Different phosphorylation rates among vertebrate cone visual pigments with different spectral sensitivities.

Cone photoreceptor subtypes having different spectral sensitivities exhibit different recovery kinetics in their photoresponses in some vertebrates. Phosphorylation by G protein-coupled receptor kinase (GRK) is essential for the rapid inactivation of light-activated visual pigment, which is the rate-limiting step of the cone photoresponse recovery in salamander. In this study we compared the rate of light-dependent phosphorylation by GRK7 of carp green- and blue-sensitive cone visual pigments. Blue pigment was phosphorylated significantly less effectively than green pigment, suggesting that the difference in the pigment phosphorylation rate is responsible for the difference in photoresponse kinetics among cone photoreceptor subtypes

Neuronal calcium sensor proteins are direct targets of the insulinotropic agent repaglinide.

The NCS (neuronal calcium sensor) proteins, including neurocalcins, recoverins and visinin-like proteins are members of a family of Ca2+-sensitive regulators, each with three Ca2+-binding EF-hand motifs. In plants, lily CCaMK [chimaeric Ca2+/CaM (calmodulin)-dependent protein kinase] and its PpCaMK ( Physcomitrella patens CCaMK) homologue are characterized by a visinin-like domain with three EF-hands. In the present study, in an effort to discover NCS antagonists, we screened a total of 43 compounds using Ca2+-dependent drug affinity chromatography and found that the insulinotropic agent repaglinide targets the NCS protein family. Repaglinide was found to bind to NCS proteins, but not to CaM or S100 proteins, in a Ca2+-dependent manner. Furthermore, the drug antagonized the inhibitory action of recoverin in a rhodopsin kinase assay with IC50 values of 400 microM. Moreover, repaglinide tightly bound to the visinin-like domain of CCaMK and PpCaMK in a Ca2+-dependent manner and antagonized the regulatory function of the domain with IC50 values of 55 and 4 microM for CCaMK and PpCaMK respectively. Although both repaglinide and a potent insulin secretagogue, namely glibenclamide, blocked K(ATP) channels with similar potency, glibenclamide had no antagonizing effect on the Ca2+-stimulated CCaMK and PpCaMK autophosphorylation, mediated by their visinin-like domain. In addition, a typical CaM antagonist, trifluoperazine, had no effect on the CCaMK and PpCaMK autophosphorylation. Repaglinide appears to be the first antagonist of NCS proteins and visinin-like domain-bearing enzymes. It may serve as a useful tool for evaluating the physiological functions of the NCS protein family. In addition, since repaglinide selectively targets NCS proteins among the EF-hand Ca2+-binding proteins, it is a potential lead compound for the development of more potent NCS antagonists

Purification of OS and IS membranes from purified rods and cones.

<p>Differential interference contrast microscopic (DIC) images of the cell fractions in each step of the purification are shown. Purified carp rods (A) and cones (B) were passed through a 27-gauge needle to dissociate the OS from the IS, and the resultant broken rods (C) and cones (D) were layered on a sucrose density gradient made in a test tube (drawings in the left of E/G and F/H) to centrifuge. The number in the drawings shows the density of sucrose (%, w/v). Separated membranes at upper (E, F) and lower (G, H) interfaces were collected. Scale bar, 20 μm throughout.</p

Estimation of separation of OS and IS membranes using TOM20, Na⁺/K⁺ ATPase α subunit and calnexin as marker proteins.

<p>(A) Specificity of antibodies used to detect TOM20, Na⁺/K⁺ ATPase α subunit and that of anti-calnexin antiserum. Purified rod membranes containing 200 pmol of rhodopsin and cone membranes containing 6 pmol of cone total pigments were subjected to SDS-PAGE and were stained with Coomassie Brilliant Blue (left panel) or probed with antibodies or antiserum against each protein (right three panels). (B-D) Quantitative immunoblot analyses of TOM20 (B), Na⁺/K⁺ ATPase α subunit (C) and calnexin (D). In the upper panels in each of (B)—(D), purified rod membranes containing 200 pmol of rhodopsin or purified cone membranes containing 6 pmol of total cone pigments (Initial), upper and lower membrane fractions obtained from the same number of the purified cells (Upper and Lower, respectively), and a diluted series of initial rod and cone membranes were subjected to SDS-PAGE. These membranes were probed with antibodies or antiserum against each marker protein. To detect the amounts of target proteins precisely, 4 times volume of samples were applied when necessary (4×). The amount of a target protein in each of the membranes was determined with a calibration line obtained from immunoblot signals obtained in a diluted series of initial rod or cone membranes. In the lower panels in each of (B)—(D), examples of quantification are shown. The quantity of a target protein in each fraction is indicated by an arrow in lower panels. With this estimation, one can determine how much % of the target protein is present in each of the membranes as compared with the amount in the initial rod or cone membranes of the same cell number.</p

Purification of cone outer segment for proteomic analysis on its membrane proteins in carp retina

<div><p>Rods and cones are both photoreceptors in the retina, but they are different in many aspects including the light response characteristics and, for example, cell morphology and metabolism. These differences would be caused by differences in proteins expressed in rods and cones. To understand the molecular bases of these differences between rods and cones, one of the ways is to compare proteins expressed in rods and cones, and to find those expressed specifically or dominantly. In the present study, we are interested in proteins in the outer segment (OS), the site responsible for generation of rod- or cone-characteristic light responses and also the site showing different morphology between rods and cones. For this, we established a method to purify the OS and the inner segment (IS) of rods and also of cones from purified carp rods and cones, respectively, using sucrose density gradient. In particular, we were interested in proteins tightly bound to the membranes of cone OS. To identify these proteins, we analyzed proteins in some selected regions of an SDS-gel of washed membranes of the OS and the IS obtained from both rods and cones, with Liquid Chromatography-tandem Mass Spectrometry (LC-MS/MS) using a protein database constructed from carp retina. By comparing the lists of the proteins found in the OS and the IS of both rods and cones, we found some proteins present in cone OS membranes specifically or dominantly, in addition to the proteins already known to be present specifically in cone OS.</p></div