Transgenic expression of a dominant-negative ASIC3 subunit leads to increased sensitivity to mechanical and inflammatory stimuli

Abstract

Molecular and behavioral evidence suggests that acid-sensing ion channels (ASICs) contribute to pain processing, but an understanding of their precise role remains elusive. Existing ASIC knock-out mouse experiments are complicated by the heteromultimerization of ASIC subunits. Therefore, we have generated transgenic mice that express a dominant-negative form of the ASIC3 subunit that inactivates all native neuronal ASIC-like currents by oligomerization. Using whole-cell patch-clamp recordings, we examined the response properties of acutely isolated dorsal root ganglion neurons to protons (pH 5.0). We found that whereas 33% of the proton-responsive neurons from wild-type mice exhibited an ASIC-like transient response, none of the neurons from the transgenic mice exhibited a transient inward current. Capsaicin-evoked responses mediated by the TRPV1 receptor were unaltered in transgenic mice. Adult male wild-type and transgenic mice were subjected to a battery of behavioral nociceptive assays, including tests of thermal, mechanical, chemical/inflammatory, and muscle pain. The two genotypes were equally sensitive to thermal pain and to thermal hypersensitivity after inflammation. Compared with wild types, however, transgenic mice were more sensitive to a number of modalities, including mechanical pain (von Frey test, tail-clip test), chemical/inflammatory pain (formalin test, 0.6% acetic acid writhing test), mechanical hypersensitivity after zymosan inflammation, and mechanical hypersensitivity after intramuscular injection of hypotonic saline. These data reinforce the hypothesis that ASICs are involved in both mechanical and inflammatory pain, although the increased sensitivity of transgenic mice renders it unlikely that they are direct transducers of nociceptive stimuli.

Figure 1.

Dominant-negative properties of the mutant ASIC3 G439R subunit in transiently transfected HEK293 cells. Representative inward currents mediated by wild-type homomeric ASIC3 receptor activated by pH 5 either expressed alone (A) or in the presence of the ASIC3 G439R subunit at a 1:2 ratio (B) are shown. C, Normalized inhibition of proton-gated currents mediated by homomeric ASIC1a, ASIC2a, and ASIC3 coexpressed with the ASIC3 G439R subunit at a 1:2 ratio. The numbers in parentheses indicate the number of recorded cells. The error bars indicate the mean ± SEM peak magnitude of proton-evoked currents. WT, Wild type.

Figure 2.

Transgenic expression of the ASIC3 G439R subunit in adult FVB mice. A, Schematic organization of the transgene. The relative position of the mutation is indicated by the asterisk. B, Northern blot showing NFL promoter-driven expression of ASIC3 G439R mRNA in the brain of homozygous transgenic animals. B, Whole brain; K, kidney. C, Detection of mutant ASIC3 mRNA (M) in the brain and DRG isolated from homozygous transgenic animals using RT-PCR. Wild-type FVB mice were used as negative controls. Tg, Transgenic; NFL, neurofilament light chain.

Figure 3.

Responses of DRG neurons from wild-type and transgenic mice to protons. A, Examples of inward current evoked by pH 5.0 in two different neurons from wild-type mice and one from a transgenic mouse. B, Percentage of neurons responding to pH 5.0. The numbers inside the bars indicate the n tested. C, Percentage of proton-evoked responses that were transient, followed by a sustained or sustained-only current. D, The bars represent the mean ± SEM average peak magnitude of proton-evoked total current (open bars), sustained-only current (gray bars), transient-only current (black bar), and sustained after transient current (hatched bar) in DRG neurons from wild-type and transgenic mice. Tg, Transgenic.

Figure 4.

Responses of DRG neurons from wild-type and transgenic mice to 1 μm capsaicin. A, Examples of inward current evoked by 1 μm capsaicin in neurons from a wild-type and transgenic mouse. B, Percentage of neurons responding to 1 μm capsaicin. The numbers inside the bars indicate the n tested. C, The bars represent the mean ± SEM peak magnitude of capsaicin-evoked inward current in DRG neurons from wild-type and transgenic mice. The numbers inside the bars indicate the n that responded. Tg, Transgenic.

Figure 5.

Sensitivity of wild-type (+/+) and transgenic (Tg/Tg) mice on a variety of acute and tonic nociceptive assays. See Materials and Methods for experimental details. The bars represent the following: A-C, mean ± SEM latency to withdraw from the thermal stimulus (in seconds); D, mean ± SEM 50% withdrawal threshold (in grams); E, mean ± SEM latency to attack/bite clip (in seconds); F, mean ± SEM total number of abdominal constrictions; G, mean ± SEM percentages of 5 s samples showing licking/biting behavior. Mutant mice are equisensitive to wild-type mice in assays of thermal nociception (A-C) but significantly more sensitive to mechanical stimuli (D, E) and chemical/inflammatory stimuli (F, G). ^*p < 0.05 compared with +/+.

Figure 6.

Increased mechanical, but not thermal, hypersensitivity in transgenic (Tg/Tg) mice compared with wild-type mice (+/+) after injection of 1 mg/ml zymosan into the right hindpaw. See Materials and Methods for experimental details. A, Symbols represent the mean ± SEM latency to withdraw from a thermal stimulus (paw-withdrawal test). B, Symbols represent the mean ± SEM total number of positive withdrawal responses to three von Frey fibers (#5, #7, and #9; each applied 6 times). ^*p < 0.05 compared with +/+ on the same assay.

Figure 7.

Mechanical hypersensitivity after in tramuscular hypotonic saline injections [at day 0 (Inj. #1) and day 4 (Inj. #2)] in transgenic mice (Tg/Tg) but not wild-type mice (+/+). See Materials and Methods for experimental details. The symbols represent the mean ± SEM number of positive withdrawal responses to a #5 von Frey fiber of the ipsilateral hindpaw. Responses of the contralateral hindpaw (data not shown) were almost identical. In a separate experiment using C57BL/6 mice, we confirmed the hypersensitivity of this strain as reported originally. ^*p < 0.05 compared with baseline (Tg/Tg mice).