Recoverin and Rhodopsin Kinase Activity in Detergent-resistant Membrane Rafts from Rod Outer Segments

Abstract

Cholesterol-rich membranes or detergent-resistant membranes (DRMs) have recently been isolated from bovine rod outer segments and were shown to contain several signaling proteins such as, for example, transducin and its effector, cGMP-phosphodiesterase PDE6. Here we report the presence of rhodopsin kinase and recoverin in DRMs that were isolated in either light or dark conditions at high and low Ca 2؉ concentrations. Inhibition of rhodopsin kinase activity by recoverin was more effective in DRMs than in the initial rod outer segment membranes. Furthermore, the Ca 2؉ sensitivity of rhodopsin kinase inhibition in DRMs was shifted to lower free Ca 2؉ concentration in comparison with the initial rod outer segment membranes (IC 50 ‫؍‬ 0.76 M in DRMs and 1.91 M in rod outer segments). We relate this effect to the high cholesterol content of DRMs because manipulating the cholesterol content of rod outer segment membranes by methyl-␤-cyclodextrin yielded a similar shift of the Ca 2؉ -dependent dose-response curve of rhodopsin kinase inhibition. Furthermore, a high cholesterol content in the membranes also increased the ratio of the membrane-bound form of recoverin to its cytoplasmic free form. These data suggest that the Ca 2؉ -dependent feedback loop that involves recoverin is spatially heterogeneous in the rod cell.

Tuning of a Neuronal Calcium Sensor

“…The high cholesterol content of these membranes facilitates membrane binding of recoverin and can shift its Ca 2ϩ -dependent properties to lower free Ca 2ϩ concentration, i.e. into the physiologically relevant range (36). Thus, cholesterol appears as an additional external modulator of Ca 2ϩ sensitivity.…”

Molecular mechanisms of protein and lipid targeting to ciliary membranes

“…Disruption of lipid rafts also leads to protein mislocalization (Tyler et al, 2009). An association between lipid rafts and many other cilia-targeted proteins has also been described in other systems, including vertebrate photoreceptors (Senin et al, 2004), Chlamydomonas reinhardtii (Iomini et al, 2006), mammalian spermatozoa (Travis et al, 2001) and Leishmania major (Tull et al, 2004). Similarly, certain proteins that are thought to be involved in targeting proteins to ciliary membranes have been shown to require protein-lipid interactions for their localization in non-ciliated cells.…”

Loss of Caveolin-1 Impairs Retinal Function Due to Disturbance of Subretinal Microenvironment

“…At the resolution of confocal microscopy, we are unsure if this immunoreactivity is derived solely from Müller glial cells or also from expression in photoreceptors because it is expressed in both cell types albeit enriched in the former (37,38). Based on localization in the outer retina, cofractionation with phototransduction proteins (32,33,44), and interaction, in vitro, with transducin ␣-subunits (30), we hypothesized that loss of Cav-1 might affect retinal function. Therefore, we assessed retinal light responses in…”

“…In photoreceptors, several phototransduction proteins, including transducin, RGS-9, arrestin, guanylate cyclase, rhodopsin kinase, and the cyclic nucleotide-gated channel, cofractionate in Cav-1-enriched membranes (32,33,44), and Cav-1 and the ␣-subunit of transducin co-immunoprecipitate in a cholesterol-and guanine nucleotide-dependent manner (30). Recently, Cav-1 has been identified as a component of photoreceptor disks (29), and the disk-localized protein, Rom-1, is associated with Cav-1-enriched membranes from disks (28).…”