Molecular Mechanisms for Phosphoinositide Regulation of Cone Photoreceptor CNG Channel Gating
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Photoreceptor cyclic nucleotide-gated (CNG) channels are critical for converting light inputs into electrical signals that are ultimately processed as visual information. However, it is not well understood how exactly photoreceptor CNG channels are regulated. Since the first study showing the direct activation of inward rectifier K+ channels by phosphoinositides in 1998 (Huang et al., 1998), emerging evidences have revealed that phosphoinositides (PIPn), which are low-abundance membrane-bound phospholipids, have tremendous effects on membrane ion channels. Previous studies on rod, cone and olfactory CNG channels have shown that the activities of CNG channels are inhibited by phosphoinositides [phosphatidylinositol 4,5-biphosphate (PIP2) and phosphatidylinositol 3,4,5-triphosphate (PIP3)], exhibiting a decrease in apparent cGMP affinity. Using electrophysiology combined with biochemistry and molecular manipulations, we further investigated the structural elements that are critical for PIP2 and PIP3 regulation of cone CNG channels. We have discovered two phosphoinositide-regulation sites within the amino (N) -terminal and the carboxyl (C) -terminal regions of CNGA3 subunits, respectively; whereas CNGB3 subunits do not contain PIPn-sensitive elements. Both of these two phosphoinositide-regulation sites are necessary for PIPn-regulation of heteromeric CNGA3+CNGB3 channels. Furthermore, by studying an achromatopsia-associated mutation (L633P) within CNGA3, we determined that an intersubunit, rather than intrasubunit, N-C terminal interaction controls the regulation of cone CNG channels by PIPn. Disruption of this intersubunit interaction either by truncation of the distal C-terminal region of CNGA3 or by CNGA3-L633P unmasked or potentiated the PIPn sensitivity of CNGA3 channels. Moreover, incorporation or exclusion of an N-terminal region within CNGA3 subunits, which is encoded by an optional e3 exon in CNGA3 transripts, significantly affected the PIPn sensitivity of cone CNG channels. Enhanced PIPn sensitivity for hetermomeric CNG channels that arises from inclusion of the e3 exon in CNGA3 transcripts depended on the C terminal PIPn-regulation module rather than the N-terminal module, presumably due to an allosteric mechanism involving changes in N-C interactions. In addition to their decisive role in channel regulation by phosphoinositides, we have determined (using subunit-specific crippling of cyclic nucleotide-binding domains) that CNGA3 subunits contribute much more than CNGB3 subunits to ligand discrimination (cGMP versus cAMP) and ligand-dependent activation of heteromeric channels.