Identification of Amino Acid Residues in the α, β, and γ Subunits of the Epithelial Sodium Channel (ENaC) Involved in Amiloride Block and Ion Permeation

Abstract

The amiloride-sensitive epithelial Nachannel (ENaC) is a heteromultimeric channel made of three αβγ subunits. The structures involved in the ion permeation pathway have only been partially identified, and the respective contributions of each subunit in the formation of the conduction pore has not yet been established. Using a site-directed mutagenesis approach, we have identified in a short segment preceding the second membrane-spanning domain (the pre-M2 segment) amino acid residues involved in ion permeation and critical for channel block by amiloride. Cys substitutions of Gly residues in β and γ subunits at position βG525 and γG537 increased the apparent inhibitory constant (K i) for amiloride by >1,000-fold and decreased channel unitary current without affecting ion selectivity. The corresponding mutation S583 to C in the α subunit increased amiloride K i by 20-fold, without changing channel conducting properties. Coexpression of these mutated αβγ subunits resulted in a nonconducting channel expressed at the cell surface. Finally, these Cys substitutions increased channel affinity for block by externalZn2+ ions, in particular the αS583C mutant showing a K i for Zn2+of 29 μM. Mutations of residues αW582L or βG522D also increased amiloride K i, the later mutation generating a Ca2+blocking site located 15% within the membrane electric field. These experiments provide strong evidence that αβγ ENaCs are pore-forming subunits involved in ion permeation through the channel. The pre-M2 segment of αβγ subunits may form a pore loop structure at the extracellular face of the channel, where amiloride binds within the channel lumen. We propose that amiloride interacts with Na+ions at an external Na+binding site preventing ion permeation through the channel pore.

A Pore Segment in DEG/ENaC Na+ Channels

“…5C); a cysteine introduced at position 536 was modified not only by MTSEA but also by the larger reagent MTSET. Our results suggest that the equivalent residues in ␣ and ␤ make a similar contribution to the pore and are consistent with previous reports regarding the accessibility of these residues to Zn 2ϩ (27) and (for mouse ␣ENaC) MTSEA (19). The contribution of this residue to the pore is also conserved in another DEG/ENaC family member; in BNC1, MTSEA inhibited the channel when a cysteine was introduced at Gly 437 (equivalent to ␥ Gly-536 ) (31).…”

“…5, A and B) (21). This is interesting since mutation of Gly 536 decreased the affinity of ENaC for amiloride (27) and benzamil. 2 The predicted equivalent to Gly 533 (Gly 79 in KcsA) is part of the selectivity filter of the K ϩ channel (Fig.…”

Extracellular Zn2+ Activates Epithelial Na+ Channels by Eliminating Na+ Self-inhibition

“…Furthermore, we observed similar activation of the whole cell Na ϩ currents and elimination of Na ϩ self-inhibition by external Zn 2ϩ in oocytes expressing human ␣␤␥ ENaCs. 2 Our results are also consistent with a previous report (22) demonstrating a lack of an effect of external Zn 2ϩ at the concentrations up to 1 mM on inward Li ϩ currents in oocytes expressing ␣␤␥ rENaC and bathed in a low Li ϩ (20 mM) bath.…”

Acid-sensing ion channels (ASICs) as pharmacological targets for neurodegenerative diseases

“…Based on the studies of ENaC [47], it is believed that amiloride inhibits the ASIC current by a direct blockade of the channel, and that the pre-TM II region of the channel is critical for its effect. Mutation of Gly-430 in this region, for example, dramatically increases the sensitivity of ASIC2a current to amiloride [48].…”