Question: 

Cyclic nucleotide-gated (CNG) channels play a central role in vertebrate transduction of olfaction and vision. Olfactory CNG channels are heterotetramers composed of three homologue subunits, CNGA2, CNGA4, CNGB1b with a stoichiometry 2:1:1. It is well established that binding of cGMP or cAMP to the intracellular binding domain of the CNGA2 subunits triggers conformational changes of the channel which finally lead to the opening of the pore. In contrast, the contribution of the CNGA4 and CNGB1b subunits to channel activation has not been elucidated so far.

Methods: 

To gain insight into the functional role of the subunits, the time courses of ligand binding and channel activation were studied in CNGA2 channels and CNGA2:A4:B1b channels in response to ligand concentration-jumps, which were applied by means of a piezo driven multi-barrel pipette. Ligand binding was measured by high-speed confocal patch-clamp fluorometry (up to 277 frames per second). The data were kinetically analyzed.

Results & Conclusions: 

The results show that the CNGA4 and CNGB1b subunit promote faster binding and activation as well as unbinding and deactivation. We also show that cGMP binding to only two out of the four CNGA2 subunits in the homotetrameric CNGA2 channel suffices to induce maximal activation. The faster activation of the CNGA2:A4:B1b channel (< 10 ms) might suggest that already the first binding step promotes maximal activation. Global fit analyses are underway. In additional experiments in channels with differentially disabled CNBDs, our results indicate that cAMP activates CNGA2:A4:B1b channels by binding to all four subunits whereas cGMP induces channel activation only by binding to the CNGA2 and CNGA4 subunits, suggesting a role of the CNGB1b subunit in distinguishing between cAMP and cGMP. Our results provide new insight into the functional role the CNGA4 and CNGB1b subunit in the gating of olfactory CNG channels.