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Department of Physiology and Biophysics, Finch University of the Health Sciences/ The Chicago Medical School, North Chicago, Illinois
1 Dept. of Pharmacology and Physiology, MCP Hahnemann School of Medicine, Philadelphia, PA
Volume-regulated anion currents are ubiquitous and contribute to volume recovery in response to cell swelling under hypotonic conditions. Glutamate (Glut-) and other amino acids are involved in volume regulatory processes (e.g., 1–5), and one pathway for volume-regulated amino acid release is through a volume-regulated anion conductance (6,7). Glut-, unlike many other volume-sensitive amino acids, is negatively charged at physiological pH; and Glut- current may therefore be measured using whole cell patch clamp recording (3). This study addresses the relative permeability of Glut- and Cl- through a volume-regulated anion conductance in the presence and absence of an ionic strength gradient.
Human embryonic kidney cells (tsA201a) were maintained as previously described (8,9). Extracellular solutions contained (in mM): 1 EGTA, 2 CaCl2, 10 HEPES, adjusted to pH 7.2 with 1M HCl or N-methyl D-glutamine. High ionic strength external solutions contained 150 Cl- or Glut-; low ionic strength solutions contained 50 Cl- or Glut-. Intracellular solutions contained (in mM): 1.1 EGTA, 0.1 CaCl2, 10 HEPES, 4 ATP, pH 7.2 and molar fractions (MF = [Glut-]/{[Glut-] + [Cl-]}) of Cl- and Glut- ranging from 0.0 to 1.0 at high (150 mM) or low (50 mM) ionic strength. At least 0.2 mM Cl- was present in all solutions for electrode stability. Solution osmolality was measured with a vapor pressure osmometer (Wescor, Logan, Utah) and adjusted such that the intracellular osmolarity was 100 ± 5 mOsm greater than the external osmolarity. Isotonic osmolarity = 313 ± 5 mM. In some experiments, a transmembrane ionic strength gradient was established with high extracellular and low intracellular ionic strength solutions. The difference between high and low ionic strength solutions was 100 mM, as determined by 
= o - in = 0.5(
ciz2i)o - 0.5(ciz2i)in, where = ionic strength, ci = concentration of ion1 and zi = valance of ion1.
Whole cell recording and analysis was performed as described in (3). Swelling activated anion currents were elicited by a voltage ramp from -60 to +100 mV. Reversal potentials (Vr) were measured when the maximal current amplitude was no longer changing and were corrected for liquid junction potentials using JPCalc (Cell MicroControls, Virginia Beach, VA). Corrected Vrs were substituted into a standard form of the Goldman-Hodgkin-Katz equation, rearranged to solve for the permeability ratio:
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Figure 1A shows that PGlut/PCl was always less than 1.0, indicating a greater relative chloride permeability when the intra- and extracellular ionic strength was the same on both sides of the membrane. The PGlut/PCl, however, varied as the intracellular molar fraction of Glut- moved from 0 toward 1.0, indicating that the anions compete for the permeation pathway of the anion channel and interact within the pore (10).
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The major finding of this study was that the anion permeability associated with the volume-regulated anion conductance is dependent on both extra- and intracellular ionic composition and on a transmembrane ionic strength gradient. These results are consistent with an ion conductive pathway that can be occupied simultaneously by more than one permeant species, and with interactions between the permeant anions within this pathway.
The combined effect of an ionic strength gradient and extracellular Cl- results in a switch in the relative permeability of the volume-regulated anion current, from preferring Cl-, to Glut-. Our hypothesis is that this effect is due to a change in the ion channel protein underlying the current, as our results indicate ion-ion interaction. It is possible that an additional glutamate transport pathway is activated under these conditions. A change in the intracellular ion composition as a result of swelling may alter the volume-regulated anion conductance so that Glut- passes more easily than Cl- during volume recovery. This may increase the Glut--induced excitotoxicity found in neuronal and glial cell populations.
Supported by NIDDK46672 and AHA94002340.
Literature Cited
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) ionic strength on both sides of the membrane and was less than 1.0 over the range of MFs tested. (B) PGlut/PCl also varies with intracellular MF but was greater than 1.0 over the range of MFs tested when determined in the presence of a transmembrane ionic-strength gradient and extracellular Cl- containing solutions (•). PGlut/PCl was always less than 1.0 in the presence of a transmembrane ionic strength gradient and extracellular Glut- containing solutions (
). Intracellular MF was varied as indicated. n 
5, ± SEM.