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 pseudosubunits either adjacent (cis) or diagonally opposite (trans). The cis tetramer showed con action, with a mean ratio of steady-state conductances g((30 mM cGMP))/g((3 mM cGMP)) = 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((30 mM cGMP))/g((3mM cGMP))= 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.