Abstract
Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.
Highlights
Acid-sensing ion channels (ASICs)1 are non-voltage-gated Naϩ channels that are transiently activated by a rapid drop in extracellular pH [1,2,3]
It is known that epithelial Naϩ channel (ENaC), which belongs to the same family as ASICs, is the target of serine proteases, which either inactivate [16, 17] or activate [18, 19] this channel
Trypsin exposure of ASIC1a-injected oocytes led to the appearance of an additional, lower molecular band (ϳ55 kDa), which corresponds to the cleaved channel protein
Summary
Recombinant Expression of ASICs—For the expression of heteromeric ASICs we performed transient transfections in COS cells by using the PerFectin reagent (Gene Therapy Systems, San Diego, CA), as described previously [22]. Cell lines that stably expressed ASICs were established for the homomultimeric ASIC1a, 1b, 2a, and 3. The original cDNA constructs were kindly provided by D. The cDNAs were subcloned into the pEAK8 expression vector (Edge Biosystems, Gaithersburg, MD). A unique EcoRV restriction site within the N-terminal part of the coding region of ASIC1b was used for the subcloning, to obtain a construct that corresponds to ASIC1b-M3, as described by Bassler et al [23]. Stable cell lines were established in CHO cells as described previously [22]
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