Bimodal agonism in heteromeric cyclic nucleotide-gated channels

Direct binding of cGMP or cAMP to tetrameric cyclic nucleotide-gated (CNG) channels will normally promote the open (conductive) conformation. However, the catfish CNGA2 subtype exhibits bimodal agonism, whereby open probability (P_o) increases with initial cGMP binding events ("pro" action) but decreases with subsequent cGMP binding events ("con" action) that occur at concentrations above 3 mM. We constructed, and heterologously expressed, chimeric CNG channel subunits with sequence substitutions in the binding domain (BD), and tested their activation using patch-clamp of cell-free membranes. A normal subunit with the rat CNGA4 BD (with only pro action) could be converted into a bimodal subunit (both pro and con action) by replacing the N-terminal portion of the BD with catfish CNGA2 sequence. We then fused two bimodal and two normal subunits in tandem tetramers, to form heteromeric CNG channels with bimodal pseudo-subunits either adjacent (cis) or diagonally opposite (trans). The cis tetramer showed con action, with a mean ratio of steady-state conductances g_(30mMcGMP) / g_(3mMcGMP) = 0.87, demonstrating bimodal agonism in a heteromeric CNG channel for the first time. In contrast, trans tetramers showed normal cGMP agonism up to 30 mM cGMP with mean g_(30mMcGMP) / g_(3mMcGMP) = 1.02, although a minority of oocytes (4 of 15) expressed anomalous channel populations with con action. Rearranging subunits in a heteromer thus influences a channel's P_o at high cGMP concentration. The sensitivity of con action to neighbouring subunits implies a cooperative mechanism.