Rhodopsin Crowding in Model Lipid Bilayers - Functional Implications

Abstract

In the rod outer segment disks of the retina, rhodopsin is densely packed in phospholipid bilayers with a high content of polyunsaturated acyl chains. It has been hotly debated if oligomerizationof rhodopsin is a critical step for efficient activation of G-protein. Here, we investigated the effect of rhodopsin density in synthetic membranes on the interaction with its cognate G-protein transducin (Gt). Experiments were conducted at rhodopsin:lipid ratios ranging from 1:4000 to 1:70 with sn1-stearoyl sn2-oleoyl phosphatidylcholine (18:0-18:1 PC) model bilayers at ambient temperature.The amount of metarhodopsin-II (MII) formed after photoactivation was determined by UV-visible spectroscopy. Guanine nucleotide exchange rate measured using labeled GTPγS was used to monitor Gt affinity for activated rhodopsin, the rate of rhodopsin catalyzed Gt activation, and the decay rate of the active photointermediate.At low rhodopsin density (1:1000 and below), MII concentration was the highest and independent of rhodopsin concentration. Increasing rhodopsin packing density correlated with a shift in the metarhodopsin-I (MI)/MII equilibrium towards MI. After photoactivation, rhodopsin decayed at two different rates (t1/2 ∼ 3 min and > 60 min) and the proportion of the fast decaying photointermediate decreased with increasing rhodopsin density. Finally, MII-catalyzed GDP-GTPγS nucleotide exchange rates for Gt were crucially affected by rhodopsin density. The enzymatic power of rhodopsin (Vmax/Km for Gt) was higher by 2 orders of magnitude at low rhodopsin density as compared to high rhodopsin density.Our results suggest that, in model membranes, rhodopsin exists as an equilibrium between at least two populations: monomeric at low rhodopsin density with rapid decay and high catalytic efficiency and oligomeric at high rhodopsin density with slow decay and inefficient catalysis of nucleotide exchange.