Endogenous Arginine-Phenylalanine-Amide-related Peptides Alter Steady-state Desensitization of ASIC1a

Thomas W Sherwood,Candice C Askwith

doi:10.1074/jbc.m705118200

Abstract

The acid-sensing ion channels (ASICs) are proton-gated, voltage-insensitive cation channels expressed throughout the nervous system. ASIC1a plays a role in learning, pain, and fear-related behaviors. In addition, activation of ASIC1a during prolonged acidosis following cerebral ischemia induces neuronal death. ASICs undergo steady-state desensitization, a characteristic that limits ASIC1a activity and may play a prominent role in the prevention of ASIC1a-evoked neuronal death. In this study, we found exogenous and endogenous arginine-phenylalanine-amide (RF-amide)-related peptides decreased the pH sensitivity of ASIC1a steady-state desensitization. During conditions that normally induced steady-state desensitization, these peptides profoundly enhanced ASIC1a activity. We also determined that human ASIC1a required more acidic pH to undergo steady-state desensitization compared with mouse ASIC1a. Surprisingly, steady-state desensitization of human ASIC1a was also affected by a greater number of peptides compared with mouse ASIC1a. Mutation of five amino acids in a region of the extracellular domain changed the characteristics of human ASIC1a to those of mouse ASIC1a, suggesting that this region plays a pivotal role in neuropeptide and pH sensitivity of steady-state desensitization. Overall, these experiments lend vital insight into steady-state desensitization of ASIC1a and expand our understanding of the structural determinants of RF-amide-related peptide modulation. Furthermore, our finding that endogenous peptides shift steady-state desensitization suggests that RF-amides could impact the role of ASIC1a in both pain and neuronal damage following stroke and ischemia.

Highlights

Of the Asic1 gene eliminates the vast majority of proton-gated current in central neurons [4, 11, 15]
PcTx1 limits neuronal damage following cerebral ischemia in mice and inhibits inflammatory pain and hyperalgesia [4, 6, 23]. These results indicate that increasing the pH sensitivity of steady-state desensitization of ASIC1a can reduce pain perception, neuronal damage, and death following cerebral ischemia and suggest that modulation of steady-state desensitization can affect ASIC1a-dependent neuronal activity
We show that the biophysical properties of mouse and human ASIC1a are not identical

Summary

EXPERIMENTAL PROCEDURES

Recombinant DNA Construction and Expression in Xenopus Oocytes—Human and mouse nucleotide sequence corresponding to GenBankTM accession numbers NM_001095 and NM_009597, respectively, were cloned into the pMT3 mammalian expression plasmid as described previously [24]. Peptide, nor venom at the indicated concentrations induced current in oocytes not injected with ASIC1a. Oocytes were washed with 5 ml of pH 7.4 solution and allowed to recover for 2 min between pH applications This amount of time, determined experimentally, allowed full recovery from desensitization of both mouse and human ASIC1a. Application of submaximal acidic pH solutions to oocytes expressing either mouse or human ASIC1a revealed that the pH sensitivity of activation was subtly different between species (Fig. 1A). Determined by application of pH 5.0 following pH 7.4 (pH dose The fact that the pH dose-response curve was different response of activation) or conditioning pH 7.9 (steady-state between mouse and human ASIC1a suggested that pH sensitivdesensitization). I/Imax ϭ 1/{1 ϩ (EC50/[peptide])n}, where n is the Hill coefficient; EC50 is the peptide concentration inducing half of the saturating peptide effect (Imax)

RESULTS

Respond Differently to Endogenous

Findings

DISCUSSION