Letters to the Editor

    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.

    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 × 10-3 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.

    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[Abstract/Free Full Text].

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

    REPLY
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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 × 10-3 pA.

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 × 10-3 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.

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).

    REFERENCES
Top
Abstract
Letter
References

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[Abstract/Free Full Text].

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[Abstract/Free Full Text].

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 


AJP Cell Physiol 273(4):C1437-C1439
0363-6143/97 $5.00 Copyright © 1997 the American Physiological Society




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