloride; open probability; sodium; subunit THE EPITHELIAL NA ϩ CHANNEL (ENaC)/degenerin family encodes a group of structurally related ion channels that are highly selective for Na ϩ and sensitive to amiloride and its derivatives (16,21). Members of the ENaC/degenerin family are involved in many fundamental biological processes, including Na ϩ absorption and volume regulation (ENaCs), mechanosensation (ENaCs and Caenorhabditis elegans degenerins), and nociception [acid-sensing ion channels (ASICs); Refs. 11,16,44]. ENaCs are heterotrimeric channels composed of three homologous subunits, termed ␣, ␤, and ␥. The second transmembrane helix from each subunit contributes to the channel pore where the gate, selectivity filter, and amiloride binding site likely reside (16,32,37,43).ENaCs with different subunit compositions have been reconstituted in heterologous expression systems (4,12,14,27). Previous studies by Fyfe and Canessa (12) and by McNicholas and Canessa (27) have shown that rat ENaCs composed solely of ␣-and ␤-subunits exhibit functional properties that differ from channels including the ␥-subunit (i.e., ␣␤␥-channels or ␣␥-channels). When expressed in Xenopus oocytes, ␣␤-channels were more permeable to Na ϩ than Li ϩ and less sensitive to amiloride and related analogs (27). At the single channel level, the ␣␤-channel displayed a smaller conductance and higher open probability (P o ; Ref. 12). These observations suggest that the ␥-subunit has roles in restraining ENaC activity and enhancing the potency of amiloride block on channel activity. However, the sites within the ␥-subunit that account for distinct properties of ␣␤␥-and ␣␥-channels, compared with ␣␤-channels, have not been elucidated.The function...

Estrogen, progesterone, and genistein differentially regulate levels of expression of α-, β-, and γ-epithelial sodium channel (ENaC) and α-sodium potassium pump (Na+/K+-ATPase) in the uteri of sex steroid–deficient rats

“…a-Epithelial sodium channel but not b-and g-ENaC was involved in aldosterone-mediated regulation of Na þ balance in the kidneys [9]. Meanwhile, g-ENaC plays an important role for proper channel gating [28]. b-and g-Epithelial sodium channel could form a selective filter that accommodates Na þ and Li þ entry [29] and could interact with Nedd4, a ubiquitin-protein ligase that is involved in regulating ENaC function [30].…”

ENaC in Salt-Sensitive Hypertension: Kidney and Beyond

“…ENaC regulation by Na+ self-inhibition (NaSI) enables the distal nephron to control Na + reabsorption based on fluctuating urinary Na + concentrations. Numerous studies have found that many of the human ENaC nonsynonymous single nucleotide variants affect channel function by altering the NaSI response [38][39][40][41][42].…”

New insights regarding epithelial Na+ channel regulation and its role in the kidney, immune system and vasculature

“…This response, referred to as Na + self-inhibition [2], provides a mechanism for cells in the distal nephron to rapidly tune the rate of Na + influx according to fluctuations of urinary [Na + ]. Numerous sites in the different domains of the extracellular regions of ENaC have been identified where amino acid substitutions alter channel activity, primarily by changing the Na + self-inhibition response [9,12,13,[16][17][18][19][20][21].…”