Distinct Contributions of CNGA3 and CNGB3 Subunits to Ligand-Specific Activation of Cone CNG Channels

Abstract

Cyclic nucleotide-gated (CNG) ion channels regulate the electrical activity of retinal photoreceptors, rods and cones, by sensing the light-induced changes of intracellular cGMP levels. Cone CNG channels consist of CNGA3 and modulatory CNGB3 subunits, both of which contain a cyclic nucleotide-binding domain (CNBD). CNGB3 subunits confer enhanced responses to cAMP and support several aspects of channel regulation. However, it is not fully understood how CNGB3 (and CNGA3) are specialized to contribute to ligand-specific activation of cone CNG channels. using patch-clamp recordings, we characterized several mutations located within the CNBD of CNGA3, each of which produced dramatic, ligand-specific effects on channel gating. In particular, D609M in CNGA3 reversed ligand selectivity, making cAMP a better agonist than cGMP, similar to equivalent mutations in paralogous channel subunits. These experiments suggest that mechanisms underlying ligand interaction with CNGA3 are well conserved. However, parallel mutations within the CNBD Cα-helix of CNGB3 had no effect on the ligand selectivity of heteromeric channels, consistent with the large decrement in sequence conservation in this region of CNGB3. CNGB3 appear to lack features supporting ligand discrimination. Next, we examined subunit contributions to ligand-dependent activation using CNBD “knock out” (R564E in CNGA3; R604E in CNGB3). CNGB3 R604E decreased relative cAMP efficacy, but only had a subtle effect on the cGMP activation for heteromeric channels (with wild-type or R564E CNGA3). In contrast, CNGA3 R564E caused an approximately 500-fold decrease in apparent cGMP affinity and nearly eliminated cAMP-dependent gating. Similar results were observed with analogous experiments using mutations of T565A in CNGA3 and T605A in CNGB3. Together, we propose that CNGA3 is the principal subunit mediating both ligand discrimination and ligand-dependent stabilization of the open state, while CNGB3 makes only a minor contribution to cGMP-dependent gating.