From the Department of Physiology and Biophysics, Cornell University Medical College, New York, New York 10021
Epithelial Na channels mediate Na reabsorption in the
distal segments of the kidney, gut, and other organs
(Garty and Palmer, 1997 The question of how the subunits interacted with
each other was unresolved in the earlier studies. On the
one hand, many properties of the holochannels were
similar to those of the In the January issue of The Journal of General Physiology
Schild et al. reported results which argue strongly in favor of the three subunits making similar contributions
to the formation of the pore (Schild et al., 1997 In an article appearing in this month's issue of the
Journal, McNicholas and Canessa add support to this general idea (McNicholas and Canessa, 1997 Which parts of the subunits form the pore itself? Previous studies had identified serine residues in the M2
domain of the The subunit stoichiometry of the channel is a major
question yet to be answered. McNicholas and Canessa
found that optimal expression of ). They are vital to the control
of blood volume and arterial blood pressure, as evidenced by various forms of hypertension involving defects in the channels themselves or the renin-angiotensin-aldosterone axis regulating them (Lifton, 1996
).
Several years ago the molecules comprising these channels were cloned and sequenced (Canessa et al., 1993
;
Lingueglia et al., 1993
; Canessa et al., 1994
). The first
clone, called
rENaC, was sufficient to produce amiloride-sensitive Na currents when expressed in Xenopus
oocytes. The physiological and pharmacological properties of these channels resembled those in the kidney
and other native epithelia, but the magnitude of the
currents was small. Much larger currents were obtained
when
rENaC was coexpressed with two additional subunits termed
rENaC and
rENaC. The
and
subunits themselves did not produce measurable currents.
A molecular basis for this synergism was suggested by measurements of the surface expression of the subunits
(Firsov et al., 1996
). Coexpression of all three subunits
was essential to have a significant number of any of the
subunits in the plasma membrane of the oocyte.
subunit expressed by itself.
These included ion selectivity (Li > Na >> K), current-voltage relationship, and the affinity for the canonical blocker amiloride (KI ~ 100 nM). This suggests
that the
subunit might form the pore by itself, while
the other subunits could serve to help transport
to or
stabilize it in the membrane. On the other hand, the
three subunits are very similar in structure. They are all
about the same size, they have two predicted membrane-spanning regions (M1 and M2) separated by a large extracellular domain, and share ~30% overall homology.
This situation is more reminiscent of the nicotinic ACh
receptor, in which 5 subunits arrange themselves
pseudo-symmetrically around a central pore (Brisson
and Unwin, 1985
). A similar structure was proposed
early on for ENaC ( Jentsch, 1994
), but until recently there was little or no direct support for this model.
). The
basic finding involved the identification of a location in
the presumed extracellular domain of all three subunits which affects channel conduction and channel
block in qualitatively similar ways. The specific amino
acids are S583 in the
subunit, G525 in
, and G537 in
. These residues are located just before M2, presumably in contact with the extracellular fluid. Substitution
of a cysteine at this position of any one of the subunits
reduced both the conductance and the sensitivity to
amiloride, although these effects were much larger for
the
and
subunits than for the
. These results suggest that the residues could form part of the pore itself; amiloride is thought to bind within the lumen of the
channel (Garty and Palmer, 1997
). More strikingly, introduction of these mutations created blocking sites for
Zn2+ ions, presumably the result of a direct interaction
with the sulfhydryl group of the cysteine. Zn2+ had little
effect on the wild-type channel but blocked the
S583C,
G525C, and
G537C mutants. In the case of
S583C the block was voltage dependent, consistent
with the idea that the blocking site resides within the
pore. These results greatly strengthen the notion that
the three subunits contribute in similar ways to the formation of the channel. In particular, it is difficult to
imagine how the mutations could produce such effects
if the
and
subunits were acting just as chaperones
or stabilizing agents.
). They report
experiments defining the properties of channels formed
from only
+
or only
+
subunits. Whereas the
+
channels had properties rather similar to the wild-type holochannel, the
+
channels were much less
sensitive to amiloride and had a very different concentration-conductance relationship with a larger apparent Km for Na. Construction of chimeric subunits suggested that the key regions involved in these differences were once again in the extracellular domain. The
region affecting amiloride block was near the M2 domain, in a region including the residue studied by
Schild et al. The region affecting Na affinity was closer
to the M1 domain. The general conclusion is that the
and
subunits can substitute for each other in the formation of the holochannel, but that these substitutions
affect channel properties. Thus the subunits must have
similar but distinct roles within the channel structure.
McNicholas and Canessa (1997)
raise the intriguing
possibility that channels with different subunit composition might exist in nature. This kind of mixing and
matching of subunits could account for some of the
variability in the properties of amiloride-sensitive channels found in different tissues (Smith and Benos, 1991
;
Palmer, 1992
).
subunit which when mutated altered
the single-channel conductance, Na:Li selectivity and
amiloride affinity (Waldmann et al., 1995
). M2 is therefore a good candidate for a pore-lining structure. The
results of Schild et al. on the effects of Zn2+ block
strongly implicate the pre-M2 domain of all three subunits as another possible contributor. The results of
McNicholas and Canessa on the Na affinity of the channels suggest that a third region, just outside M1, may
also play a role in the conduction system. However, a
caveat to this conclusion is that older experiments on
frog skin implied the existence of allosteric binding
sites for Na that might modify channel activity and contribute to the apparent Km for Na transport (Lindemann and Van-Driessche, 1978
).
+
and
+
channels occurred with the injection of equivalent
doses of cRNA. This provides indirect evidence that roughly equal numbers of each subunit might be required. However, neither the exact proportions nor the
absolute number of molecules needed to form a functioning channel has been determined. This information will be required in order to advance more detailed models of how the various subunits might interact with
each other as well as with Na ions moving through the
pore.