EDITORIAL FOCUS
A potential second ion permeability
barrier of the epithelial
Na+ channel
Focus on "Point
mutations in the post-M2 region of human
-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
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ARTICLE |
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:
,
, and
. 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 (
3,
3, and
3)
(3) or 2) one composed of four subunits
(
2,
1, and
1)
(9). Studies of the expression of the subunits of ENaCs,
,
, and
, mainly in the Xenopus oocyte expression system, have shown that although heterologous expression of the
-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
-subunit of the ENaC constitutes the main conductive moiety or pore region of the multimeric ENaC, whereas the
- and
-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
-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 (
,
, and
) may
participate in forming the selectivity filter and pore of the ion
channel (7, 12, 13).
Studies with the
-subunit of rat ENaC (
-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
-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
-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
-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
-hENaC that were
identical in
-ENaCs from five different mammalian species but were
absent in
- and
-ENaCs. This arginine-rich region is localized
just downstream to the M2 region in the cytoplasmic COOH-terminal
domain of the
-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
- and
-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 (
and
) 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.
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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).
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