Acid-sensing ion channel (ASIC) 1a/2a heteromers have a flexible 2:1/1:2 stoichiometry

Abstract

Acid-sensing ion channels (ASICs) are widely expressed proton-gated Na(+) channels playing a role in tissue acidosis and pain. A trimeric composition of ASICs has been suggested by crystallization. Upon coexpression of ASIC1a and ASIC2a in Xenopus oocytes, we observed the formation of heteromers and their coexistence with homomers by electrophysiology, but could not determine whether heteromeric complexes have a fixed subunit stoichiometry or whether certain stoichiometries are preferred over others. We therefore imaged ASICs labeled with green and red fluorescent proteins on a single-molecule level, counted bleaching steps from GFP and colocalized them with red tandem tetrameric mCherry for many individual complexes. Combinatorial analysis suggests a model of random mixing of ASIC1a and ASIC2a subunits to yield both 2:1 and 1:2 ASIC1a:ASIC2a heteromers together with ASIC1a and ASIC2a homomers.

Keywords: BLINaC; ENaC; single-molecule imaging; subunit counting.

Fig. 1.

Electrophysiological characterization of heteromeric ASICs. (A) H⁺ concentration–response curves of homomeric ASIC1a, ASIC2a, GFP-ASIC1a, and GFP-ASIC2a, respectively, expressed in Xenopus oocytes, illustrating identical apparent H⁺ affinity of GFP-tagged and untagged ASICs. Lines represent fits to a single Hill function. (B and C) H⁺ concentration–response curves for oocytes expressing ASIC1a and ASIC2a at different ratios. In B, lines represent fits to a single Hill function. In C, dashed lines represent fits to the sum of three Hill functions: two with the properties of ASIC1a and ASIC2a homomers and one for the ASIC1a/ASIC2a heteromer. Solid lines represent fits of the sum of four Hill functions: two for the ASIC1a and ASIC2a homomers and two for different heteromer species. Data points in B and C were scaled with different values for I₀ from the Hill curve fits. Error bars in A–C represent SEM. (D) Fractions of homo- and heteromers from the best fits of three (Left) or four (Right) Hill curves. Absolute current amplitudes are given in Table S1.

Fig. 2.

Single-molecule subunit counting of homomeric GFP-ASIC1a and GFP-ASIC2a. (A) Image from a TIRF movie with GFP-ASIC1a expressed at the cell surface of a X. laevis oocyte. Green circles indicate immobile spots suitable for counting of bleaching steps. (Scale bar: 5 μm.) (B) Fluorescence intensity from two GFP-ASIC1a spots exemplary of a three-step photobleaching process. (C and D) The distribution of observed bleaching steps (green bars) for (C) GFP-ASIC1a and (D) GFP-ASIC2a and fits of binomial distributions (gray bars).

Fig. 3.

Principle of assembly of red- and green-tagged subunits into homo- and heteromers. (A) Recorded fluorescence from single channels is green, red, or a colocalization of green and red, depending on the channel composition and the functionality of the tags. When none of the tags is functional, channels stay dark (Left). For certain channels, the fluorescence does not reflect the composition (gray boxes). Of channels with functional GFP tags, the bleaching steps can be counted. (B) From the observed distribution of green-only, red-only, and colocalized spots and the bleaching steps, the underlying fractions of homo- and heteromeric channels can be reconstructed.

Fig. 4.

Assembly of differentially tagged ASIC1a subunits. (A) Microscope image of an oocyte patch with GFP-ASIC1a and ttCherry-ASIC1a. Arrowheads mark colocalized GFP and ttCherry spots. (Scale bars: 5 μm.) (B) Fluorescence intensity profile over time from a representative colocalized spot. ttCherry was excited first (red bar) followed by GFP (green bar). In-between, lasers are off and the intensity drops to zero. (C) Histograms for observed spots with green-only, red-only, and colocalized green and red fluorescence, and for GFP bleaching steps of green-only spots and of spots where green and red colocalized. All four fractions of homo- and heteromers were used as free parameters. Filled bars indicate the observed distribution and white bars are the results of the least-squares fit. (D) Fitted fractions of homo- and heteromeric channels of stoichiometry are as indicated. (E) Experimental histograms were fitted with homo- and heteromer ratios as given by a binomial distribution, with the ratio of ASIC2a to ASIC1a as a free parameter. (F) Reconstructed fractions of homo- and heteromers.

Fig. 5.

Assembly of ASIC1a/2a heteromers. (A) Histograms for counted spots of GFP-ASIC1a/ttCherry-ASIC2a with the properties as indicated and the fit values. (B) Fit for fractions of homo- and heteromers with independent fractions and fractions related by a binomial distribution, implying random assembly. (C) Histograms for counted spots of GFP-ASIC2a/ttCherry-ASIC1a with the properties as indicated and the fit values. (D) Fit for fractions of homo- and heteromers with independent fractions and fractions related by a binomial distribution.