Two structural components in CNGA3 support regulation of cone CNG channels by phosphoinositides

Cyclic nucleotide-gated (CNG) channels in retinal photoreceptors play a crucial role in vertebrate phototransduction. The ligand sensitivity of photoreceptor CNG channels is adjusted during adaptation and in response to paracrine signals, but the mechanisms involved in channel regulation are only partly understood. Heteromeric cone CNGA3 (A3) + CNGB3 (B3) channels are inhibited by membrane phosphoinositides (PIP(n)), including phosphatidylinositol 3,4,5-triphosphate (PIP(3)) and phosphatidylinositol 4,5-bisphosphate (PIP(2)), demonstrating a decrease in apparent affinity for cyclic guanosine monophosphate (cGMP). Unlike homomeric A1 or A2 channels, A3-only channels paradoxically did not show a decrease in apparent affinity for cGMP after PIP(n) application. However, PIP(n) induced an ∼2.5-fold increase in cAMP efficacy for A3 channels. The PIP(n)-dependent change in cAMP efficacy was abolished by mutations in the C-terminal region (R643Q/R646Q) or by truncation distal to the cyclic nucleotide-binding domain (613X). In addition, A3-613X unmasked a threefold decrease in apparent cGMP affinity with PIP(n) application to homomeric channels, and this effect was dependent on conserved arginines within the N-terminal region of A3. Together, these results indicate that regulation of A3 subunits by phosphoinositides exhibits two separable components, which depend on structural elements within the N- and C-terminal regions, respectively. Furthermore, both N and C regulatory modules in A3 supported PIP(n) regulation of heteromeric A3+B3 channels. B3 subunits were not sufficient to confer PIP(n) sensitivity to heteromeric channels formed with PIP(n)-insensitive A subunits. Finally, channels formed by mixtures of PIP(n)-insensitive A3 subunits, having complementary mutations in N- and/or C-terminal regions, restored PIP(n) regulation, implying that intersubunit N-C interactions help control the phosphoinositide sensitivity of cone CNG channels.