Native and recombinant ASIC1a receptors conduct negligible Ca2+ entry

Abstract

Acid Sensing Ion Channels (ASICs) are a family of proton-gated cation channels that play a role in the sensation of noxious stimuli. Of these, ASIC1a is the only family member that is reported to be permeable to Ca 2+ , although the absolute magnitude of the Ca 2+ current is unclear. Here, we used patch-clamp photometry to determine the contribution of Ca 2+ to total current through native and recombinant ASIC1a receptors. We found that acidification of the extracellular medium evoked amiloride and psalmotoxin 1-sensitive currents in isolated chick dorsal root ganglion neurons and human embryonic kidney cells, but did not alter fura-2 fluorescence when the bath concentration of Ca 2+ was close to that found in normal physiological conditions. Further, activation of recombinant ASIC1a receptors also failed to produce measurable changes in fluorescence despite of the fact that the total cation current through the over-expressed receptor was ten-fold larger than that of the native channels. Finally, we imaged a field of intact DRG neurons loaded with the Ca 2+ -sensing dye Fluo-4, and found that acidification increased [Ca 2+ ]i in a small population of cells. Thus, although our whole-field imaging data agree with previous studies that activation of ASIC1a receptors can potentially cause elevations in intracellular free Ca 2+ , our single cell data strongly challenges the view that Ca 2+ entry through the ASIC1a receptor itself contributes to this response.

ENaCs and ASICs as therapeutic targets

“…In vivo animal data regarding ASIC1 suggest that inhibition of this channel will be effective up to 5 h after the stroke rather than the narrow one hour window of N-methyl-D-aspartate antagonists (353). However, there is some slight controversy to this calcium-based hypothesis, as Samways et al (336) were unable to see a significant increase in intracellular calcium in physiological conditions in a large number of cells natively or exogenously expressing ASIC1. The authors observed increases in intracellular calcium in a small population of chicken dorsal root ganglia neurons, but the majority of the chicken DRG cells and the entirety of the HEK293 and COS7 cells did not show increases in intracellular [Ca 2ϩ ] when expressing chicken ASIC1 or human ASIC1b (336).…”

“…However, there is some slight controversy to this calcium-based hypothesis, as Samways et al (336) were unable to see a significant increase in intracellular calcium in physiological conditions in a large number of cells natively or exogenously expressing ASIC1. The authors observed increases in intracellular calcium in a small population of chicken dorsal root ganglia neurons, but the majority of the chicken DRG cells and the entirety of the HEK293 and COS7 cells did not show increases in intracellular [Ca 2ϩ ] when expressing chicken ASIC1 or human ASIC1b (336). The difficulty in showing significant calcium permeability of ASIC1 channels, combined with the transient and desensitizing nature of ASIC1 currents, suggests that there may be more than a calcium conductance playing a role in the neuronal death during stroke.…”

International Union of Basic and Clinical Pharmacology. XCI. Structure, Function, and Pharmacology of Acid-Sensing Ion Channels and the Epithelial Na+Channel

“…Recently it was shown, however, that the synthetic compound GMQ activates ASIC3 at pH 7.4 (Yu et al, 2010). The spider toxin PcTx1 shifts ASIC pH dependencies, inhibiting ASIC1a under physiologic conditions and activating cASIC1 and rat ASIC1b at pH 7.4 and slightly acidic pH, respectively (Chen et al, 2005(Chen et al, , 2006Samways et al, 2009;Baconguis and Gouaux, 2012). The recently identified MitTx activates ASICs by a currently unknown mechanism (Bohlen et al, 2011).…”

“…MitTx is an ASIC activator and PcTx1 had been known to be a gating modifier that shifts the pH dependence of activation and desensitization to more alkaline values, thereby inhibiting human and rat ASIC1a. However, PcTx1 opens cASIC1 at pH 7.4 and rat ASIC1b at slightly acidic pH (see section VI) (Chen et al, 2005(Chen et al, , 2006Samways et al, 2009). Both functional cASIC1-toxin complexes showed an incomplete desensitization at the pH used for crystallization.…”

Binding Site and Inhibitory Mechanism of the Mambalgin-2 Pain-relieving Peptide on Acid-sensing Ion Channel 1a

“…The spider peptide psalmotoxin 1 (PcTxl), which blocks rodent ASIC1a homomeric (7) and ASIC1a/2b heteromeric (8) channels but can also act as an agonist of ASIC1b and chicken ASIC1a (9,10), has been used to explore the role of ASIC1a in normal and pathophysiological conditions in brain (5) and to demonstrate a role for the central ASIC1a in pain modulation (11,12). The MitTx identified from the venom of the Texas coral snake does not inhibit but potently activates several homomeric and heteromeric ASIC channels and helped to identify a role for peripheral ASIC1a-containing channels in cutaneous pain (13) and to define the structure of the open state of ASIC1a (14).…”