Abstract
The exact subunit combinations of functional native acid-sensing ion channels (ASICs) have not been established yet, but both homomeric and heteromeric channels are likely to exist. To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a-, ASIC1b-, ASIC2a-, ASIC2b-, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current. Analyzing and comparing data for these three features demonstrated unique heteromeric channels in a number of co-expression experiments. Formation of heteromeric ASIC1a+2a and ASIC1b+2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has. Several lines of evidence supported formation of ASIC1a+3, ASIC1b+3, and ASIC2a+3 heteromeric channels. The most compelling was the desensitization characteristics, which, besides being proton-independent, were faster than those of any of the respective homomeric channels. ASIC2b, which homomerically expressed is not activated by protons per se, did not appear to form unique heteromeric combinations with other subunits and in fact appeared to suppress the function of ASIC1b. Co-expression of three subunits such as ASIC1a+2a+3 and ASIC1b+2a+3 resulted in data that could best be explained by coexistence of multiple channel populations within the same cell. This observation seems to be in good agreement with the fact that ASIC-expressing sensory neurons display a variety of acid-evoked currents.
Highlights
It is well established that tissue acidification, which may be present in inflammatory and ischemic conditions, causes pain [1,2,3]
To determine the ability of different subunits to assemble into heteromeric channels, a number of ASIC1a, ASIC1b, ASIC2a, ASIC2b, and ASIC3-containing homo- and heteromeric channels were studied by whole-cell patch clamp recordings with respect to pH sensitivity, desensitization kinetics, and level of sustained current normalized to peak current
Formation of heteromeric ASIC1a؉2a and ASIC1b؉2a channels was foremost supported by the desensitization characteristics that were independent of proton concentration, a feature none of the respective homomeric channels has
Summary
Cloning of ASICs—Poly(A)ϩ mRNA was purified from fetal rat dorsal root ganglion (DRG) neurons using the Oligotex® direct mRNA mini kit (Qiagen) according to the manufacturer’s protocol. CHO-K1 cells were co-transfected with the plasmids containing ASICs and a plasmid encoding enhanced green fluorescent protein using the LipofectAMINE PLUS kit (Invitrogen) according to the manufacturer’s protocol. Test solutions were made from standard Na-R, adjusting pH with HCl. Rapid pH changes were achieved by placing a piezo-driven doublebarreled application pipette (theta tube) in front of the cell. Rapid pH changes were achieved by placing a piezo-driven doublebarreled application pipette (theta tube) in front of the cell Using this system, complete solution exchange can be achieved in Ͻ1 ms, as measured by the liquid junction potential shift (data not shown). A p value of less than 0.05 was considered to be significant
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