Alternative Splicing and Interaction with Di- and Polyvalent Cations Control the Dynamic Range of Acid-sensing Ion Channel 1 (ASIC1)

Abstract

belong to the Deg/ENaC super family of ion channels (1). Members of this super gene family form Na ϩ -selective ion channels (P Na /P K , 8 -100) that can be blocked by amiloride (IC 50 , 0.2-20 M). All family members show some common hallmarks including two hydrophobic domains, short intracellular N and C termini, and a large extracellular loop containing conserved cysteines. Channels of this gene family probably form tetramers (2, 3).To date, six different members of the ASIC subfamily have been cloned (ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4), which are encoded by four genes. ASIC1a and ASIC1b are alternative splice products of the ASIC1 gene (4, 5). Splicing exchanges approximately the first third of the protein, including the first transmembrane domain and the proximal part of the large ectodomain. In contrast, the C-terminal twothirds are identical. We will use the term ASIC1 when we do not refer to a specific splice variant. All ASICs with the exception of ASIC4 (6) are expressed in sensory neurons of the dorsal root ganglion. Proposed functions in sensory neurons include peripheral pain perception (1,7,8) and mechanotransduction (9, 10). Although some evidence suggests that some of the native H ϩ -gated currents in sensory neurons are mediated by heteromeric ASICs (11, 12), part of these currents are probably mediated by homomeric ASIC1 and ASIC3 (8, 11).ASIC1a is expressed in sensory neurons and throughout the brain (13), whereas ASIC1b is specifically expressed in sensory neurons (5). Both subunits are Na ϩ -selective (P Na /P K Ϸ 10 -15), and only ASIC1a has a low Ca 2ϩ permeability (P Na /P Ca Ϸ 15) (4). ASIC1a and ASIC1b form rapidly activating and completely desensitizing ion channels ( act , ϳ10 msec; desens , ϳ1 s) (4). The expression of ASIC1 in small diameter, capsaicinsensitive sensory neurons (5, 11, 14, 15) has led to the proposal that ASIC1a mediates excitation during tissue acidosis, which accompanies inflammation and ischemia. However, complete desensitization of ASIC1 makes it difficult to imagine how this channel can sense H ϩ signals during inflammation and ischemia when the pH persistently falls.Here we show that both ASIC1a and ASIC1b undergo steady-state inactivation. The steady-state inactivation curve for ASIC1b is shifted by 0.25 pH units to more acidic values as compared with ASIC1a, showing that ASIC1b can operate at a more acidic resting pH. pH activation is shifted by 0.7 pH units. Differences in the sensitivity of activation and inactivation by protons are intimately linked, as both are controlled by only two amino acids in the ectodomain. These two amino acids are exchanged by alternative splicing. Moreover, we show that Ca 2ϩ , Mg 2ϩ , and spermine, when applied during the steadystate, shift the steady-state inactivation curves of both ASIC1a and ASIC1b. This leads to a potentiation of the current. Modulation by di-and polyvalent cations may be a means to adapt ASIC1 activity to changes in the extracellular concentration of these ions. Our results show that ASIC1b...

Protons are a neurotransmitter that regulates synaptic plasticity in the lateral amygdala

“…Increased interstitial pH after neurostimulation has also been reported in other preparations (11,42,47). Previous studies showed that raising extracellular pH before an acid stimulus reduced steady-state inactivation and increased the amplitude of ASIC currents (43,44,48). Thus, alkalinization might prepare postsynaptic ASICs for subsequent stimuli, and thereby maximize their current.…”

“…The alkalinization that followed the transient acidification has been reported in other preparations (11). Because ASICs inactivate in the continued presence of protons (39,(42)(43)(44), alkalinization between synaptic release stimulations might function to resensitize the receptor.…”

Deactivation kinetics of acid-sensing ion channel 1a are strongly pH-sensitive

“…However, probing deactivation kinetics at more acidic pHs is hampered by steady-state inactivation (20,38). For example, in a standard theta-tube application experiment, a resting pH of 7.2 produces extensive steady-state inactivation, making responses in outside-out patches small and difficult to evaluate.…”

“…The deactivation kinetics of ASIC1a to brief applications of acidic solution were recently examined by jumping ASIC1a-containing outside-out patches from a baseline pH of 8.0, followed by a jump to pH 5.0 for 1 ms and then a return to pH 8.0 (20). Under such conditions, ASIC1a deactivation kinetics were surprisingly brief, given their reported single-channel open times (37) and high apparent affinity for protons [pH evoking half the maximal response (pH 50 ) of 6.4-6.6, EC 50 of 400-250 nM (38)(39)(40)]. However, as mentioned above, during intense neuronal activity or stimulation, the interevent synaptic cleft pH does not toggle between neutral and acidic pH in a binary fashion.…”

Extracellular acidosis increases neuronal cell calcium by activating acid-sensing ion channel 1a

“…In some cases, the molecular identity of the channels was uncertain. Moreover, in some cases, permeability was measured by varying Ca 2ϩ and Na ϩ concentrations in the presence of other monovalent and divalent cations; this result could limit the accuracy of the determinations, especially for a channel that may not only conduct Ca 2ϩ but also be regulated by it (38,39). Our studies of recombinant channels indicate that ASIC1a can carry Ca 2ϩ current; an advantage of these studies is that Ca 2ϩ was the only abundant extracellular cation.…”