Naked mole-rat acid-sensing ion channel 3 forms nonfunctional homomers, but functional heteromers

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.

Acid and inflammatory sensitisation of naked mole-rat colonic afferent nerves

“…In the peripheral nervous system, subcutaneous injection of acid (pH 3.5), capsaicin or histamine does not cause the nocifensive or pruriceptive behaviours in NMR that such stimuli characteristically induce in mice [11,12]. This acid insensitivity is a function of altered ASIC responses compared to mouse [19] and a variation in NMR NaV1.7, which renders the channel hypersensitive to proton-mediated block and therefore prevents acid-driven action potential initiation from the skin [18]. Such intrinsic differences in the sensitivity of NMR to acid may explain our observation of significantly lower firing rates in response to application of acid to NMR colonic afferents compared mouse.…”

“…Recent single-cell RNA-sequencing analysis of colonic sensory neurones shows discrete expression of such acid-sensitive ion channels with differing populations of mouse colonic afferents, suggesting functional specialism [16]. Compared to mice, NMRs display a similar expression profile of ASICs throughout the nervous system [17], and of those analysed, with the exception of ASIC3, NMR acid sensors show similar activation profiles to those of mice [13,18,19]. NMR TRPV1 is also expressed in sensory afferents and shows similar proton sensitivity to mouse TRPV1 [18].…”

Independent evolution of pain insensitivity in African mole-rats: origins and mechanisms

“…Acid, or more specifically protons, can activate sensory neurons by the modulation or activation of several different classes of ion channels including activation of TRPV1, acidsensing ion channels (ASICs) and proton-sensing G-proteincoupled receptors (GPCR) and inhibition of certain twopore K + channels (Holzer 2009;Pattison et al 2019). Direct electrophysiological studies have shown that naked mole-rat 1 3 DRG neurons display depolarizing currents to proton application, indeed both TRPV1-like and ASIC-like inward currents have been observed (Smith et al 2011;Schuhmacher et al 2018) and expression of different ASIC subunits appears to be roughly equivalent between mouse and naked mole-rat sensory neurons (Schuhmacher and Smith 2016). When examining the proton sensitivity of cloned acid-sensitive proteins, naked mole-rat TRPV1, ASIC1a and ASIC1b have a similar proton sensitivity to their mouse orthologues (Smith et al 2011); however, the ASIC3 subunit is proton insensitive and appears to shift the proton sensitivity when present in heterotrimers, which may alter sensory neuron acid sensitivity to some extent (Schuhmacher et al 2018).…”

“…Direct electrophysiological studies have shown that naked mole-rat 1 3 DRG neurons display depolarizing currents to proton application, indeed both TRPV1-like and ASIC-like inward currents have been observed (Smith et al 2011;Schuhmacher et al 2018) and expression of different ASIC subunits appears to be roughly equivalent between mouse and naked mole-rat sensory neurons (Schuhmacher and Smith 2016). When examining the proton sensitivity of cloned acid-sensitive proteins, naked mole-rat TRPV1, ASIC1a and ASIC1b have a similar proton sensitivity to their mouse orthologues (Smith et al 2011); however, the ASIC3 subunit is proton insensitive and appears to shift the proton sensitivity when present in heterotrimers, which may alter sensory neuron acid sensitivity to some extent (Schuhmacher et al 2018). It was thus puzzling that isolated naked mole-rat sensory neurons can be excited by protons, but why did we observe no acid driven action potentials in nociceptors and no pain behavior?…”

Naked mole-rat cortical neurons are resistant to acid-induced cell death

“…It is striking that NMRs are resistant to many pathological conditions known to involve ASICs. Recordings of ASIC-mediated currents in dorsal root ganglion (DRG) sensory neurons demonstrated an increased frequency and magnitude of ASIC responses in NMR neurons compared to mouse neurons [ 43 ], with APETx2, an inhibitor of ASIC3-containing ASICs, demonstrating a key role for ASIC3, even though nmrASIC3 does not appear to form functional homotrimers [ 50 ]. Previously we mapped out ASIC expression in different NMR brain regions and observed similar expression between mouse and NMR, a key exception being much lower ASIC4 levels throughout the NMR brain [ 51 ], however, no one has yet studied the function of ASICs in NMR brain neurons.…”