Epithelial Na + Channels

Epithelial Na ⁺ channels (ENaCs) have a key role in the regulation of extracellular fluid volume and blood pressure. Over the two decades since the initial characterization of ENaCs at a molecular level, investigators have identified key structural features within channel subunits and have defined mechanisms by which hormones and other factors regulate ENaC activity. Mutations within ENaC subunits have been identified that result in a gain or loss of function, which have profound effects on blood pressure. The identification of Liddle syndrome mutations led to the elucidation of a network of proteins, including Sgk1, GILZ, and Nedd4-2, which have important roles in the regulation of ENaC activity. The high-resolution structure of ASIC1, in combination with functional studies, has provided new insights regarding the role of the extracellular region in the regulation of ENaC gating.

Keywords

ENaC; Epithelia; Amiloride, Sodium; ASIC; Aldosterone; Nedd4-2; kinase

Introduction

From the late distal convoluted tubule, connecting tubule, and throughout the collecting duct, Na ⁺ exits the urinary space by passive diffusion through an apical membrane epithelial Na ⁺ channel, referred to as ENaC. Na ⁺ exits cells across basolateral membranes via the Na ⁺ ,K ⁺ -ATPase. Koefoed-Johnson and Ussing initially proposed this model of electrogenic transepithelial Na ⁺ transport, with diffusion of Na ⁺ across an apical membrane conductance pathway, in 1958. Studies using both noise analysis and single channel recordings demonstrated the presence of apical membrane Na ⁺ selective ion channels. Subsequent to the cloning of ENaC, knockout studies have confirmed the role of ENaC in the reabsorption of filtered Na ⁺ in the distal nephron, as well as its role in facilitating renal K ⁺ secretion.

Electrophysiologic Characteristics

The basic electrophysiologic characteristics of epithelial Na ⁺ channels have been defined using both macroscopic and single channel studies, and are presented in Figure 30.1 . ENaCs are Na ⁺ - and Li ⁺ -permeable channels that exhibit negligible K ⁺ conductance, with a single channel conductance of 4 to 5 pS at room temperature with Na ⁺ as the charge carrier. These channels exhibit a slight increase in open probability under hyperpolarizing membrane potentials. ENaCs characteristically exhibit long open and closed times, in the order of seconds to tens of seconds, although a population of ENaCs has been described that has brief open times and long closed times that likely represent channels that have not been processed by proteases. Channels are blocked by submicromolar concentrations of amiloride, a pyrazinoylguanidine derivative. Amiloride is a weak base and has a pK _a of 8.8 in water. It is the charged, or protonated, form of amiloride that blocks ENaC. Other organic weak bases, including triamterene, trimethoprim, and pentamidine also inhibit ENaC.

Figure 30.1, Biophysical properties of epithelial Na + channels.

Biochemical and Molecular Characteristics of ENaC

Amiloride was first demonstrated to inhibit electrogenic transepithelial Na ⁺ transport in 1968. With an IC ₅₀ of ~100 nM, amiloride and several related compounds proved to be highly selective Na ⁺ channel inhibitors. Prior to the cloning of ENaC subunits, several different approaches were used to isolate a Na ⁺ channel complex. The relationship of this channel complex to ENaC is still unclear, although antibodies raised against a subunit of the cloned Na ⁺ channel have been observed to recognize a polypeptide within the purified channel complex.

The molecular characteristics of this channel were elucidated in studies from Canessa and co-workers and Lingueglia and co-workers in 1993 and 1994. An expression cloning technique led to the identification of a cDNA, termed α ENaC, whose cRNA induced expression of amiloride-sensitive Na ⁺ currents when injected into Xenopus oocytes. However, Na ⁺ currents were lower than expected. Two subsequent cDNA clones were isolated based on their ability to complement α ENaC cRNA in the expression of amiloride-sensitive Na ⁺ currents in Xenopus oocytes, and were termed β and γ ENaC. Xenopus oocytes co-injected with the three cRNA species expressed amiloride-sensitive Na ⁺ channels with characteristics nearly identical to that of Na ⁺ channels expressed in renal cortical collecting tubules and in cultured cell lines derived from the distal nephron. Na ⁺ currents were not observed when either the β- or γ-subunits were expressed alone.

The three ENaC subunits are likely derived from a common ancestral gene, given their limited (~30 to 40%) sequence identity. ENaC subunits have been cloned from a variety of species, including rat, human, mouse, rabbit, guinea pig, chicken, cattle, sheep, salamander, clawed African frog ( Xenopus laevis ), and bullfrog. The human α-subunit gene Scnn1a spans 17 kb on human chromosome 12p13. The β- and γ-subunit genes Scnn1b and Scnn1g are closely linked on human chromosome 16p12-p13. The genes encoding the three ENaC subunits have a conserved exon–intron architecture, with 13 exons. Splice variants have been described that alter the cytoplasmic N-termini or alter the extracellular domains. Variants that result in a termination codon in the region coding the extracellular domain of the α-subunit have also been reported. Some of these variants are associated with a reduction or loss of channel activity when expressed in heterologous systems, and with an autosomal recessive loss of function phenotype (pseudohypoaldosteronism) in humans.

ENaC/Degenerin Gene Family

Canessa and co-workers noted that ENaC subunits were related to genes identified in Caenorhabditis elegans that participate in mechanosensation specific mutations of which result in degeneration of selected neurons. ENaCs belong to the ENaC/Degenerin gene family ( Figure 30.2 ). There are two additional ENaC subunits, referred to as δ and ε, that appear to be functionally related to the α-subunit. δ ENaC is a proton-activated channel expressed in primates, although its physiologic role is still unclear. The ε-subunit was identified in Xenopus , and has an altered Na ⁺ self-inhibition response suggesting altered gating properties. Members of this family also include genes identified in C. elegans that are involved in mechanosensation ( mecs and degs ) or control of defecation rhythm ( flrs ); H ⁺ -gated channels (referred to as acid-sensing ion channels (or ASICs)) that are expressed in mammalian central and peripheral nervous systems and have a role in nociception, mechanosensation, fear-related behavior, and seizure termination; a family of 16 genes expressed in Drosophila , referred to as pickpocket, that may have roles in airway fluid clearance, mechanosensation, salt sensation, and detection of pheromones; and peptide-gated channels expressed in marine snails.

Figure 30.2, Phylogenetic tree of the ENaC/DEG superfamily.

Structure and Function of ENaC

Each ENaC subunit has only two predicted membrane spanning domains, similar to the topology of members of the Kir family of K ⁺ channels, and members of the P2X family of purinergic receptors that are ligand-gated ion channels ( Figure 30.3 ). Three independent groups published topologic analyses of ENaC subunits. Each subunit has intracellular amino and carboxyl termini and two α helical membrane spanning domains connected by a large extracellular domain. The cytoplasmic domains have kinase phosphorylation sites, as well as protein–protein and protein–lipid interaction motifs that affect channel gating or trafficking.

Figure 30.3, Structural features of ENaC subunits.

Jasti and colleagues resolved the crystal structure of ASIC1, a member of the ENaC/Degenerin family. Although initially lacking the N- and C-terminal cytoplasmic regions, this structure provided important insights into the structural organization of ASIC and related family members, including ENaC. ASIC1 is a trimer, suggesting that ENaC has a subunit stoichiometry of α1β1γ1, in contrast to the higher ordered stoichiometry that had been proposed based on biophysical and biochemical studies. The extracellular region is a highly ordered structure that resembles an outstretched hand containing a ball, and has clearly defined domains termed wrist, finger, thumb, palm, knuckle, and β-ball (see Figure 30.3 ). Jasti et al. suggested that ASIC1 proton-dependent gating occurs in conjunction with conformational changes within the thumb and finger domains, which are transmitted to the transmembrane domains. The three ENaC subunits contribute residues that line the channel’s pore. The resolved ASIC1 structure suggests that the channel’s gate resides in the outer part of the pore, in the vicinity of the “degenerin” site where the introduction of bulky residues in: (1) ENaC subunits; (2) mec4 or mec10 (a component of the mechanosensitive channel in C. elegans ); and (3) ASIC subunits result in a dramatic increase in channel open probability.

ASIC1 is the only member of the ENaC/Degenerin gene family whose structure has been resolved. This structure provides a starting point to generate models of ENaC subunits. Kashlan and co-workers have built models of the extracellular region of the α-subunit of ENaC based, in part, on homology to ASIC1. The extracellular regions of ENaC subunits and ASIC1 are reasonably homologous, except in the finger domain. For example, the α-subunit of ENaC has 73 additional residues when compared to ASIC (see ). The α-subunit is proteolytically processed at specific sites within its finger domain, releasing an inhibitory tract. To determine the molecular architecture of the α-subunit finger domain, sites within α ENaC that interact with an 8 residue inhibitory peptide derived from the portion of the α-subunit that is removed by furin cleavage were determined. The working hypothesis was that the inhibitory tract within a non-cleaved α-subunit and the 8 residue inhibitory peptide share common binding sites. Distance constraints were introduced to construct a model of the α-subunit. The model places the inhibitory tract at an interface of the finger and thumb domains. Kashlan and co-workers suggested that the inhibitory tract blocks channel activity by stabilizing the movement of the finger domain relative to the thumb domain.

Stockand and co-workers have also generated a model of an ENaC trimer, based on the structure of ASIC1. To generate the model areas that lacked sequence similarity within the finger regions of ENaC subunits were removed. Although this limits the utility of the model, the model provides important insights regarding sites of intersubunit interactions.

ENaC Biogenesis

Na ⁺ channel subunits likely undergo assembly in the endoplasmic reticulum (ER), where core, high mannose Asn- (or N)-linked glycans are added at specific sites. Each subunit is modified by N-linked glycosylation at multiple sites. For example, rat α-, β-, and γ-subunits have six, twelve, and five consensus sites (Asn-X-Ser/Thr) for N-linked oligosaccharide addition, respectively. Exit of assembled channels from the ER appears to be inefficient. A motif that facilitates the exit of ENaC from the ER was found in the proximal cytoplasmic carboxyl terminus of the α-subunit.

Channel subunits that do not exit the ER are likely degraded via proteasome-mediated ER associated degradation (ERAD). Integral membrane proteins such as ENaC are co-translationally inserted into the ER. The proper folding and assembly of polypeptides synthesized in the ER involves interactions with a variety of chaperone proteins. Specific chaperones that participate in ENaC folding and assembly or targeting misfolded subunits for degradation are starting to be defined. These include members of the luminal Hsp40s, the small heat shock protein alpha A-crystallin, and calreticulin. Cytoplasmic Hsp70s also have roles in channel trafficking, although at present it is unclear whether their roles are related to facilitating ER exit or post-ER trafficking events.

The half-life of newly synthesized subunits, determined by metabolic labeling/pulse-chase experiments, is approximately an hour, consistent with the notion that the majority of ENaC subunits synthesized within the ER are targeted for degradation via ERAD. A longer-lived pool of channels is apparent in pulse-chase studies, which likely reflects properly assembled channels that have exited the ER. Several groups have determined the half-life of channels that have reached the surface. The rate of degradation of channels that have reached the surface may be in the order of many hours to days, although some investigators have reported a shorter half-life for channels that have reached the cell surface of A6 cells and MDCK cells expressing exogenous ENaCs. These differences in the half-life of channels that have reached the cell surface could result from differences in cell type, and whether ENaCs are expressed endogenously or exogenously.

ENaC Processing in the Biosynthetic Pathway

As assembled ENaCs exit the ER, it has been thought that channels follow the route used by other proteins in the secretory pathway. This involves trafficking through the Golgi where most N-glycans are processed, and the trans-Golgi network where channels are sorted into endosomes that are delivered to the apical membrane. N-glycan processing is often monitored by the enzyme endoglycosidase H, an enzyme that removes high mannose N-linked glycans prior to processing events that occur in the medial Golgi complex.

N-glycans on all three subunits of assembled channels are modified to complex-type Endo H-resistant forms. Surprisingly, subunits with endoglycosidase H sensitive N-glycans have also been described. Hughey and co-workers reported that two distinct pools of ENaC subunits were expressed at the plasma membrane: subunits with processed N-glycans and cleaved α- and γ-subunits; and full-length subunits that have non-processed N-glycans. Processing of subunits within a channel complex appears to be an all-or-nothing event. These findings suggest that a population of channel complexes exiting the ER transits through Golgi and post-Golgi compartments where subunits are processed ( Figure 30.4 ). These processed channels likely represent the pool of active, functional channels. A distinct population of channels exiting the ER appears to bypass Golgi and post-Golgi processing events. This distinct pool of non-processed channels is likely a functionally inactive pool, as proteolysis of ENaC subunits appears to have a dramatic effect on increasing channel open probability. Proteolytic cleavage of these inactive channels provides a potential mechanism to increase rates of Na ⁺ transport in the distal nephron. The role of proteolysis of ENaC subunits in regulating channel gating is discussed in detail later in this chapter.

Figure 30.4, Model of ENaC biogenesis and intracellular trafficking.

Intracellular Trafficking of ENaC

Functional channels are delivered to the apical plasma membrane via the traditional secretory pathway ( Figure 30.4 ). The exocytic insertion of channels into the apical plasma membrane occurs as a regulated process that is increased in response to a variety of hormones, including vasopressin, aldosterone, and insulin. Vesicle and target SNAREs (soluble N-ethylmaleimide sensitive factor attachment protein receptors) participate in this process, and overexpression of specific SNARE proteins disrupts the intracellular trafficking of ENaC subunits. Channels at the plasma membrane appear to reside within specific compartments. Several groups have reported that a population of channels resides within lipid-rich microdomains, referred to as lipid rafts, although other groups have not confirmed this finding. While lipid rafts have been reported to facilitate the co-localization of membrane proteins and signaling molecules, the functional consequences of ENaCs within lipid rafts are still unclear. ENaCs interact with cytoskeletal elements, including actin and α-spectrin, which may have a role in localizing the channel to the plasma membrane and in modulating ENaC activity.

The residency time of channels at the plasma membrane has been examined by several groups, with reported half-lives in the order of minutes to hours. Mutations within a carboxyl-terminal PY motif within the β- and γ-subunits are associated with increases in the half-life of the channel at the plasma membrane via mechanisms that are discussed later in this chapter. Internalization of channels from the plasma membrane has been proposed to occur via a dynamin-dependent process. Dynamin is required for clathrin-dependent endocytosis, as well as for caveolae-dependent endocytosis. Ubiquitin conjugation of defined lysine residues within ENaC subunits at the plasma membrane targets the channels for endocytosis, presumably via a clathrin-dependent mechanism ( Figure 30.4 ). Once internalized, some channels are targeted for degradation via proteasomes or possibly lysosomes. A significant fraction of the pool of endocytosed channels may undergo recycling to the plasma membrane in a regulated manner ( Figure 30.4 ). Specific deubiquitination enzymes have a role in removing ubiquitin from internalized channels, facilitating the recycling of channels to the plasma membrane.

Localization within the Kidney and Other Organs

The aldosterone-sensitive distal nephron is the final site of Na ⁺ reabsorption within the nephron. ENaCs are expressed in principal cells in the late distal convoluted tubule, connecting tubule, and through the collecting duct, and are the major pathway for Na ⁺ entry across the apical plasma membrane. In the more proximal segments of the aldosterone-sensitive distal nephron, Na ⁺ reabsorption via ENaC is coupled to K ⁺ secretion mediated by apical membrane K ⁺ channels, including Kir1.1 (or ROMK) and the large conductance Ca ⁺ -activated K ⁺ channel (maxi-K). ENaC-dependent reabsorption of Na ⁺ in the distal nephron has a major role in the control of extracellular fluid volume, blood pressure, and renal K ⁺ secretion.

The cellular localization of individual ENaC subunits may differ within the nephron. When maintained on normal laboratory diet, β- and γ-subunits were localized within an intracellular compartment in principal cells within the cortical and outer medullary segments of the rat aldosterone-sensitive distal nephron. One group reported that the α-subunit was localized primarily to apical part of principal cells, whereas other groups have observed either modest cytoplasmic localization or have failed to detect the α-subunit. Within the inner medullary collecting duct, all three ENaC subunits were localized primarily within an intracellular compartment. When placed on a low-Na ⁺ diet or following administration of aldosterone, all three subunits were expressed at the apical membrane of principal cells.

Mice lacking expression of the β- or γ-subunits or that have reduced expression of the α-subunit exhibit renal Na ⁺ -wasting. Mice that lack expression of the α-subunit are unable to clear airway fluids at birth, leading to death in the early postnatal period. Recent work suggests that the density of ENaC expression may be greatest in the connecting tubule, an early segment within the aldosterone-sensitive distal nephron that connects the distal convoluted tubule to the collecting tubule. Mice that lack expression of α ENaC beyond the connecting tubule are able to maintain Na ⁺ and K ⁺ balance, even in the setting of dietary Na ⁺ restriction or K ⁺ -loading.

In addition to its expression in the nephron, ENaCs are expressed within numerous other organs. They are expressed throughout the airways, as well as in both type I and type II alveolar cells. ENaC has a key role in the reabsorption of airway fluids. Maintaining an appropriate volume of airway surface liquids has an important role in facilitating mucociliary clearance. ENaCs are also expressed in the distal colon, sweat ducts, salivary ducts, inner ear, lingular epithelium, keratinocytes, lymphocytes, and vascular smooth muscle. ENaC expression has also been reported in endothelia and in various sites within the eye, including epithelia within the retina, lens, and pigmented ciliary body and iris. The functional roles of ENaCs within many of these tissues are unclear.

ENaC Structure and Function

Specific structural features within channel subunits have key roles in defining the biophysical properties of ENaC. All three ENaC subunits contribute to the formation of the conduction pore, as pore properties are altered by mutations within each of the three subunits. The published structures of ASIC1 reveal a homomeric trimer ( Figure 30.3 ). The structures of the extracellular domain (ECD) and transmembrane domain (TM) are well-defined, while the cytoplasmic amino- and carboxyl-terminal domains are not resolved in the crystal structures. The cASIC1 structures confirm a longstanding notion that the channel pore is formed largely by the second transmembrane domain (TM2s) that determines the basic biophysical properties of ENaC. Both the extracellular and cytoplasmic domains have roles in modulating channel-gating and trafficking. The functional roles of individual domains within ENaC subunits, and potential mechanisms of ion permeation, ion selectivity, channel-gating, and amiloride block are reviewed below with insights from the ASIC1 structures.

Functional Domains within ENaC Subunits

Amino-Terminus

The cytoplasmic amino-termini have regions that affect channel-gating, trafficking, and regulation by intracellular factors. Chalfant and co-workers identified a Lys-Gly-Asp-Lys tract within the rat α-subunit corresponding to residues 47–50, that may function as an endocytic signal that regulates the number of channels in the plasma membrane. A domain that affects channel-gating has been characterized within the distal portion of the amino-terminus of the α-subunit, which includes a highly-conserved His-Gly tract. A mutation in the corresponding Gly in the β-subunit was described in a patient with pseudohypoaldosteronism. Reduced channel activity attributed to a decreased open probability was observed with a mutation of the conserved Gly in each subunit, suggesting that the His-Gly tract within the amino termini of all three subunits influences channel-gating. A recent study showed that mutations of this Gly in the α and γ ENaC subunits induced a strong inward rectification in the whole cell currents, reflecting voltage-dependent gating. Mutations at other sites have also been found to reduce channel activity and induce voltage-dependent gating.

Phosphatidylinositol 4,5-bisphosphate (PIP2) activates ENaCs, an effect that reflects an increase in channel open probability as a result of direct interactions between PIP2 and ENaC. Several groups have proposed that the amino-termini of the β- and γ-subunits of ENaC harbor putative PIP2-binding domains containing basic amino acid residues. In addition, Helms and co-workers suggest that PIP3 mediates aldosterone-induced ENaC activity and trafficking, and interacts with γ ENaC. The ENaC amino-termini also contain Cys residues that may interact with intracellular metals (Cd ²⁺ and Zn ²⁺ ) and thiol-reactive chemicals, leading to a reduction in channel open probability. Palmitoylation of amino-terminal Cys-43 and carboxyl-terminal Cys-557 of mouse β ENaC enhances channel open probability (see below).

First Transmembrane Domain (TM1)

The ASIC1 structures were apparently resolved in a desensitized state. In these structures, the channel pore is primarily lined by TM2 helices from three identical subunits, with the three TM1 helices packed tangentially behind the TM2 helices and contacting the lipid bilayer. The TM1 helix from an individual subunit also makes extensive interactions with the TM2 helix of the same subunit, as well as specific contacts with helices from an adjacent subunit. The closed-pore conformation of ENaC may be similar to that of ASIC1. A Trp scanning of the TM1 of α ENaC showed that mutations within the amino-terminal portion of αTM1 alter channel activity, selectivity, and gating, consistent with the extensive intrasubunit interaction between TM1 and TM2 helices revealed in the ASIC1 structures. A recent Cys scanning of ASIC1 TM domains is also consistent with the relative locations of TM1 and TM2 helices in the resolved structures.

Extracellular Domain (ECD)

Each ENaC subunit has a large, ~450 residue extracellular region with 16 conserved Cys residues clustered within two Cys-rich domains. Other members of the ENaC/DEG family also possess extracellular Cys-rich domains. The size and the apparently conserved structural organization of the extracellular domain suggest that this region has important functional roles. Recent studies have examined the role of the ECD in modulating channel-gating in response to proteases, sheer stress, external Na ⁺ , metals, H ⁺ , and Cl ⁻ . These functional studies and insights from the ASIC1 structure suggest that the ECD of ENaC functions as a sensor or receptor for a variety of extracellular signals. The extracellular domains also facilitate the assembly of the heteroligomeric channel complex within the endoplasmic reticulum, and influence intracellular trafficking.

Several proteases, including prostasin and related channel-activating proteases, furin, trypsin, chymotrypsin, and elastase have been shown to activate ENaC. Activation of ENaC by proteases is a result of proteolytic cleavage of ENaC subunits within their extracellular domains at defined sites, releasing intrinsic inhibitory tracts. Mechanisms by which proteases activate ENaCs are discussed below.

ENaCs are inhibited by extracellular Na ⁺ , which presumably binds to sites within the ECD. This response, referred to as self-inhibition, reflects a reduction in channel open probability. The structural basis of Na ⁺ self-inhibition has begun to emerge from mutagenesis studies. At present, the sites of Na ⁺ -binding, as well as the subsequent allosteric changes that lead to a reduction in channel open probability, have not been defined. In addition to Na ⁺ , other external cations such as Ni ²⁺ , Zn ²⁺ , Cu ²⁺ , Cd ²⁺ , and Hg ²⁺ affect ENaC activity, presumably by binding to sites within the ECDs and altering channel open probability. The effects of these metals on ENaC are both metal- and species-specific.

Extracellular anions also interact at sites within the extracellular domains of ENaC subunits and alter ENaC gating. Collier and Snyder found that external Cl ⁻ regulates ENaC activity in part through enhancing Na ⁺ self-inhibition. Their findings provide a mechanism by which changes in extracellular Cl ⁻ modulates epithelial Na ⁺ absorption. They identified two Cl ⁻ inhibitory sites in ENaC, one formed by residues in the thumb domain of the α-subunit and the palm domain of the β-subunit, and the other formed by residues at the interface of the thumb domain of the β-subunit and the palm domain of the γ-subunit. Based on the effects of mutagenesis on Cl ⁻ inhibition, the additive nature of mutations, and on differences in the mechanisms of Cl ⁻ inhibition, the authors propose a model in which ENaC subunits assemble in an αγβ-orientation when viewed from above ( Figure 30.3 ). This subunit arrangement is also suggested by Chen et al., based on the identification of a putative Cu ²⁺ -binding site at the α- and β-subunit interface within the ECD of human ENaC. However, the model is based on the assumption that the functional ENaC complex is a heterotrimer. The possibility that multiple arrangements co-exist needs to be tested.

Within the ENaC extracellular domains are 16 Cys residues, which are largely conserved among other members of the ENaC/degenerin family. Firsov and colleagues proposed that specific Cys residues (the first and sixth Cys in all three subunits, as well as the eleventh and twelfth Cys in the α- and β-subunits) have an essential role in the efficient transport of assembled channels to the plasma membrane. A mutation of the first cysteine residue in the human α-subunit (aC133Y) is associated with PHA-1. Sheng et al. proposed that there are several intrasubunit disulfide bonds, based on analyses of additivity of double Cys mutations and responses to sulfhydryl reagent of single and double mutants. These predicted disulfide bonds (Cys1-Cys6, Cys4-Cys5, Cys7-Cys16, Cys11-Cys12, Cys10-Cys13, and Cys8-Cys15 (conserved Cys numbered sequentially)) were present in the resolved ASIC1 structure. ASIC subunits have 14 conserved Cys residues within the ECD that form seven intrasubunit disulfide bonds, one in β-ball, one in palm, and five in thumb domain. The locations of the disulfide bonds are consistent with their roles in stabilizing the conformations of these domains.

Second Transmembrane Domain (TM2)

The TM2 region is thought to be the segment that lines the conduction pore and contains important functional sites, including the degenerin (DEG) site, amiloride-binding site, and selectivity filter site ( Figure 30.5 ). The structure of the ENaC pore is expected to be similar to that of ASIC1, given the high degree of sequence homology and similar biophysical properties. For example, three ENaC TM2s likely line the channel pore, while TM1s contact the lipid bilayer. As the resolved ASIC1 structures likely represent non-conducting states, they provide limited information regarding the roles of TM2 residues in ion permeation and selectivity. Two distinct TM domain structures of ASIC1 have been described, adding an additional layer of complexity in interpreting structure and function relationships of the TM2 domains from the resolved ASIC1 structures. Here we discuss mutagenesis results, mainly in reference to the most recent ASIC1 structure.

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