The TRPM1 channel in ON-bipolar cells is gated by both the α and the βγ subunits of the G-protein G _o

Abstract

Transmission from photoreceptors to ON bipolar cells in mammalian retina is mediated by a sign-inverting cascade. Upon binding glutamate, the metabotropic glutamate receptor mGluR6 activates the heterotrimeric G-protein Gα _oβ3γ13, and this leads to closure of the TRPM1 channel (melastatin). TRPM1 is thought to be constitutively open, but the mechanism that leads to its closure is unclear. We investigated this question in mouse rod bipolar cells by dialyzing reagents that modify the activity of either Gα _o or Gβγ and then observing their effects on the basal holding current. After opening the TRPM1 channels with light, a constitutively active mutant of Gα _o closed the channel, but wild-type Gα _o did not. After closing the channels by dark adaptation, phosducin or inactive Gα _o (both sequester Gβγ) opened the channel while the active mutant of Gα _o did not. Co-immunoprecipitation showed that TRPM1 interacts with Gβ3 and with the active and inactive forms of Gα _o. Furthermore, bioluminescent energy transfer assays indicated that while Gα _o interacts with both the N- and the C- termini of TRPM1, Gβγ interacts only with the N-terminus. Our physiological and biochemical results suggest that both Gα _o and Gβγ bind TRPM1 channels and cooperate to close them.