Reversible modulation of rhodopsin photolysis in pure phosphatidylserine membranes

Abstract

Bovine rhodopsin is reconstituted into artificial membranes of bovine spinal cord phosphatidylserine (PS). The influence of cations (La 3+, Ca 2+, Mn 2+, Mg 2+) and variation in lipid/protein ratio (100:1:30:1:10:1) on structure and photolytic behaviour (in particular the metarhodopsin I → II transition) of these reconstituted preparations is investigated. For comparison, rhodopsin reconstituted in endogenous photoreceptor membrane lipids has been studied under similar conditions. The following observations have been made: (1) Photolytic behaviour or structure of rhodopsin reconstituted in endogenous lipids is not affected by the cations studied. Lowering the lipid/protein ratio below 30, however, markedly affects the metarhodopsin I → II transition. Decreasing the lipid/protein ratio then simultaneously increases the percentage of very slowly decaying metarhodopsin I. (2) In the absence of cations, rhodopsin reconstituted in PS shows normal photolytic behaviour. However, lowering the lipid/protein ratio has much less influence on the metarhodopsin I → II transition than incase of endogenous lipids. (3) At the high lipid/rhodopsin ratio (100:1), Ca 2+ induces a phase separation in preparations reconstituted in PS and formation of so-called ‘cochleate cylinders’, a highly condensed Ca 2+-PS phase. A concomitant effect on the metarhodopsin I → II transition rate is observed. This effect is reasonably specific for Ca 2+ (Ca 2+ ⪢ La 3+ > Mn 2+ ∼ Mg 2+) and is completely reversed by incubation with EDTA. (4) At the lower lipid/rhodopsin ratios, Ca 2+ affects the structure and photolytic behaviour of rhodopsin reconstituted in PS only slightly. Now, only La 3+ shows any significant effect (La 3+ ⪢ Mn 2+ > Ca 2+ > Mg 2+). (5) We conclude that interaction of PS with rhodopsin has a strong electrostatic component. Solvation of rhodopsin in the lipid bilayer is enhanced by PS relative to the average endogenous lipid and consequently PS minimizes protein-protein contacts, even at low lipid volumes.