A current paradigm for visual function centers on the metarhodopsin I (MI) to metarhodopsin II (MII) conformational transition as the trigger for an intracellular enzyme cascade leading to excitation of the retinal rod. We investigated the influences of the membrane lipid composition on this key triggering event in visual signal transduction using flash photolysis techniques. Bovine rhodopsin was combined with various phospholipids to form membrane recombinants in which the lipid acyl chain composition was held constant at that of egg phosphatidylcholine (PC), while the identity of the lipid headgroups was varied. The ratio of MII/MI produced in these recombinants by an actinic flash at 28 degrees C was studied as a function of pH. The results were compared to the photochemical function observed for rhodopsin in native retinal rod outer segment (ROS) membranes, in total native ROS lipid recombinants, and in dimyristoylphosphatidylcholine (DMPC) recombinants. In membrane recombinants incorporating lipids derived from egg PC, as well as in the total ROS lipids control and the native ROS disk membranes, MI and MII were found to coexist in a pH-dependent, acid-base equilibrium on the millisecond time scale. The recombinants of rhodopsin with egg PC, either alone or in combination with egg PC-derived phosphatidylethanolamine (PE) or phosphatidylserine (PS), exhibited substantially reduced photochemical activity at pH 7.0. However, all recombinants comprising phospholipids with unsaturated acyl chains were capable of full native-like MII production at pH 5.0, confirming previous results [Gibson, N.J.. & Brown, M.F. (1990) Biochem. Biophys. Res. Commun. 169, 1028-1034]. It follows that energetic constraints on the MI and MII states imposed by egg PC-derived acyl chains can be offset by increased activity of H+ ions. The data reveal that the major effect of the membrane lipid composition is to alter the apparent pK for the MI-MII conformational equilibrium of rhodopsin [Gibson, N.J., & Brown, M.F. (1991) Biochem. Biophys. Res. Commun. 176, 915-921]. Recombinants containing only phosphocholine headgroups exhibited the lowest apparent pK values, whereas the presence of either 50 mol % PE or 15 mol % PS increased the apparent pK. The inability to obtain full native-like function in recombinants having egg PC-derived chains and a native-like headgroup composition indicates a significant role of the polyunsaturated docosahexaenoic acid (DHA) chains (22:6 omega 3) of the native retinal rod membrane lipids. Temperature studies of the MI-MII transition enabled an investigation of lipid influences on the thermodynamic parameters of a membrane protein conformational change linked directly to function.(ABSTRACT TRUNCATED AT 400 WORDS)

Rhodopsin is an important example of a G protein-coupled receptor (GPCR) in which 11-cis-retinal is the ligand and acts as an inverse agonist. Photolysis of rhodopsin leads to formation of the activated meta II state from its precursor meta I. Various mechanisms have been proposed to explain how the membrane composition affects the meta I-meta II conformational equilibrium in the visual process. For rod disk membranes and recombinant membranes containing rhodopsin, the lipid properties have been discussed in terms of elastic deformation of the bilayer. Here we have investigated the relation of nonlamellar-forming lipids, such as dioleoylphosphatidylethanolamine (DOPE), together with dioleoylphosphatidylcholine (DOPC), to the photochemistry of membrane-bound rhodopsin. We conducted flash photolysis experiments for bovine rhodopsin recombined with DOPE/DOPC mixtures (0:100 to 75:25) as a function of pH to explore the dependence of the photochemical activity on the monolayer curvature free energy of the membrane. It is well-known that DOPC forms bilayers, whereas DOPE has a propensity to adopt the nonlamellar, reverse hexagonal (H(II)) phase. In the case of neutral DOPE/DOPC recombinants, calculations of the membrane surface pH confirmed that an increase in DOPE favored the meta II state. Moreover, doubling the PE headgroup content versus the native rod membranes substituted for the polyunsaturated, docosahexaenoic acyl chains (22:6 omega 3), suggesting rhodopsin function is associated with a balance of forces within the bilayer. The data are interpreted by applying a flexible surface model, in which the meta II state is stabilized by lipids tending to form the H(II) phase, with a negative spontaneous curvature. A simple theory, based on principles of surface chemistry, for coupling the energetics of membrane proteins to material properties of the bilayer lipids is described. For rhodopsin, the free energy balance of the receptor and the lipids is altered by photoisomerization of retinal and involves curvature stress/strain of the membrane (frustration). A new biophysical principle is introduced: matching of the spontaneous curvature of the lipid bilayer to the mean curvature of the lipid/water interface adjacent to the protein, which balances the lipid/protein solvation energy. In this manner, the thermodynamic driving force for the meta I-meta II conformational change of rhodopsin is tightly controlled by mixtures of nonlamellar-forming lipids having distinctive material properties.

The nitric oxide (NO)-cGMP signaling system is thought to play important roles in the function of the olfactory system in both vertebrates and invertebrates. One way of studying the role of NO in the nervous system is to study the distribution and properties of NO synthase (NOS), as well as the soluble guanylyl cyclases (sGCs), which are the best characterized targets of NO. We study NOS and sGC in the relatively simple and well characterized insect olfactory system of the hawkmoth, Manduca sexta. We have cloned Manduca sexta nitric oxide synthase (MsNOS) and two sGCs (MsGC␣1 and MsGC␤1), characterized their basic biochemical properties, and studied their expression in the olfactory system. The sequences of the Manduca genes are highly similar to their mammalian homologs and show similar biochemical properties when expressed in COS-7 cells. In particular, we find that MsGC functions as an obligate heterodimer that is stimulated significantly by NO. We also find that MsNOS has a Ca 2ϩ -sensitive NO-producing activity similar to that of mammalian neuronal NOS. Northern and in situ hybridization analyses show that MsNOS and the MsGCs are expressed in a complementary pattern, with Ms-NOS expressed at high levels in the antennae and the MsGCs expressed at high levels in a subset of antennal lobe neurons. The expression patterns of these genes suggest that the NOsGC signaling system may play a role in mediating communication between olfactory receptor neurons and projection neurons in the glomeruli of the antennal lobe.

During development of the adult olfactory system of the moth Manduca sexta, olfactory receptor neurons extend axons from the olfactory epithelium in the antenna into the brain. As they arrive at the brain, interactions with centrally derived glial cells cause axons to sort and fasciculate with other axons destined to innervate the same glomeruli. Here we report studies indicating that activation of the epidermal growth factor receptor (EGFR) is involved in axon ingrowth and targeting. Blocking the EGFR kinase domain pharmacologically leads to stalling of many axons in the sorting zone and nerve layer as well as abnormal axonal fasciculation in the sorting zone. We also find that neuroglian, an IgCAM known to activate the EGFR through homophilic interactions in other systems, is transiently present on olfactory receptor neuron axons and on glia during the critical stages of the sorting process. The neuroglian is resistant to extraction with Triton X-100 in the sorting zone and nerve layer, possibly indicating its stabilization by homophilic binding in these regions. Our results suggest a mechanism whereby neuroglian molecules on axons and possibly sorting zone glia bind homophilically, leading to activation of EGFRs, with subsequent effects on axon sorting, pathfinding, and extension, and glomerulus development.

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