Fluorescence resonance energy transfer analysis of subunit assembly of the ASIC channel

Abstract

Acid-sensing ion channels (ASICs) are believed to be homo-or heteromeric complexes, which have been verified by classical methods such as co-immunoprecipitation or electrophysiological assays. However, the exact subunit combinations of ASICs in living cells have not been established yet. Here, we apply assays based on fluorescence resonance energy transfer (FRET) between GFP color mutants CFP and YFP to investigate ASIC assembly directly in living cells. Homomerization as well as heteromerization of different combinations of ASIC subunits were found. In addition, our results suggest the formation of heteromeric 1a/2a channels of stoichiometry consisting of at least two 1a subunits and two 2a subunits. Similar stoichiometry was observed from heteromeric 1a/2b and 2a/ 2b channels. Our results imply that these heteromeric ASIC channels contain at least four subunits. KeywordsFRET; ASIC; Assembly; Homomer; Heteromer; Stoichiometry; CHO Acid-sensing ion channels (ASICs), a class of ligand-gated cation channels activated by protons and highly expressed in both peripheral sensory neurons and central neurons, play important roles in mechanosensation, nociception, learning-memory, and brain ischemia [1,2]. ASICs belong to the ENaC/DEG superfamily, which also includes the amiloride-sensitive epithelial sodium channels (ENaCs), the mechanically gated degenerins of Caenorhabditis elegans (DEGs), and a neuropeptide-gated channel of Helix aspersa (FaNaC). The transmembrane topology of all channels in this superfamily features cytosolic amino and carboxy termini, two hydrophobic transmembrane segments and a large cysteine-rich extracellular domain [3]. So far, six ASIC subunits encoded by four genes have been cloned, ASIC1a (BNaC2α), ASIC1b(BNaC2β), ASIC2a (MDEG1), ASIC2b (MDEG2), ASIC3 (DRASIC) and ASIC4 (SPASIC). Homo-and heteromeric ASIC complexes have been verified by classical methods such as co-immunoprecipitation, or functional assays applying electrophysiology [4][5][6][7][8][9]. However, it is at present largely unknown how many subunits are Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. To directly determine the assembly of ASIC subunits of intact channels in living cells, we have used a biophysical approach based on fluorescence resonance energy transfer (FRET). FRET is a physical phenomenon in which energy absorbed by a fluorophore (CFP, the donor) is transferred to another fluorophore (YFP, the acceptor) through a nonradiative process. Because distances closer than about 10 nm between appropriate fluorophores are required to elicit FRET, it is a sensitive measure o...

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

“…Fluorescence resonance energy transfer analysis suggested that the ASIC1a/2a heteromer contains at least two ASIC1a and two ASIC2a subunits (17). In agreement, several studies reported that the related epithelial Na + channel (ENaC) is composed of four (18)(19)(20)(21) or nine subunits (22)(23)(24).…”

Regulation of Neuronal M-Channel Gating in an Isoform-Specific Manner: Functional Interplay between Calmodulin and Syntaxin 1A

“…in which D and A are molar extinction coefficients for the donor and acceptor, respectively, at the donor excitation wavelength (Takanishi et al, 2006;Gao et al, 2007). For the control groups, Kv2.1⌬C1a-Yϩ C-syx and Kv2.1⌬331-Yϩ C-syx, data presented were extracted from eCFP/eYFP molar ratio Ͼ3 at which saturation of FRET occurred.…”

Presynaptic Release Probability Is Increased in Hippocampal Neurons From ASIC1 Knockout Mice

“…ASIC1a, ASIC2a, and ASIC2b are expressed throughout the brain with particularly high abundance in the cerebral cortex, hippocampus, amygdala, olfactory bulb, and the cerebellum (GarciaAnoveros et al 1997;Lingueglia et al 1997;Price et al 1996;Waldmann et al 1996Waldmann et al , 1997. ASIC1a forms not only heteromeric channels with ASIC2a, but also Ca 2ϩ -permeable homomeric channels Bassilana et al 1997;Benson et al 2002;Gao et al 2007;Hesselager et al 2004;Yermolaieva et al 2004). ASIC1a is enriched in synaptosomal fractions and expressed in dendritic spines Wemmie et al 2002).…”