Technical Comments

Response to Comments 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.1253146

Abstract

We appreciate the interest in our paper and the opportunity to clarify theoretical and technical aspects describing the influence of Donnan equilibria on neuronal chloride ion (Cl) distributions.

We first address the concerns of Voipio et al. (1) about Donnan equilibria and then the concerns of Luhmann et al. (2) on technical aspects of our study (3). Regarding Donnan energetics, Voipio et al. state: “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”. The first section of the quote is a concise description of Donnan effects on the Cl distribution, and the resultant electrochemical potential is the Donnan potential (4). The last part of the quote—“thereby leading to a persistent driving force for Cl fluxes”—is the point of confusion.

The Donnan potential is the membrane voltage at which the system is at equilibrium— i.e., at which there is no free energy available to do work such as moving Cl across the membrane. The free energy to drive membrane Cl currents is supplied by the process that shifts the membrane potential away from the Donnan potential and thereby shifts the system away from equilibrium. The driving force for electrogenic Cl flux across the membrane depends on the difference between the shifted membrane potential and the Donnan potential, not the Donnan potential itself. Without the addition of energy to the system to shift the membrane potential, there will be no net ion flux across the membrane in a system at equilibrium.

Regarding the distribution of impermeant charges, Voipio et al. express two concerns. First, they cite charge screening studies to support their argument that impermeant anion distributions do not alter either the bulk [Cl] or the free energy of Cl flux across the membrane. For sufficiently thin anion layers, we agree. Charge screening studies concern the effects of the charged polar heads of phospholipids comprising lipid bilayers. These are not the distributions that we describe.

Intracellularly, most intracellular anions cannot permeate γ-aminobutyric acid type A (GABAA) receptor–operated channels, and these impermeant anions are not confined to a thin layer along the membrane. Nevertheless, Voipio et al. are concerned that “[a] consequence of the logic of Glykys et al. is that local charges could even reverse ‘the polarity of local GABAAR signaling.’” Since the classic studies of Coombs, Eccles, and Fatt (5), it has been routine to manipulate the Cl equilibrium potential in intracellular recordings by replacing various amounts of Cl in the recording electrode solution with less permeant anions. Thus, it is widely accepted that (exogenous) intracellular impermeant local charges can displace [Cl]i and thereby change the polarity of GABAAR signaling.

Extracellularly, bulk cerebrospinal fluid has a high [Cl] and few impermeant charges. However, neurons are not apples bobbing in a sea of cerebrospinal fluid. Rather, they are embedded in a gelatinous extracellular matrix composed of polyanionic biopolymers that are sufficiently dense to impart the matrix with a tortuosity that far exceeds that of cerebrospinal fluid (6). Although the exceptionally high charge density of these anionic biopolymers is well established (7), the actual spatial distribution of fixed anionic charges in the brain’s extracellular matrix has only rarely been considered (8) and merits more study.

Voipio et al. express a second concern regarding the distribution of intracellular impermeant anions: “...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.” This was our point also. If the electrochemical potential for [Cl]i is uniform within the neuron, then oppositely directed Cl cotransport is not required to maintain differences in subcellular [Cl]i (9). Although the electrochemical potential of Cl would be uniform within the cell, the electrochemical potential of Cl across the cell membrane would not be uniform at subcellular locations containing differing concentrations of impermeant anions, as has been repeatedly observed (10, 11).

Luhmann et al. raise several technical questions for which we provide the following clarifications. Regarding the sensitivity of Clomeleon, with a dissociation constant (Kd) of ~100 mM, the change in the fluorescence ratio is 1% Δ ratio per 1- to 2-mM change in Cli for Cli between 1 and 20 mM [figure 3C in (12); figure 3C in (11); and supplemental figure 7B in (13)]. This sensitivity is sufficient to test our hypotheses. [Cl]i and volume stability experiments can be found in figure S4 in (3).

Regarding the variance in [Cl]i, including immature preparations: A key finding driving the current study is the substantial variance in neuronal [Cl]i, which has also been reported by other groups using Clomeleon (11, 14), as well as perforated patch (15) and dual cell–attached recordings (16). Intraneuronal [Cl]i is also variable (10, 11). Rather than being an experimental deficiency, we propose that the variability of [Cl]i is a fundamental feature of the brain’s composition.

Regarding the effects of NKCC1 inhibition, our data are consistent with the cited studies. Data in figure 3, H and I, in (3) are from two different populations of neurons and are well within the range of values shown in figure 1, B and C, in (3). NKCC1 inhibition reduces [Cl]i in neurons with high initial [Cl]i and increases [Cl]i in neurons with low initial [Cl]i [figures S1B, S2B, and S3 in (3)] (17, 18). Fluorometric techniques sample dozens to hundreds of neurons. Electrophysiological studies, including our earlier studies, report a handful of recorded cells selected based on the experimenter’s preferences for cell turgor. In light of our findings regarding the relation between neuronal volume and [Cl]i, such selection could readily bias small samples of neurons.

Regarding knockout studies of transporters, as stated in the concluding sentence of the summary of our study, cation-chloride transporters are critically important for restoring [Cl]i and volume after signaling transients. The sequelae of chronic cation-chloride cotransport inhibition [e.g., (19)] do not invalidate our hypotheses.

Regarding Na/K/ATPase (adenosine triphosphatase), the suggested experiment was not included because we had previously reported that perforated patch measurement of [Cl]i during Na/K/ATPase inhibition showed only very modest changes in [Cl]i that were well within the range we would predict (20).

Regarding the permeability of gluconate, this anion permeates a variety of Cl channels with permeabilities ranging from 10 to 40% of Cl (2123), which is ample for the experiment we performed. The interesting hypotheses put forward as to why the experiment using weak organic acids might not work would only be valid if proton buffering were purely passive—i.e., only in the absence of proton pumps and exchangers. We and others, including Luhmann’s group [e.g., (24)], have also altered the cytoplasmic concentration of relatively impermeant anions by introducing gluconate directly from the recording pipette solution. This approach of altering [A]i eliminates the dependence on membrane transport or permeation. Much larger reductions in [Cl]i can be demonstrated with this technique, and the data robustly support the idea that [A]i and [Cl]i are inversely related.

Regarding the seizure experiments, our confirmation of the predicted correlation between neuronal volume and [Cl]i changes during seizures has not been previously reported. As indicated in our publication, cation-chloride cotransporters are important elements in [Cl]i homeostasis, but, based on our experiments, what they do not do is determine the [Cl] set point.

We accept that this is a complex topic to introduce in a short communication, and we appreciate the opportunity to provide clarifications based on the theoretical and technical questions raised here. These questions do not affect the validity of our conclusions. Cl homeostasis and GABA signaling are more complex than we initially envisioned. Our hypothesis and data have provided an explanation for the inter- and intraneuronal variance of [Cl]i observed in different preparations and experimental approaches and create broad opportunities for further research.

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