Differential Contribution of the Olfactory CNG Channel Subunits to the Activation Process

Abstract

In olfactory receptor neurons, the cyclic nucleotide-gated (CNG) channels play an important role in converting sensory stimuli into electrical signals. The CNG channels are heterotetrameric proteins composed of three homologue subunits, 2xCNGA2, CNGA4 and CNGB1b. Each subunit has an intracellular cyclic nucleotide binding domain. This suggests that the binding of cGMP or cAMP to each subunit is involved in channel opening. The CNGA4 and CNGB1b subunits produce functional channels only if expressed together with CNGA2. Their contribution to the activation process, in particular their ability to bind cyclic nucleotides, has not been elucidated so far. Herein, heterotetrameric CNGA2A4B1b channels were expressed in Xenopus oocytes and studied in excised patches by monitoring ligand binding and gating under both steady-state and non-steady state conditions. Ligand binding was measured by confocal patch-clamp fluorometry using a fluorescent cGMP analogue (fcGMP). Similar to the homotetrameric CNGA2 channels, we observed in CNGA2A4B1b channels a crossover of the normalized steady-state binding and steady-state activation. Also, the concentration-binding relationships were similar in homo- and heterotetrameric channels. By expressing TFP-labelled CNGA4 and CNGB1b subunits alone we show that these subunits do reach the plasma membrane also in the absence of CNGA2 subunits. In order to study the binding to the CNGA4 and CNGB1b subunits we coexpressed either of them with a mutated CNGA2 subunit (T539M) that has an increased sensitivity to cAMP and is not able to bind fcGMP. Under these conditions, we observed significant binding of fcGMP to the CNGA4, but not to the CNGB1b subunit. The binding to the CNGA4 subunit is rapid (∼30 ms) and opens the channels. Our results suggest that CNGA2A4B1b channels are activated by ligand binding to CNGA2 and CNGA4 subunits, but not to the CNGB1b subunit.