EDITORIAL FOCUS
A potential second ion permeability barrier of the epithelial Na+ channel
Focus on "Point mutations in the post-M2 region of human alpha -ENaC regulate cation selectivity"

Pradeep K. Dudeja

Department of Medicine/Physiology, University of Illinois at Chicago and Westside Division, Veterans Affairs Medical Center, Chicago, Illinois 60612


    ARTICLE
TOP
ARTICLE
REFERENCES

AMILORIDE-SENSITIVE Na+ channels constitute a new class of superfamily of ion channel proteins known as the degenerin/epithelial Na+ channel (Deg/ENaC) superfamily (1, 2). This superfamily is rapidly expanding with new members identified from vertebrates and invertebrates. These channels are ubiquitous in nature and have been shown to be expressed in a variety of epithelial and nonepithelial cell types (1, 2). These ion channels play a vital role in Na+ homeostasis and extracellular volume control. Derangement of these proteins has been implicated in the pathophysiology of a number of human genetic diseases, e.g., Liddle's syndrome (salt-sensitive hypertension) and PHA-1 (pseudohypoaldosteronism type I) (1, 2, 5). Interactions of ENaC with CFTR (cystic fibrosis transmembrane conductance regulator) have been implicated in yet another human genetic disease, cystic fibrosis (2).

Among various members of the Deg/ENaC superfamily, ENaC represents the most characterized prototype member (1, 2). Epithelial Na+ channels exhibit high selectivity for Na+ and Li+, inhibition by submicromolar concentrations of amiloride, small conductance, and voltage-independent slow kinetics (1, 2). ENaCs are expressed at the apical membrane of cells in many epithelial tissues, e.g., colon, lung, distal nephron, and secretory glands (1, 2). Because of the critical role of these ion channels in the physiology as well as pathophysiology of various human diseases, recent studies have intensely focused on elucidating the structure-function relationship of these proteins. ENaCs have been shown to be composed of three nonidentical but homologous subunits: alpha , beta , and gamma . Each subunit has two membrane-spanning hydrophobic domains (M1 and M2), a large extracellular domain, and cytoplasmic NH2- and COOH-terminal domains (1, 2). For the stoichiometry of ENaC or other members of the Deg/ENaC superfamily, two models have been proposed: 1) one composed of nine subunits (alpha 3, beta 3, and gamma 3) (3) or 2) one composed of four subunits (alpha 2, beta 1, and gamma 1) (9). Studies of the expression of the subunits of ENaCs, alpha , beta , and gamma , mainly in the Xenopus oocyte expression system, have shown that although heterologous expression of the alpha -subunit alone was sufficient for the ion conductance and amiloride inhibition characteristics of ENaCs, the expression of all three subunits was, however, required for the optimal targeting and functioning of these channels (2). On the basis of the above findings, it has been proposed that the alpha -subunit of the ENaC constitutes the main conductive moiety or pore region of the multimeric ENaC, whereas the beta - and gamma -subunits are auxiliary proteins that enhance the function of the ion channel (2). However, the detailed characteristics of the conductive pore of ENaCs have not been defined. Therefore, a number of recent studies have focused on elucidating the structural determinants of ion conductance, selectivity, and amiloride inhibition of the ENaC alpha -subunit (4, 7, 8, 12, 13). The current article in focus (Ref. 6; see page C64 in this issue) represents a significant contribution from a group of investigators who for many years have been focusing on molecular characterization of the epithelial Na+ channels. Their study provides strong evidence that a cation has to interact with at least two selectivity barriers in a series during its passage through the channel and greatly advances our knowledge with respect to characterization of the channel pore.

The critical function of ENaC in Na+ absorption depends on its ability to discriminate between Na+ and other cations (e.g., K+, Ca2+) and its inability to conduct anions. In an attempt to characterize this selectivity filter and conductive pore, recent studies utilizing mutagenesis and subsequent functional analysis have indicated that amino acid residues of the hydrophobic region (H2) preceding the M2 region of the ENaC may form the selectivity filter of the channel (7, 8, 12, 13). It has been suggested that all three subunits (alpha , beta , and gamma ) may participate in forming the selectivity filter and pore of the ion channel (7, 12, 13).

Studies with the alpha -subunit of rat ENaC (alpha -rENaC) suggested a P-loop model similar to K+ channels in which the pre-M2 (H2) region of the channel dips into the membrane, presumably forming the ion pore (11). Additionally, previous studies utilizing alpha -ENaC splice variants and chimeras indicated that the M2 region was important for channel function (14). Studies from the laboratory of the authors of the current article in focus (6) have recently demonstrated that the M2 region of alpha -hENaC was critical to channel function (10). In that study, reversing the negative charges of the three amino acids of the M2 region (E568R, E571R, and D575R) significantly decreased the channel conductance without affecting ion selectivity. In contrast, similar mutation (E108R) of the M1 region of alpha -hENaC showed no functional consequences (10).

In the current article in focus (6), the authors have identified a novel arginine-rich sequence of conserved positively charged amino acids between residues 586 and 597 of alpha -hENaC that were identical in alpha -ENaCs from five different mammalian species but were absent in beta - and gamma -ENaCs. This arginine-rich region is localized just downstream to the M2 region in the cytoplasmic COOH-terminal domain of the alpha -hENaC. The current study was designed to test the hypothesis that this arginine-rich domain may comprise part of the inner mouth of the hENaC pore and may play a role in the conduction and/or ion selectivity of this channel. Two adjacent double point mutations of this region were generated, and the functional studies were performed in Xenopus oocytes at both the macroscopic and single-channel level by utilizing various concentrations of Na+, Li+, and K+. The functional analysis of the double point mutants showed significant differences with respect to steady-state kinetics and biophysical properties compared with the wild-type ENaC. The differences were noted in macroscopic current, open probability, apparent equilibrium dissociation constant (Km) and maximal amiloride-sensitive current (Imax) for Na+, and ion selectivity. On the basis of these data, the authors have concluded that this region of the positive charge is a novel and important domain for ion permeation, serves as the potential second barrier for the ions moving through the channel, and may belong to the inner mouth of the conduction pore of the channel. The authors concluded that the ion selectivity resides at multiple sites, e.g., pre-H2, H2, proximal portion of the M2, and the arginine-rich post-M2 region. It is possible that the post-M2 regions of the beta - and gamma -subunits also may be similarly involved in ion selectivity.

In summary, the elegant studies of Ji et al. (6) provide strong evidence for a novel second ion selectivity barrier, presumably at the inner mouth of the channel pore, and raise interesting questions with respect to the role of this region in the other ENaC subunits (beta  and gamma ) and their possible contributions to the channel conductance and selectivity filter. Future studies focusing on further characterization of the structure/function of the human ENaC pore region should greatly increase our understanding of the physiology of the ENaC as well as the pathophysiological basis of the malfunctions of these channels in various human diseases.


    FOOTNOTES

Address for reprint requests and other correspondence: P. K. Dudeja, Univ. of Illinois at Chicago, Medical Research Service (600/151), Veterans Affairs Medical Center, 820 South Damen Ave., Chicago, Illinois 60612 (E-mail: pkdudeja{at}uic.edu).


    REFERENCES
TOP
ARTICLE
REFERENCES

1.   Alvarez de la Rosa, D, Canessa CM, Fyfe GK, and Zhang P. Structure and regulation of amiloride-sensitive sodium channels. Annu Rev Physiol 62: 573-594, 2000[ISI][Medline].

2.   Benos, DJ, and Stanton BA. Functional domains within the degenerin/epithelial sodium channel (Deg/ENaC) superfamily of ion channels. J Physiol (Lond) 520: 631-644, 1999[Abstract/Free Full Text].

3.   Eskandari, S, Snyder PM, Kreman M, Zampighi GA, Welsh MJ, and Wright EM. Number of subunits comprising the epithelial sodium channel. J Biol Chem 274: 27281-27286, 1999[Abstract/Free Full Text].

4.   Fyfe, GK, Zhang P, and Canessa CM. The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels. J Biol Chem 274: 36415-36421, 1999[Abstract/Free Full Text].

5.   Hummler, E, and Horisberger JD. Genetic disorders of membrane transport. V. The epithelial sodium channel and its implication in human diseases. Am J Physiol Gastrointest Liver Physiol 276: G567-G571, 1999[Abstract/Free Full Text].

6.   Ji, HL, Parker S, Langloh AL, Fuller CM, and Benos DJ. Point mutations in the post-M2 region of human alpha -ENaC regulate cation selectivity. Am J Physiol Cell Physiol 281: C64-C74, 2001[Abstract/Free Full Text].

7.   Kellenberger, S, Gautschi I, and Schild L. A single point mutation in the pore region of the epithelial Na+ channel changes ion selectivity by modifying molecular sieving. Proc Natl Acad Sci USA 96: 4170-4175, 1999[Abstract/Free Full Text].

8.   Kellenberger, S, Hoffmann-Pochon N, Gautschi I, Schneeberger E, and Schild L. On the molecular basis of ion permeation in the epithelial Na+ channel. J Gen Physiol 114: 13-30, 1999[Abstract/Free Full Text].

9.   Kosari, F, Sheng S, Li J, Mak DO, Foskett JK, and Kleyman TR. Subunit stoichiometry of the epithelial sodium channel. J Biol Chem 273: 13469-13474, 1998[Abstract/Free Full Text].

10.   Langloh, AL, Berdiev B, Ji HL, Keyser K, Stanton BA, and Benos DJ. Charged residues in the M2 region of alpha -hENaC play a role in channel conductance. Am J Physiol Cell Physiol 278: C277-C291, 2000[Abstract/Free Full Text].

11.   Renard, S, Lingueglia E, Voilley N, Lazdunski M, and Barbry P. Biochemical analysis of the membrane topology of the amiloride-sensitive Na+ channel. J Biol Chem 269: 12981-12986, 1994[Abstract/Free Full Text].

12.   Sheng, S, Li J, McNulty KA, Avery D, and Kleyman TR. Characterization of the selectivity filter of the epithelial sodium channel. J Biol Chem 275: 8572-8581, 2000[Abstract/Free Full Text].

13.   Snyder, PM, Olson DR, and Bucher DB. A pore segment in DEG/ENaC Na+ channels. J Biol Chem 274: 28484-28490, 1999[Abstract/Free Full Text].

14.   Tucker, JK, Tamba K, Lee YJ, Shen LL, Warnock DG, and Oh Y. Cloning and functional studies of splice variants of the alpha-subunit of the amiloride-sensitive Na+ channel. Am J Physiol Cell Physiol 274: C1081-C1089, 1998[Abstract/Free Full Text].


Am J Physiol Cell Physiol 281(1):C15-C16




This Article
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Dudeja, P. K.
Articles citing this Article
PubMed
PubMed Citation
Articles by Dudeja, P. K.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online