Abstract
Acid-sensing ion channels (ASICs) are neuronal Na+-permeable ion channels that are activated by extracellular acidification and are involved in fear sensing, learning, neurodegeneration after ischemia, and in pain sensation. We have recently found that the human ASIC1a (hASIC1a) wild type (WT) clone which has been used by many laboratories in recombinant expression studies contains a point mutation that occurs with a very low frequency in humans. Here, we compared the function and expression of ASIC1a WT and of this rare variant, in which the highly conserved residue Gly212 is substituted by Asp. Residue 212 is located at a subunit interface that undergoes changes during channel activity. We show that the modulation of channel function by commonly used ASIC inhibitors and modulators, and the pH dependence, are the same or only slightly different between hASIC1a-G212 and -D212. hASIC1a-G212 has however a higher current amplitude per surface-expressed channel and considerably slower current decay kinetics than hASIC1a-D212, and its current decay kinetics display a higher dependency on the type of anion present in the extracellular solution. We demonstrate for a number of channel mutants previously characterized in the hASIC1a-D212 background that they have very similar effects in the hASIC1a-G212 background. Taken together, we show that the variant hASIC1a-D212 that has been used as WT in many studies is, in fact, a mutant and that the properties of hASIC1a-D212 and hASIC1a-G212 are sufficiently close that the conclusions made in previous pharmacology and structure-function studies remain valid.
Highlights
A rare variant of the human acid-sensing ion channel 1a has been used as ‘‘wild type’’ (WT) in many functional studies involving recombinant expression of Acid-Sensing Ion Channel (ASIC), including those carried out by our laboratory
HASIC1a-G212 and -D212 were expressed in chinese hamster ovary (CHO) cells, and their function was assessed by whole-cell patch-clamp
We found here the following differences in biophysical properties between human acid-sensing ion channel 1a (hASIC1a)-D212 and the WT hASIC1a-G212: (1) the midpoint of steady-state desensitization (SSD) is shifted by 0.08 pH units to more acidic values in hASIC1a-G212; (2) with the same amount of transfected DNA, hASIC1a-G212 shows ∼20-fold higher current amplitudes, and a ∼2-fold higher cell-surface expression than -D212; (3) the current decay kinetics are slower in hASIC1a-G212 (∼5-fold at pH close to the pH of half-maximal activation (pH50), and ∼2-fold at more acidic pH in whole-cell experiments)
Summary
A rare variant of the human acid-sensing ion channel 1a (hASIC1a) has been used as ‘‘wild type’’ (WT) in many functional studies involving recombinant expression of ASICs, including those carried out by our laboratory. Crystal structures of chicken ASIC1a (cASIC1a), whose sequence shares ∼90% homology with hASIC1a, were obtained in conformations corresponding to the desensitized, the open and the closed state (Jasti et al, 2007; Gonzales et al, 2009; Baconguis and Gouaux, 2012; Dawson et al, 2012; Baconguis et al, 2014; Yoder et al, 2018) They show that the shape of a subunit is comparable to a hand, with the domains palm (yellow in Figure 1A), β-ball (orange), knuckle (cyan), finger (purple) and thumb (blue); the transmembrane domain would correspond to the forearm (Jasti et al, 2007). ASICs are the target of rather nonspecific small molecule inhibitors such as diminazene (Chen et al, 2010) and amiloride (Waldmann et al, 1997), of modulators such as 2-guanidine-4-methylquinazoline (GMQ; Yu et al, 2010) and of several high affinity toxins (Baron and Lingueglia, 2015; Rash, 2017)
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