Intracellular Cl− Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

Hogan, Emilia M.; Davis, Bruce A.; Boron, Walter F.

doi:10.1085/jgp.110.5.629

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Article| November 01 1997

Intracellular Cl⁻ Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions

Emilia M. Hogan,

Emilia M. Hogan

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

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Bruce A. Davis,

Bruce A. Davis

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

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Walter F. Boron

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

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Author and Article Information

Emilia M. Hogan

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Bruce A. Davis

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Walter F. Boron

From the Department of Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06520

Address correspondence to Walter F. Boron, Department of Cellular and Molecular Physiology, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520. Fax: 203-785-7678; E-mail: [email protected]

Portions of this work have been published in preliminary form (Hogan, E.M., B.A. Davis, and W.F. Boron. 1995. Biophys. J. 9:A356).

1

Abbreviations used in this paper: ASW, artificial seawater; BMF, barnacle muscle fiber; CTX, cholera toxin; DF, dialysis fluids; NMDG⁺, N-methyl-d-glucammonium.

2

The slope of the pH_i recovery in bc reflects not only the Na-H exchange rate, which tends to increase pH_i, but also the presence of background acid loading, which tends to decrease pH_i. In experiments in which the cell was later exposed to a hypertonic solution (c–f), it was not possible to inhibit the bc pH_i increase with amiloride because the amiloride is not fully reversible. Thus, we can draw no conclusions about the Na-H exchange rate in bc in this experiment.

3

Because we added the 1 mM GTPγS as the tetra-lithium salt, the DF containing GTPγS also contained 4 mM Li⁺. Although previous work from this laboratory (Davis et al., 1992b) showed that 100 mM Li⁺ stimulates Na-H exchange, here we found that dialyzing with 4 mM Li⁺ (in the absence of GTPγS) fails to stimulate Na-H exchange at pH_i ∼7.2 (not shown).

6

In principle, introducing GTPγS could also result in the de novo generation of ATPγS within the muscle fiber. Such ATPγS could lead to the generation of long lived phosphoproteins. However, our demonstration that cholera toxin and AlF₃ also activate the Na-H exchanger argues that, regardless of a possible phosphorylation, activation of a G protein can lead to activation of the exchanger.

4

We would also predict that this “basal” shrinkage signal is also G-protein dependent so that dialyzing with GDPβS should have the same effect as dialyzing with a Cl⁻-free solution: inhibition of basal Na-H exchange.

5

A recent report shows that AlF₃ can bind to a ras-GAP-GDP complex, inducing a shift in the absorbance spectrum of a fluorescent GDP analog (Mittal et al., 1996). The crystal structure of this ras-GAP-GDP-AlF₃ complex has been solved (Scheffzek et al., 1997). However, there is no evidence that AlF₃ activates a ras-related protein.

Received: February 27 1997

Accepted: September 12 1997

Online ISSN: 1540-7748

Print ISSN: 0022-1295

1997

J Gen Physiol (1997) 110 (5): 629–639.

https://doi.org/10.1085/jgp.110.5.629

We previously showed that shrinking a barnacle muscle fiber (BMF) in a hypertonic solution (1,600 mosM/kg) stimulates an amiloride-sensitive Na-H exchanger. This activation is mediated by a G protein and requires intracellular Cl⁻. The purpose of the present study was to determine (a) whether Cl⁻ plays a role in the activation of Na-H exchange under normotonic conditions (975 mosM/kg), (b) the dose dependence of [Cl⁻]_i for activation of the exchanger under both normo- and hypertonic conditions, and (c) the relative order of the Cl⁻- and G-protein-dependent steps. We acid loaded BMFs by internally dialyzing them with a pH-6.5 dialysis fluid containing no Na⁺ and 0–194 mM Cl⁻. The artificial seawater bathing the BMF initially contained no Na⁺. After dialysis was halted, adding 50 mM Na⁺ to the artificial seawater caused an amiloride-sensitive pH_i increase under both normo- and hypertonic conditions. The computed Na-H exchange flux (J_Na-H) increased with increasing [Cl⁻]_i under both normo- and hypertonic conditions, with similar apparent K_m values (∼120 mM). However, the maximal J_Na-Hincreased by nearly 90% under hypertonic conditions. Thus, activation of Na-H exchange at low pH_i requires Cl⁻ under both normo- and hypertonic conditions, but at any given [Cl⁻]_i, J_Na-H is greater under hyper- than normotonic conditions. We conclude that an increase in [Cl⁻]_i is not the primary shrinkage signal, but may act as an auxiliary shrinkage signal. To determine whether the Cl⁻-dependent step is after the G-protein-dependent step, we predialyzed BMFs to a Cl⁻-free state, and then attempted to stimulate Na-H exchange by activating a G protein. We found that, even in the absence of Cl⁻, dialyzing with GTPγS or AlF₃, or injecting cholera toxin, stimulates Na-H exchange. Because Na-H exchange activity was absent in control Cl⁻-depleted fibers, the Cl⁻-dependent step is at or before the G protein in the shrinkage signal-transduction pathway. The stimulation by AlF₃ indicates that the G protein is a heterotrimeric G protein.

1997

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Intracellular Cl⁻ Dependence of Na-H Exchange in Barnacle Muscle Fibers under Normotonic and Hypertonic Conditions