The Journal of Neurophysiology Vol. 77 No. 2 February 1997, pp. 639-653
Copyright ©1997 The American Physiological Society

Differential Sensitivity to Intracellular pH Among High- and Low-Threshold Ca²⁺ Currents in Isolated Rat CA1 Neurons

Geoffrey C. Tombaugh and George G. Somjen

Department of Cell Biology, Duke University Medical Center, Durham, North Carolina 27710

Tombaugh, Geoffrey C. and George G. Somjen. Differential sensitivity to intracellular pH among high- and low-threshold Ca²⁺ currents in isolated rat CA1 neurons. J. Neurophysiol. 77: 639-653, 1997. The effects of intracellular pH (pH_i) on high-threshold (HVA) and low-threshold (LVA) calcium currents were examined in acutely dissociated rat hippocampal CA1 neurons with the use of the whole cell patch-clamp technique (21-23°C). Internal pH was manipulated by external exposure to the weak base NH₄Cl or in some cases to the weak acid Na-acetate (20 mM) at constant extracellular pH (7.4). Confocal fluorescence measurements using the pH-sensitive dye SNARF-1 in both dialyzed and intact cells confirmed that NH₄Cl caused a reversible alkaline shift. However, the external TEA-Cl concentration used during I_Ca recording was sufficient to abolish cellular acidification upon NH₄Cl wash out. With 10 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES) in the pipette, NH₄Cl exposure reversibly enhanced HVA currents by 29%, whereas exposure to Na-acetate markedly and reversibly depressed HVA Ca currents by 62%. The degree to which NH₄Cl enhanced HVA currents was inversely related to the internal HEPES concentration but was unaffected when internal ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) was replaced by equimolar bis-(o-aminophenoxy)-N,N,N,N-tetraacetic acid (BAPTA). When depolarizing test pulses were applied shortly after break-in (V_h = 100 mV), NH₄Cl caused a proportionally greater increase in the sustained current relative to the peak. The dihydropyridine Ca channel antagonist nifedipine (5 µM) blocked nearly all of this sustained current. A slowly inactivating nifedipine-sensitive (L-type) HVA current could be evoked from a depolarized holding potential of 50 mV; NH₄Cl enhanced this current by 40 ± 3% (mean ± SE) and reversibly shifted the tail-current activation curve by +6-8 mV. L-type currents exhibited more rapid rundown than N-type currents; HVA currents remaining after prolonged cell dialysis, or in the presence of nifedipine, inactivated rapidly and were depressed by -conotoxin (GVIA). NH₄Cl enhanced these N-type currents by 76 ± 9%. LVA Ca currents were observed in 32% of the cells and exhibited little if any rundown. These amiloride-sensitive currents activated at voltages negative to 50 mV, were enhanced by extracellular alkalosis and depressed by extracellular acidosis, but were unaffected by exposure to either NH₄Cl or NaAC. These results demonstrate that HVA Ca currents in hippocampal CA1 neurons are bidirectionally modulated by internal pH shifts, and that N-type currents are more sensitive to alkaline shifts than are L- or T-type (N > L > T). Our findings strengthen the idea that distinct cellular processes governed by different Ca channels may be subject to selective modulation by uniform shifts in cytosolic pH.

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