Molecular physiology and biophysics of cyclic nucleotide-gated channels

Project Summary Cyclic nucleotide-gated (CNG) channels transduce chemical signals to electrical signals in photoreceptors and olfactory receptor cells and are essential for vision and smell. Mutations in CNG channel genes cause retinitis pigmentosa and achromatopsia. CNG channels are activated cooperatively by intracellular cGMP or cAMP. Although rich knowledge has been gained from extensive functional studies of CNG channels, structural underpinnings of CNG channel properties and mechanisms are limited. Recently, closed- and open- state cryo-EM structures of the homomeric C. elegans TAX-4 CNG channel and human CNGA1 channel have been reported. These structures reveal many interesting and unique features that explain some CNG channel properties. However, these structures do not faithfully represent native mammalian CNG channels, which are heteroterameric complexes formed by CNGA1 and CNGB1 in rod photoreceptors and by CNGA3 and CNGB3 in cone photoreceptors. It is also unclear how disease-associated mutations (DAMs) alter CNG channel structure and function. We have recently obtained a 3.5 Å-resolution cryo-EM structure of the full-length human CNGA3/CNGB3 channel in the apo closed state. The structure shows that the CNGA3/CNGB3 channel is composed of 3 CNGA3 and 1 CNGB3, with an overall structure similar to that of TAX-4 and CNGA1, but strikingly, R403 (located in the pore helix) and R442 (located at the cytoplasmic end of S6) of CNGB3 protrude into the pore. We have also found that R410W, a DAM in CNGA3, opens the channel in the absence of cGMP. Building on these advances, we propose three specific aims to elucidate the molecular mechanisms of allostery, cooperativity and channelopathy of CNGA3/CNGB3 and TAX-4 channels. (1) We will obtain high- resolution structures of CNGA3/CNGB3 channels in closed and open states, in the absence and presence of Ca2+. These structures are likely to shed light on the unique role of CNGB3 and on the fundamental question of why native cone CNG channels have a CNGB3 subunit. (2) We will elucidate the molecular mechanisms of allosteric cGMP activation of CNGA3/CNGB3 and TAX-4 channels through functional and structural characterization of partially liganded channels. Partial cGMP occupancy will be achieved by mixing CNGA3 or CNGB3 with a mutant CNGB3 or CNGA3 subunit unable to bind cGMP, by concatenating CNGA3 and/or CNGB3 into tandem-dimers that contain a subunit unable to bind cGMP, and by concatenating TAX-4 into tandem-dimers and tandem-tetramers that contain varying numbers of WT TAX-4 and TAX-4_EA, a mutant subunit incapable of binding cGMP. (3) We will investigate the molecular mechanisms of CNG channelopathy, focusing on a systematic examination of the effect of DMAs in S4 and pore helix on the structure and function of CNGA3/CNGB3 and TAX-4 channels. These studies will enhance our understanding of CNG channel ion permeation, gating and channelopathy and provide guidance for the development of therapeutic strategies to treat degenerative visual disorders.