Molecular Mechanisms for Phosphoinositide Regulation of Cone Photoreceptor Cng Channel Gating
Year: 2014
Type: dissertation
Abstract: 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 (PIP n ), 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 (PIP 2 ) and phosphatidylinositol 3,4,5-triphosphate (PIP 3 )], exhibiting a decrease in apparent cGMP affinity.Using electrophysiology combined with biochemistry and molecular manipulations, we have investigated the structural elements that are critical for PIP 2 and PIP 3 regulation of cone CNG channels.We have discovered two phosphoinositideregulation sites within the amino (N) -terminal and the carboxyl (C) -terminal regions of CNGA3 subunits, respectively; CNGB3 subunits do not contain PIP n -sensitive elements.Both of these two phosphoinositide-regulation modules are necessary for PIP nvi 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 PIP n .Disruption of this intersubunit interaction either by truncation of the distal C-terminal region of CNGA3 or by CNGA3-L633P unmasked or potentiated the PIP n 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 transcripts, significantly altered the PIP n sensitivity of cone CNG channels.Enhanced PIP n sensitivity for heteromeric CNG channels that arises from inclusion of e3 exon in CNGA3 transcripts depended on the C terminal PIP nregulation 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 liganddependent activation of heteromeric channels.
Author: Gucan Dai
Language: English
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W2290903384