Technical Comments

Comment on “Local impermeant anions establish the neuronal chloride concentration”

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Science  05 Sep 2014:
Vol. 345, Issue 6201, pp. 1130
DOI: 10.1126/science.1252978

Abstract

Glykys et al. (Reports, 7 February 2014, p. 670) conclude that, rather than ion transporters, “local impermeant anions establish the neuronal chloride concentration” and thereby determine “the magnitude and direction of GABAAR currents at individual synapses.” If this were possible, perpetual ion-motion machines could be constructed. The authors’ conclusions conflict with basic thermodynamic principles.

The magnitude and polarity of chloride (Cl) currents mediated by γ-aminobutyric acid type A receptor (GABAAR) channels depend on the transmembrane Cl electrochemical gradient. It is generally thought that cation-chloride cotransporters (CCCs) (1)—and, to a lesser extent, bicarbonate-coupled chloride transporters (2, 3)—are the primary mechanisms that generate and maintain nonequilibrium transmembrane distributions of Cl and thereby generate the driving force for Cl currents.

In their recent Report, Glykys et al. (4) conclude that immobile negative charges near the extracellular surface of the cell membrane and impermeant negative charges in the cytosol play a central role in generating the driving force for Cl currents through GABAARs. Notably, however, any hypothetical mechanism based on immobile or impermeant charges that, without consuming energy, would maintain a driving force for channel-mediated Cl currents or for transporter-mediated Cl fluxes would conflict with basic thermodynamics.

The driving force of a channel-mediated Cl flux depends on the difference in electrochemical potential (free energy) of Cl ions between the intracellular and extracellular solutions. If immobile negative charges are present near the membrane, they cause a negative shift in local electrical potential that repels Cl ions (57). Although the resulting fall in local Cl concentration (or, more precisely, in local Cl activity) decreases the chemical component of the electrochemical potential of Cl ions, the change in local electrical potential has exactly the opposite effect on the electrical component (57). In more general terms, mobile ions such as Cl affected by electrostatic effects will move until they attain an equilibrium distribution. Consequently, the electrochemical potential gradient of Cl ions between the intracellular and extracellular solutions is not sensitive to local immobile charges. Consider the consequences if Glykys and co-workers were correct. If Cl were initially in equilibrium across a membrane, then the mere introduction of immobile negative charges (a passive element) at one side of the membrane would, according to their line of thinking, cause a permanent change in the local electrochemical potential of Cl, thereby leading to a persistent driving force for Cl fluxes with no input of energy.

A comparable argument, based on Donnan theory, can be made for impermeant negative charges distributed throughout the cytosol. A change in the concentration of impermeant cytosolic negative charge would—in the absence of further energy input—cause both a change in intracellular [Cl] and an opposing change in membrane potential (Vm), so that the electrochemical potential for Cl is unchanged. Similarly, a gradient in cytosolic impermeant charge density would create opposing [Cl] and electrical potential gradients within the cell. However, under these conditions, the electrochemical potential of Cl would be uniform within the cell. It is possible that a change in impermeant cytosolic negative charge could lead to a secondary change in Vm that would alter transmembrane driving forces of ion species. However, producing and maintaining such a shift in Vm would require the input of energy. Moreover, in the absence of active ion transport, a persistent change in Vm would lead to the dissipation of electrochemical gradients for ions across a membrane with a finite leak. Therefore, we focus next on Cl transporters.

The free energy of Cl transport mediated by CCCs and other secondary active Cl transporters depends on the product of the activities of the transported ion species on each side of the membrane (8). Immobile charges influence the local activities of these ion species, as well as the local electrical potential. The net effect is no change in the product of local ion activities (ai). Taking as an example the K+-Cl cotransporter 2 (KCC2), the product aK · aCl remains constant, as can be readily shown using the free energy function electrochemical potential or the Boltzmann equation (7, 8). Qualitatively, this lack of effect is a consequence of the attracting and repelling action of immobile charges on cations and anions. Therefore, transmembrane driving forces calculated for CCCs using bulk ion activities are not affected by immobile charges present in the vicinity of membranes, and these driving forces correctly predict the direction in which CCCs transport Cl, which is important in setting the transmembrane electrochemical gradient of Cl ions. The same considerations apply to mobile impermeant cytosolic charges, which have no effect on electrochemical potentials of cytosolic ions. If the above arguments were not true, perpetual ion-motion machines could be constructed along the lines outlined above for Cl channels.

In summary, a fundamental misconception in the paper by Glykys et al. is their central assumption that local immobile or impermeant charges could, by altering local Cl concentrations, affect the driving forces for channel-mediated Cl currents or transporter-mediated Cl fluxes and thereby (in their words) “determine the homeostatic set point for [Cl]” in neurons. However, by themselves, changes in the density of immobile or impermeant charges cannot cause maintained changes in the energy of any mobile ion species, including HCO3, to which Glykys et al. ascribed special properties (“[HCO3] is fixed by pH requirements”). A consequence of the logic of Glykys et al. is that local charges could even reverse “the polarity of local GABAAR signaling.” This is energetically impossible.

Given these theoretical objections to their interpretations, we choose not to comment here on the experimental results of Glykys et al.

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