1.A.6 The Epithelial Na⁺ Channel (ENaC) Family

ENaC family members are from animals with no recognizable homologues in other eukaryotes or bacteria. The vertebrate ENaC proteins from epithelial cells cluster tightly together on the phylogenetic tree; voltage-insensitive ENaC homologues are also found in the brain. The many sequenced C. elegans proteins, including the worm degenerins, are distantly related to the vertebrate proteins as well as to each other. At least some of these proteins form part of a mechano-transducing complex for touch sensitivity. D. melanogaster also has many ENaC family paralogues, some closely related to each other, others very distant in sequence. Other members of the ENaC family, the acid-sensing and/or mechanosensory ion channels, ASIC1-4, are homo- or hetero-oligomeric neuronal Zn²⁺ and H⁺-gated, mechanosensitive channels that mediate pain sensation in response to tissue acidosis. Two extracellular histidines (his-162 and his-339) potentiate Zn²⁺ activation while another (his-72) mediates pH sensitivity (Baron et al., 2001). ASIC1-4 also mediate light touch sensation and are excited by hair movement. The homologous Helix aspersa (FMRF-amide)-activated Na⁺ channel is the first peptide neurotransmitter-gated ionotropic receptor to be sequenced. Salty taste is mediated by an ENaC channel in the fungiform papillae in the dorsal epithelium of the anterior tongue.

Protein members of this family all exhibit the same apparent topology, each with N- and C-termini on the inside of the cell, two amphipathic transmembrane spanning segments, M1 and M2, and a large extracellular loop. The extracellular domains contain numerous highly conserved cysteine residues. They are proposed to serve a receptor function. Welsh et al. (2002) present three models whereby members of the ENaC family sense mechanostimulation. Their preferred model involves tethering the channel protein to extracellular matrix proteins such as collagens and/or intracellular cystoskeletal proteins such as α- and β-tubulins. Carnally et al., 2008 have presented evidence, based on the X-ray crystal structure, that ASIC1a assembles as a trimer.

Mammalian ENaC is important for the maintenance of Na⁺ balance and the regulation of blood pressure. Three homologous ENaC subunits, α, β and γ, have been shown to assemble to form the highly Na⁺-selective channel. Only the dehydrated form of Na⁺ (or Li⁺) is transported. The stoichiometry of the three subunits is possibly α₂βγ in a heterotetrameric architecture. A structural model has been proposed in which the properties of the channel are conferred by the second TMS together with the preceding hydrophobic region that may loop into the membrane as do the P-regions of VIC family members. The selectivity filter of the epithelial Na⁺ channel α-subunit is at least in part determined by residues Ser⁵⁸⁰ to Ser⁵⁹² following the second TMS. Residues conferring cation selectivity are in both M2 and the preceding loop. Negatively charged residues in M2 of the mammalian α-subunit are important as two substitutions, αE595C and αD602C confer K⁺ permeability (Sheng et al., 2001b).

The C-terminus of each ENaC subunit contains a PPXY motif which when mutated or deleted in either the β- or γ-ENaC subunit leads to Liddle's syndrome, a human autosomal dominant form of hypertension. In this disease, the mutation induces abnormally high levels of channel expression due to a loss of interaction with the inhibitory Nedd4 protein. Nedd4 regulates the activity of the epithelial Na⁺ channel in normal people but not in those suffering from Liddle's syndrome. Multiple WW domains in Nedd4 mediate the interaction with all three subunits of ENaC, α, β and γ, and WW domains 2-4 are most important for this interaction (Snyder et al., 2001).

Acid-sensing ion channels (ASICs) have been implicated in perception of pain, ischaemic stroke, mechanosensation, learning and memory. Jasti et al. (2007) reported the low-pH crystal structure of a chicken ASIC1 deletion mutant at 1.9 Å resolution. Each subunit of the chalice-shaped homotrimer is composed of short amino and carboxy termini, and two transmembrane helices. A bound chloride ion is present. A disulphide-rich, multidomain extracellular region is enriched in acidic residues with carboxyl-carboxylate pairs, suggesting that at least one carboxyl group bears a proton. Electrophysiological studies on aspartate-to-asparagine mutants confirmed that these carboxyl-carboxylate pairs participate in proton sensing. Between the acidic residues and the transmembrane pore lies a disulphide-rich 'thumb' domain poised to couple the binding of protons to the opening of the ion channel. The results demonstrated that proton activation involves long-range conformational changes. The Akt and Sgk protein kinases are components of an insulin signaling pathway that increases Na+ absorption by up-regulating membrane expression of ENaC via a regulatory system that involves inhibition of Nedd4-2 (Lee et al., 2007).

Gonzales et al. (2009) presented the structure of a functional acid-sensing ion channel in a desensitized state at 3 Å resolution, the location and composition of the approximately 8 Å thick desensitization gate, and the trigonal antiprism coordination of caesium ions bound in the extracellular vestibule. Comparison of the acid-sensing ion channel structure with the ATP-gated P2X(4) receptor revealed similarity in pore architecture and aqueous vestibules, suggesting that there are unanticipated yet common structural and mechanistic principles (Gonzales et al., 2009).

The generalized transport reaction for Na⁺ channels is:

Na⁺ (out) Na⁺ (in).

That for the degenerins is:

Cation (out) cation (in).