RESUMO
Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks â¼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of â¼27 min), whereas it accelerates in Arr1(ox) rods (τ of â¼8 min) and Grk1(-/-) rods (τ of â¼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo.
Assuntos
Arrestinas/metabolismo , Adaptação à Escuridão/fisiologia , Receptor Quinase 1 Acoplada a Proteína G/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Animais , Arrestinas/genética , Receptor Quinase 1 Acoplada a Proteína G/genética , Camundongos , Camundongos Knockout , Fosforilação , Estimulação Luminosa , Ligação Proteica , Rodopsina/metabolismoRESUMO
We have fabricated a stack of five 1,2-dipalmitoyl-sn-3-phosphatidylethanolamine (DPPE) bilayers supported on a polished silicon substrate in excess water. The density profile of these stacks normal to the substrate was obtained through analysis of x-ray reflectivity. Near the substrate, we find the layer roughness and repeat spacing are both significantly smaller than values found in bulk multilayer systems. The reduced spacing and roughness result from suppression of lateral fluctuations due to the flat substrate boundary. The layer spacing decrease then occurs due to reduced Helfrich repulsion.