Acid-sensing ion channels (ASICs) in mouse skeletal muscle afferents are heteromers composed of ASIC1a, ASIC2, and ASIC3 subunits

Abstract

Acid-sensing ion channels (ASICs) are expressed in skeletal muscle afferents, in which they sense extracellular acidosis and other metabolites released during ischemia and exercise. ASICs are formed as homotrimers or heterotrimers of several isoforms (ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3), with each channel displaying distinct properties. To dissect the ASIC composition in muscle afferents, we used whole-cell patch-clamp recordings to study the properties of acid-evoked currents (amplitude, pH sensitivity, the kinetics of desensitization and recovery from desensitization, and pharmacological modulation) in isolated, labeled mouse muscle afferents from wild-type (C57BL/6J) and specific ASIC(-/-) mice. We found that ASIC-like currents in wild-type muscle afferents displayed fast desensitization, indicating that they are carried by heteromeric channels. Currents from ASIC1a(-/-) muscle afferents were less pH-sensitive and displayed faster recovery, currents from ASIC2(-/-) mice showed diminished potentiation by zinc, and currents from ASIC3(-/-) mice displayed slower desensitization than those from wild-type mice. Finally, ASIC-like currents were absent from triple-null mice lacking ASIC1a, ASIC2a, and ASIC3. We conclude that ASIC1a, ASIC2a, and ASIC3 heteromers are the principle channels in skeletal muscle afferents. These results will help us understand the role of ASICs in exercise physiology and provide a molecular target for potential drug therapies to treat muscle pain.

Figure 1.

ASICs in skeletal muscle afferents are composed of multiple subunits. A) Corresponding phase (left panel) and fluorescence (right panel) micrographs of 3 labeled skeletal muscle afferents in primary dissociated culture of DRG neurons collected 2 wk after injection of DiI into the mouse gastrocnemius muscle. B) Representative currents evoked by application of pH 5 solution to muscle afferents from mice of the indicated genotype. Currents are normalized to demonstrate differences in kinetics (current amplitudes: wild-type, 1.04 nA; ASIC1a^−/−, 2.85 nA; ASIC2^−/−, 3.55 nA; and ASIC3^−/−, 5.46 nA). C) Percentages of muscle afferents from each genotype that responded to pH 5 with an evoked current > 60 pA. Three wild-type mice (n=11–12 neurons/mouse), 6 ASIC1a^−/− mice (n=5–15 neurons/mouse), 3 ASIC2^−/− mice (n=8–9 neurons/mouse), and 7 ASIC3^−/− mice (n=5–16 neurons/mouse) were studied. *P < 0.02 vs. wild-type. D) Mean peak pH 5-evoked current amplitude of the responding neurons. *P < 0.01 vs. wild-type.

Figure 2.

Properties of acid-evoked currents in skeletal muscle afferents from wild-type and ASIC1a^−/− mice. A) Representative currents evoked by the indicated pH solutions from a control solution of pH 7.4 in muscle afferents from wild-type and ASIC1a^−/− mice. The pH 4-evoked currents were from separate cells. B) pH dose-response data for pH-evoked currents in wild-type and ASIC1a^−/− muscle afferents. Data were normalized to the peak currents evoked by pH 5. Lines are fits of the Hill equation of the means (n=5–13). P < 0.01 for pH₅₀ values calculated from the fits of the Hill equation. Right graph, normalized individual data points and means at pH 6.8. *P < 0.01 vs. wild-type. C) Mean τ of desensitization as measured from single exponential fits to the falling phase of the transient currents (dashed line over pH 5-evoked current from wild-type neuron in panel A) evoked by the indicated pH solutions in wild-type and ASIC1a^−/− muscle afferents (n=6–24). Rates were not calculated for pH 4-evoked currents from ASIC1a^−/− muscle afferents because these currents were mostly sustained. *P < 0.03 vs. wild-type pH 5-evoked currents. D) Overlay of current traces showing recovery from desensitization of a wild-type and an ASIC1a^−/− muscle afferent. Current was desensitized with a 7-s application of pH 6 (only the first 1.25 s is shown). Cells were then exposed to pH 7.4 solution for the indicated times (see x axis in panel E) before they were stimulated again with pH 6. Recovery is the percentage of current evoked by the second pH 6 application compared with the first. E) Mean recovery data as collected in panel D for wild-type and ASIC1a^−/− muscle afferents (n≥5). Lines are fits of single exponentials of the means. P < 0.01 for τ was calculated from the fits of individual cells. Right graph, individual data points and means for recovery at 0.6 s. *P < 0.01 vs. wild-type.

Figure 3.

Properties of acid-evoked currents in skeletal muscle afferents from ASIC2^−/− mice. A) Representative currents evoked by the indicated pH solutions in muscle afferents from ASIC2^−/− mice. B) pH dose-response data of currents evoked from ASIC2^−/− muscle afferents normalized to the currents evoked by pH 5 (n≥12). Line is fit of the Hill equation. Dashed line is fit of data from wild-type afferents in Fig. 2B. C) Mean τ of desensitization of the transient currents evoked by the indicated pH solutions from ASIC2^−/− muscle afferents compared with wild-type data from Fig. 2C (n≥11). D) Recovery from desensitization data for ASIC2^−/− muscle afferents (n≥5). Line is fit of single exponential of the means. Dashed line is fit of data from wild-type afferents in Fig. 2E. E) Representative pH 6.5-evoked currents in the presence and absence of 300 μM zinc in wild-type and ASIC2^−/− muscle afferents. Zinc was present only in the pH 6.5 solution and not in the bathing solution. Zinc potentiated currents in wild-type (n=14; P<0.01 vs. control using paired Student's t test) but not in ASIC2^−/− muscle afferents (n=8; P=0.16 vs. control). F) Relative change in pH 6.5-evoked currents in presence of zinc. Data are individual data points as well as means ± se. *P < 0.02 vs. wild-type.

Figure 4.

Properties of acid-evoked currents in skeletal muscle afferents from ASIC1a/3 double-null mice. A) Superimposed currents evoked by the indicated pH solutions in a muscle afferent from ASIC1a/3^−/− mice. B) Overlay of pH 4-evoked currents in the presence and absence of 300 μM amiloride. Amiloride was only present in the pH 4 test solution. All of the transient components of the currents were blocked, whereas the sustained components were not inhibited. C) pH dose-response data from ASIC1a/3^−/− muscle afferents normalized to the currents evoked by pH 3.5 (n≥4). Line is fit of the Hill equation. Dashed line is fit of data from wild-type afferents in Fig. 2B.

Figure 5.

Properties of acid-evoked currents in skeletal muscle afferents from ASIC3^−/− mice. A) Representative currents evoked by the indicated pH solutions in muscle afferents from ASIC3^−/− mice compared with those from wild-type mice. B) pH dose-response data from ASIC3^−/− muscle afferents normalized to the currents evoked by pH 5 (n≥12). Line is fit of the Hill equation. Dashed line is fit of data from wild-type afferents in Fig. 2B. C) Recovery from desensitization data for ASIC3^−/− muscle afferents (n≥5). Line is fit of single exponential of the means. Dashed line is fit of data from wild-type afferents in Fig. 2E. D) Mean ± se τ of desensitization as well as individual data points of the transient currents evoked by the indicated pH solutions from ASIC3^−/− muscle afferents compared with wild-type data from Fig. 2C (n≥11). P < 0.01 for data at each pH compared with wild-type.

Figure 6.

ASIC-like currents are absent in skeletal muscle afferents from ASIC1a/2/3 TKO mice. A) Representative currents evoked by pH 5 solution from wild-type and TKO muscle afferents. Transient ASIC-like currents were absent in all 22 TKO muscle afferents studied. B) Mean pH 5-evoked sustained current amplitudes from wild-type and the indicated ASIC^−/− muscle afferents (n≥8). *P = 0.03 vs. wild-type.