Properties of acid-induced currents in mouse dorsal root ganglia neurons

Abstract

Acid-sensing ion channels (ASICs) are cation channels that are activated by protons (H(+)). They are expressed in neurons throughout the nervous system and may play important roles in several neurologic disorders including inflammation, cerebral ischemia, seizures, neurodegeneration, anxiety, depression, and migraine. ASICs generally produce transient currents that desensitize in response to a decrease in extracellular pH Under certain conditions, the inactivation of ASICs can be incomplete and allow them to produce sustained currents. Here, we characterize the properties of both transient and sustained acid-induced currents in cultured mouse dorsal root ganglia (DRG) neurons. At pH levels between 7.3 and 7.1 they include "window currents" through ASICs. With stronger acid signals sustained currents are maintained in the absence of extracellular Na(+) or the presence of the ASIC blockers amiloride and Psalmotoxin-1(PcTx1). These sustained responses may have several different origins in these cells, including acid-induced stimulation of inward Cl(-) currents, block of outward K(+) currents, and augmentation of inward H(+) currents, properties that distinguish these novel sustained currents from the well-characterized transient currents.

Keywords: ASIC; Amiloride; Pc1Tx; Zn2+; sustained currents.

Figure 1

Proton‐evoked (pH 5.3) currents in mouse DRG neurons. (A) examples of transient plus sustained currents (top), sustained‐only currents (middle), and transient‐only currents (bottom). (B) numbers of neurons responding to low pH with patterns illustrated in A with pH ≤ 6.3. (C) Frequency histogram of peak amplitude of transient currents. The line represents the best‐fit to a Gaussian distribution with mean = 137 pA and standard deviation = 96 pA.

Figure 2

pH‐dependent activation of transient and sustained currents in mouse DRG neurons. (A) Recordings from neurons in response to a low pH challenge (responding to steps from 7.4 to 7.0, 6.5, 6.0, and 5.5, respectively). (B) pH‐dependent activation of transient currents and sustained currents. Data are normalized to values obtained at pH 5.5 and are represented as means ± SEM for four recordings. The pH required for a half‐maximal response (pH ₅₀) was 6.8 for transient currents. Sustained currents did not exhibit a maximal amplitude.

Figure 3

Activation and desensitization curves of proton‐evoked transient currents. (A) Conditioning steps of different pH between 7.4 and 6.6 are followed by a test step with pH 6.0. (B) Peak values of currents at pH 6.0 were used for the desensitization curve, and peak currents during the conditioning steps were used for the activation curve. Currents were normalized to those measured with pH 7.8 (desensitization) and pH 6.0 (activation), and are represented as mean ± SEM for seven cells (activation) and 21 cells (desensitization). Solid lines represent best fits of the Hill equation with half‐activation at pH 6.9, and half‐desensitization at pH 7.35.

Figure 4

Effects of amiloride (1 mmol/L), PcTx1 (20 nmol/L) and Na⁺‐free extracellular solution on proton‐evoked transient currents and sustained currents in mouse DRG neurons. Amiloride and PcTx1 were added only to the low‐pH solution. (A) Recordings from neurons in response to a low pH challenge and inhibition of transient currents. (B) Values of peak and sustained currents normalized to control values. Data are represented as means ± SEM for 5–6 cells. Transient currents were significantly decreased with amiloride, PcTx1, and removal of Na⁺ from extracellular solution. Amiloride and removal of Na⁺ had no effects on sustained currents, whereas PcTx1 increased them **P < 0.01 compared with control).

Figure 5

Effects of extracellular K⁺ and intracellular Cl⁻ concentrations on proton‐evoked currents. (A) Reduction in sustained current with extracellular high K⁺. (B) Increased sustained current with low extracellular K⁺. (C) Reversal of sustained current with increased extracellular K⁺ in the presence of 7 mmol/L intracellular Cl. (D) Increase in sustained currents with reduced extracellular K⁺. in the presence of 7 mmol/L (E) Values of sustained currents with high (140 mmol/L) and low (7 mmol/L) intracellular Cl‐ and high (20 mmol/L) and low (5 mmol/L) extracellular K⁺. Data are represented as means ± SEM for 3–11 cells. (*P < 0.05, **P < 0.01) (F) Comparison of I‐V curves at normal and low extracellular K⁺ conditions. Results are from a single cell, representative of five independent experiments (1 mmol/L (K) and six experiments (5 mmol/L K).

Figure 6

Effects of Zn²⁺ on activation of transient currents. (A) Currents activated by different pH with or without Zn²⁺. Thick gray lines represent best fits to exponential decay functions. (B) Time constants and maximum transient currents from experiments like those shown in panel A. Values are normalized to those of control traces. Data are represented as means ± SEM for seven cells (*P < 0.05, **P < 0.01 compared with control).

Figure 7

Activation of sustained currents by Zn²⁺ and amiloride, and amiloride‐washoff effect at pH 7.4. (A) Effect of 1 mmol/L amiloride. (B) Effect of 1 mmol/L Zn²⁺. C. Effects of amiloride + Zn²⁺. Simultaneous washoff of amiloride and Zn²⁺ produced a transient inward current. Results are from a single cell, representative of three independent experiments.