Heteromultimerization of ASIC and ENaC channels determined by FRET microscopy

Abstract

High grade glioma cells express constitutively active inward Na+ currents that are inhibited by amiloride and psalmotoxin 1, implicating Epithelial Na+ channels (ENaCs) and Acid Sensitive Ion Channels (ASICs) as core components of the active channel. ASIC and ENaC subunits have been detected in normal glia and glioma cells using rtPCR, but the functional subunit composition of the glioma Na+ channel remains unknown. In order to determine if ASIC and ENaC subunits intermix to form heteromeric channels, intersubunit associations were measured using confocal fluorescence resonant energy transfer (FRET) microscopy. Human ASIC (1b, 2a) and ENaC (α, β, γ, δ ) ion channel subunits have been fused with the fluorescent fusion proteins eCFP and eYFP at both N- and C-termini; these constructs form functional ASIC and ENaC channels in Xenopus oocytes or transiently transfected CHO cells. FRET efficiencies measured in transiently transfected CHO cells follow: eCFP fused directly to eYFP, 27.5% ± 3.8% (positive control); eCFP cotransfected with eYFP, 6.6% ± 3.6% (negative control); ASIC1-eCFP or ASIC2-eCFP cotransfected with ASIC1, ASIC2, αENaC, or γ ENaC fused to eYFP, > 20%; ASIC1-eCFP or ASIC2-eCFP cotransfected with either βENaC or δ ENaC, < 12% (error is standard deviation from >15 cells from >3 transfections). These results suggest heteromultimerization of ASIC and ENAC subunits, particularly between ASIC1, ASIC2, αENaC, and γ ENaC. Heteromeric channel assembly has also been demonstrated by immunoprecipitation and western blot analysis. Supported by NIH grants CA101952 and DK37206.