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ABSTRACT |
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The following is the abstract of the article discussed in the subsequent letter:
Blazer-Yost, Bonnie L., and Sandy I. Helman.
The amiloride-sensitive epithelial Na+ channel: binding
sites and channel densities. Am. J. Physiol. 272 (Cell
Physiol. 41): C761-C769, 1997.The amiloride-sensitive Na+ channel found in many transporting epithelia plays a
key role in regulating salt and water homeostasis. Both biochemical and biophysical approaches have been used to identify, characterize, and
quantitate this important channel. Among biophysical methods, there is
agreement as to the single-channel conductance and gating kinetics of
the highly selective Na+ channel found in native epithelia.
Amiloride and its analogs inhibit transport through the channel by
binding to high-affinity ligand-binding sites. This characteristic of
high-affinity binding has been used biochemically to quantitate channel
densities and to isolate presumptive channel proteins. Although the
goals of biophysical and biochemical experiments are the same in
elucidating mechanisms underlying regulation of Na+
transport, our review highlights a major quantitative discrepancy between methods in estimation of channel densities involved in transport. Because the density of binding sites measured biochemically is three to four orders of magnitude in excess of channel densities measured biophysically, it is unlikely that high-affinity ligand binding can be used physiologically to quantitate channel densities and
characterize the channel proteins.
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LETTER |
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Binding sites for amiloride in intact epithelia
To the Editor: I agree with Blazer-Yost and Helman (3) that selective, high-affinity ligands for epithelial Na+ channels (ENaCs) are still needed, but urgency is less than it was 25 years ago when to isolate, sequence, and clone the channel was the goal. In their review they found only a single instance in which ENaC binding data corresponded with the biophysical measurements, describing our study on the chicken coprodeum as unique (5). However, they are mistaken in dismissing other studies, as there are serious defects in their arguments, where they appear to have confused single-channel currents (i) at high Na+ concentrations ([Na+]) with those at low [Na+].The classical papers by Lindemann and Van Driessche (7, 8),
using noise analysis in frog skin, established the characteristics of
ENaCs, now confirmed by single-channel recording. Their Fig. 2 (8)
shows the linear relation between i and [Na+],
which gives a value for i of 2 × 103 pA at 1 mM [Na+]. Binding data with amiloride combined with
biophysical measurements, carried out at 1 mM [Na+]
quoted in the review (Table 1 of Ref. 3) gives values for i of
0.3-0.8 × 10
3 pA, hardly the four orders of
magnitude difference claimed by the reviewers. The channel density,
N, increases as i falls with lowered
[Na+] (1, 7, 8). Much later, in 1991, Els and Helman (6) reported similar data, showing that [Na+] could be
reduced without affecting short-circuit current
(Isc), due to an increase in N balanced by
a reduction in i, but failed to reduce
[Na+] below the [Na+] that reduces
Isc to one-half its original value. From noise analysis (7), the number of channels in frog skin epithelium was
1/µm2, rising maximally to
50/µm2 at zero [Na+]. In many studies,
binding was measured at low [Na+] to increase the
affinity of the ligand by reducing Na+/ligand competition.
It is surprising that the reviewers failed to calculate the data from
two papers describing benzamil binding in frog skin (1, 2), as they
have done for the coprodeum. At near-zero [Na+], the
binding site density was 130/µm2, i.e., less than
threefold of the value from noise, but no specific binding was seen at
111 mM [Na+]. The Isc at 1 mM
[Na+] was 6.25 µA/cm2 (Fig. 1 of Ref. 2),
giving a value for i of 0.5 × 10
3 pA, within
fourfold, not four-orders-fold, of the value from noise. Although no
specific binding was detectable at high [Na+],
Isc increased to 28 µA/cm2. This
current is supportable by a channel density of 1.5/µm2
with an i of 0.2 pA, dimensions favored by the reviewers but undetectable by binding. Thus the appearance of detectable binding is
only found when N is increased by lowering [Na+].
Ligand binding is unlikely ever to give data as definitive as with
noise but still may provide useful insights in intact epithelia. As the
reviewers point out, we showed, in 1981 (4), that once tissues are
disrupted multitudinous binding sites are revealed, with binding
properties similar to channels. We termed these acceptor, but not
receptor, sites.
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REFERENCES |
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1.
Aceves, J.,
and
A. W. Cuthbert.
Uptake of [3H]benzamil at different sodium concentrations. Inferences regarding the regulation of sodium permeability.
J. Physiol. (Lond.)
295:
491-504,
1979[Abstract].
2.
Aceves, J.,
A. W. Cuthbert,
and
J. M. Edwardson.
Estimation of the density of sodium entry sites in frog skin epithelium from the uptake of [3H]benzamil.
J. Physiol. (Lond.)
295:
477-490,
1979[Abstract].
3.
Blazer-Yost, B. L.,
and
S. I. Helman.
The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities.
Am. J. Physiol.
272 (Cell Physiol. 41):
C761-C769,
1997
4.
Cuthbert, A. W.,
and
J. M. Edwardson.
Benzamil binding to kidney cell membranes.
Biochem. Pharmacol.
30:
1175-1183,
1981[Medline].
5.
Cuthbert, A. W.,
J. M. Edwardson,
N. Bindslev,
and
E. Skadhauge.
Identification of potential components of the transport mechanism for Na+ in the hen colon and coprodaeum.
Pflügers Arch.
392:
347-351,
1982[Medline].
6.
Els, W. J.,
and
S. I. Helman.
Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action.
J. Gen. Physiol.
98:
1197-1220,
1991[Abstract].
7.
Lindemann, B.,
and
W. Van Driessche.
Sodium-specific membrane channels of frog skin are pores: current fluctuations reveal high turnover.
Science
195:
292-294,
1977[Medline].
8.
Van Driessche, W.,
and
B. Lindemann.
Concentration dependence of currents through single sodium-selective pores in frog skin.
Nature
282:
519-520,
1979[Medline].
A. W. Cuthbert Department of Pharmacology University of Cambridge Cambridge CB2 1QJ, UK |
To the Editor: Reduction of apical solution Na+
concentration ([Na+]) does indeed increase
open channel density (No) (4, 7, 9) due,
in part, to increase of open probability (Po) (4, 7), which should enhance detection of channels by ligand binding. For
comparative purposes between papers cited by Cuthbert (1, 2) and our
review (3), it must be recognized that experiments by Van Driessche and
Lindemann (9) were done with K+-depolarized tissues in
which single-channel current (iNa) is reduced
substantially (8). Furthermore, no biophysical measurements of
iNa exist below 5 mM Na+ in
nondepolarized tissues (4) or below 10.9 mM (6 mM activity) in
K+-depolarized tissues (9). Linear extrapolation of
iNa to 1 mM is at best a rough approximation as
[Na+] approaches its reversal potential difference.
Assuming linearity for nondepolarized tissues, 100-fold decreases of
[Na+] lead to 100-fold decreases of
iNa at high fractional transcellular resistances
that exist at very low rates of Na+ entry into the cells.
Compared with iNa of 0.6 pA at 111 mM
Na+ (Fig. 1 of Ref. 3), the expected calculated
iNa at 1.1 mM [Na+] is 6 × 10 In one of two papers referred to by Cuthbert and in our review,
short-circuit current (Isc; combined
benzamil-sensitive and -insensitive currents) at 1.1 mM Na+
was reported to be ~0.1 of the mean value at 110 mM Na+,
or 1.87 µA/cm2 with specific binding density of 130 sites/µm2 (2). Neglecting insensitive currents, maximum
iNa is 0.143 × 10 We believe that we are not confused or mistaken on the primary issues
of our review. It must be noted that 1) Lindemann and Van
Driessche (6) estimated by extrapolation an upper limit of
No = 50/µm2 at 0 mM Na+
for K+-depolarized tissues (exposed additionally to
relatively high concentrations of amiloride required for noise analysis
that may also autoregulate No). The upper
extrapolated limit of No for nondepolarized tissues
calculated with data from Aceves et al. (2) is
~3.4/µm2. These upper limits are considerably less than
any value of ligand binding at any Isc (3);
2) Van Driessche and Lindemann (9) did not measure or calculate
iNa at [Na+] < 10.9 mM,
recognizing, no doubt, the uncertainty in doing so; 3) no
matter how derived, iNa (and, apparently, upper
limits of No) of K+-depolarized tissues
cannot be equated with iNa of nondepolarized tissues as done by Cuthbert; 4) the Isc of
6.25 µA/cm2 at 1.1 mM Na+ cited by Cuthbert
is taken from a single experiment (1) and is not typical of a lower
mean value (near 1.87 µA/cm2) reported by his laboratory
(2); and 5) Els and Helman (4) did in fact observe a decrease
of Isc at 5 mM Na+ (nonpaired tissues)
accompanied by increases of channel density and Po.
Their goal was not a study of discrepancies in estimation of channel
densities.
The important problem remains to know how channel density is regulated
at low [Na+] in terms not only of density but also
Po (4, 7). Changes of Po
further complicate interpretation of ligand binding data and
[Na+] dependence of the macroscopic blocker equilibrium
constant (5).
REPLY
Top
Abstract
Letter
References
3 pA.
3 pA, which
underestimates by 42 times or more the expected value. Alternatively,
assuming specific ligand binding yields true open-channel density,
Isc is at least 78 µA/cm2, far
greater than Isc measured at this binding density.
Thus we agree that the enormous discrepancy between biochemical and biophysical estimates of channel densities is less at 1.1 mM but not at
111 mM Na+. Discrepancies of one to two orders of magnitude
in near-maximally autoregulated tissues are an improvement but are no
more acceptable.
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REFERENCES |
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1.
Aceves, J.,
and
A. W. Cuthbert.
Uptake of [3H]benzamil at different sodium concentrations. Inferences regarding the regulation of sodium permeability.
J. Physiol. (Lond.)
295:
491-504,
1979[Abstract].
2.
Aceves, J.,
A. W. Cuthbert,
and
J. M. Edwardson.
Estimation of the density of sodium entry sites in frog skin epithelium from the uptake of [3H]benzamil.
J. Physiol. (Lond.)
295:
477-490,
1979[Abstract].
3.
Blazer-Yost, B. L.,
and
S. I. Helman.
The amiloride-sensitive epithelial Na+ channel: binding sites and channel densities.
Am. J. Physiol.
272 (Cell Physiol. 41):
C761-C769,
1997
4.
Els, W. J.,
and
S. I. Helman.
Activation of epithelial Na channels by hormonal and autoregulatory mechanisms of action.
J. Gen. Physiol.
98:
1197-1220,
1991[Abstract].
5.
Helman, S. I.,
and
L. M. Baxendale.
Blocker-related changes of channel density. Analysis of a three-state model for apical Na channels of frog skin.
J. Gen. Physiol.
95:
647-678,
1990[Abstract].
6.
Lindemann, B.,
and
W. Van Driessche.
Sodium-specific membrane channels of frog skin are pores: current fluctuations reveal high turnover.
Science
195:
292-294,
1977[Medline].
7.
Ling, B. N.,
and
D. C. Eaton.
Effects of luminal Na+ on single Na+ channels in A6 cells, a regulatory role for protein kinase C.
Am. J. Physiol.
256 (Renal Fluid Electrolyte Physiol. 25):
F1094-F1103,
1989
8.
Tang, J.,
F. J. Abramcheck,
W. Van Driessche,
and
S. I. Helman.
Electrophysiology and noise analysis of K+-depolarized epithelia of frog skin.
Am. J. Physiol.
249 (Cell Physiol. 18):
C421-C429,
1985[Abstract].
9.
Van Driessche, W.,
and
B. Lindemann.
Concentration dependence of currents through single sodium-selective pores in frog skin.
Nature
282:
519-520,
1979[Medline].
Sandy I. Helman Department of Molecular and Integrative Physiology University of Illinois at Urbana-Champaign Urbana, IL 61801 | |||||
Bonnie Blazer-Yost Department of Biology Indiana University-Purdue University at Indianapolis Indianapolis, IN 46202 |
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