Biophysical physiology of phosphoinositide rapid dynamics and regulation in living cells

Phosphoinositide membrane lipids are ubiquitous low-abundance signaling molecules. They direct many physiological processes that involve ion channels, membrane identification, fusion of membrane vesicles, and vesicular endocytosis. Pools of these lipids are continually broken down and refilled in living cells, and the rates of some of these reactions are strongly accelerated by physiological stimuli. Recent biophysical experiments described here measure and model the kinetics and regulation of these lipid signals in intact cells. Rapid on-line monitoring of phosphoinositide metabolism is made possible by optical tools and electrophysiology. The experiments reviewed here reveal that as for other cellular second messengers, the dynamic turnover and lifetimes of membrane phosphoinositides are measured in seconds, controlling and timing rapid physiological responses, and the signaling is under strong metabolic regulation. The underlying mechanisms of this metabolic regulation remain questions for the future

Molecular Mechanisms for Phosphoinositide Regulation of Cone Photoreceptor CNG Channel Gating

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

Subunit-specific regulation of photoreceptor CNG channels by phosphoinositides

Funding agency: National Institute of HealthWashington State UniversityDai, Gucan et al. (2010, March 26). Subunit-specific regulation of photoreceptor CNG channels by phosphoinositides. Poster presented at the Washington State University Academic Showcase, Pullman, WA

Lipid signaling to membrane proteins: From second messengers to membrane domains and adapter-free endocytosis

Lipids influence powerfully the function of ion channels and transporters in two well-documented ways. A few lipids act as bona fide second messengers by binding to specific sites that control channel and transporter gating. Other lipids act nonspecifically by modifying the physical environment of channels and transporters, in particular the protein-membrane interface. In this short review, we first consider lipid signaling from this traditional viewpoint, highlighting innumerable Journal of General Physiology publications that have contributed to our present understanding. We then switch to our own emerging view that much important lipid signaling occurs via the formation of membrane domains that influence the function of channels and transporters within them, promote selected protein-protein interactions, and control the turnover of surface membrane