Abstract
Acid-sensing ion channels (ASICs) form both homotrimeric and heterotrimeric ion channels that are activated by extracellular protons and are involved in a wide range of physiological and pathophysiological processes, including pain and anxiety. ASIC proteins can form both homotrimeric and heterotrimeric ion channels. The ASIC3 subunit has been shown to be of particular importance in the peripheral nervous system with pharmacological and genetic manipulations demonstrating a role in pain. Naked mole-rats, despite having functional ASICs, are insensitive to acid as a noxious stimulus and show diminished avoidance of acidic fumes, ammonia, and carbon dioxide. Here we cloned naked mole-rat ASIC3 (nmrASIC3) and used a cell-surface biotinylation assay to demonstrate that it traffics to the plasma membrane, but using whole-cell patch clamp electrophysiology we observed that nmrASIC3 is insensitive to both protons and the non-proton ASIC3 agonist 2-guanidine-4-methylquinazoline. However, in line with previous reports of ASIC3 mRNA expression in dorsal root ganglia neurons, we found that the ASIC3 antagonist APETx2 reversibly inhibits ASIC-like currents in naked mole-rat dorsal root ganglia neurons. We further show that like the proton-insensitive ASIC2b and ASIC4, nmrASIC3 forms functional, proton-sensitive heteromers with other ASIC subunits. An amino acid alignment of ASIC3s between 9 relevant rodent species and human identified unique sequence differences that might underlie the proton insensitivity of nmrASIC3. However, introducing nmrASIC3 differences into rat ASIC3 (rASIC3) produced only minor differences in channel function, and replacing the nmrASIC3 sequence with that of rASIC3 did not produce a proton-sensitive ion channel. Our observation that nmrASIC3 forms nonfunctional homomers may reflect a further adaptation of the naked mole-rat to living in an environment with high-carbon dioxide levels.
Highlights
To determine whether nmrASIC3 can form proton-sensitive heteromers with other ASIC subunits as DRG neuron data would suggest, we cotransfected either nmrASIC3 or mASIC3 with nmrASIC1b and compared the properties of currents recorded from these cotransfected cells with those only transfected with either mASIC3 or nmrASIC1b
Sensitivity to acid as a noxious stimulus is largely conserved throughout the animal kingdom [44], but the naked mole-rat is behavioral insensitive to acid [35] due to a variation in NaV1.7, which results in acid anesthetizing, rather than activating, naked mole-rat sensory neurons [36]
We undertook to investigate the properties of nmrASIC3 because evidence supports a role for ASIC3 in a wide variety of situations, including: pain [9, 23,24,25,26], as well itch [29], mechanosensation [23, 30], and anxiety [31]
Summary
Primers for cloning mouse ASIC3 (mASIC3) and naked mole-rat ASIC3 (nmrASIC3) were designed based upon the published genome sequences and constructs were made using pIRES2-EGFP or pTarget vectors; rat ASIC3 (rASIC3 in pTracer) was a kind gift from G. To determine whether nmrASIC3 can form proton-sensitive heteromers with other ASIC subunits as DRG neuron data would suggest, we cotransfected either nmrASIC3 or mASIC3 with nmrASIC1b and compared the properties of currents recorded from these cotransfected cells with those only transfected with either mASIC3 or nmrASIC1b. Based upon biophysical characterization, nmrASIC3 forms functional heteromers with nmrASIC1b (Fig. 3), but the evidence is less clear for heteromeric formation with rASIC2a (Fig. 4) these two subunits are both present at the plasma membrane when cotransfected (Fig. 1G) and the fact that ASIC-like currents are sensitive to inhibition by APETx2 in DRG neurons from naked mole-rats (Fig. 2) strongly supports the premise that nmrASIC3 produces proton-insensitive homomers it can form functional heteromers in vivo. It would appear that neither Ala nor Arg102 are of crucial importance in proton activation of ASIC3 and when expressed in CHO cells neither nmrASIC3-E62A nor nmrASIC3-H102R resulted in the rescuing of nmrASIC3 proton-sensitivity
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