Abstracts of selected publications

1. Kalamkarov G., Pogozheva I., Shevchenko T., Koskelainen A., Hemila S. and Donner K . pH changes in frog rods upon manipulation of putative pH-regulating transport mechanisms. Vision Research. 36(19):3029-36, 1996.
   Abstract. Rod intracellular pH (pHi) in the intact frog retina was measured fluorometrically with the dye 2',7'-bis(2-carboxyethyl)-5(and-6)-carboxyfluorescein under treatments chosen to affect putative pH-regulating transport mechanisms in the plasma membrane. The purpose was to relate possible pHi changes to previously reported effects on photoresponses. In nominally bicarbonate-free Ringer, application of amiloride (1 mM) or substitution of 95 mM external Na⁺ by K⁺ or choline triggered monotonic but reversible acidifications, consistent with inhibition of Na⁺/H⁺ exchange. Bicarbonate-dependent mechanisms were characterized as follows: (1) Replacing half of a 12 mM phosphate buffer by bicarbonate caused a sustained rise of pHi. (2) Subsequent application of the anion transport inhibitor 4,4'-diisothiocyanatostilbene-2',2'-disulphonic acid (DIDS, 0.2 mM) set off a slow acidification. (3) Substitution of external Cl- by gluconate (95 mM) caused a rapid pHi rise both in normal Na⁺ and low-Na⁺ perfusion. (4) This effect was inhibited by DIDS. The results support a consistent explanation of parallel electrophysiological experiments on the assumption that intracellular acidifications reduce and alkalinizations (in a certain range) augment photoresponses. It is concluded that both Na⁺/H⁺ exchange and bicarbonate transport control rod pHi, modulating the light-sensitive current. Part of the bicarbonate transport is by Na⁺-independent HCO₃^-/Cl^-  exchange, but a further Na⁺-coupled bicarbonate import mechanism is implicated.
2. Kalamkarov G.R., Shevchenko T.F. and Pogozheva I.D. Te role of HCO₃^-/Cl^- exchange in the regulation of phototransduction in the retinal cell.  Biologicheskie membrani (USSR), 8: 1195-1197, 1991.
3. Pogozheva I.D., Shevchenko T.F., Livshits V.A. and Kalamkarov G.R. Determination of transducin binding sites by local rhodopsin SH-group modification. Biologicheskie membrani (USSR), 8: 44-49, 1991.
   Abstract.  Two different types of SH reagents were used to modify cystein residues on the cytoplasmic surface of rhodopsin molecule.  Modification of Cys¹⁴⁰, Cys³²² and Cys³²³, but not Cys³¹⁶ was shown to disturb phosphodiesterase activity.  However its inhibition by arrestin was not changed after modification of these residues.  In contract to arrestin, transducin is proposed to be bound by rhodospin near Cys¹⁴⁰, Cys³²² and Cys²³² residues.
4. Donner K., Hemila S., Kalamkarov G., Koskelainen A., Pogozheva I. and Rebrik T. Sulfhydryl binding reagents increase the conductivity of the light-sensitive channel and inhibit phototransduction in retinal rods. Experimental Eye Research. 51(1):97-105, 1990.
   Abstract.  The mechanisms by which sulfhydryl (SH-) binding reagents modulate the light-sensitive conductance of retinal rods were investigated by current recording from single rods, by patch clamp recording from the plasma membrane of the rod outer segment (ROS), and by biochemical study of their effects on the light-induced hydrolysis of cyclic GMP. The electrophysiology, as well as measurements of the reagents' ability to traverse the ROS plasma membrane, was done on amphibian (Rana and Ambystoma) rods, and the biochemistry on bovine rods. The main SH-reagents used were N-ethyl-maleimide (NEM) and iodoacetamide (IAA). Both transiently increased rod current, but part of the large current could not be turned off by light. After a few minutes' exposure, NEM, but not IAA, caused a continuous decay of the rod's light sensitivity. In patch-clamp recordings from the ROS plasma membrane, the reagents increased conductivity both in the presence and absence of cGMP, consistent with the observation that the drug-induced current increase in intact rods involved both light-sensitive and light-insensitive components. In vitro, NEM was found to be a powerful inhibitor of cGMP hydrolysis, which can explain the gradual loss of light sensitivity in the rod and could initially contribute to the increased dark current via elevated cGMP levels. Thus, SH-reagents act both by modifying the light-sensitive channel and by inhibiting phototransduction inside the rod.
5. Pogozheva I.D., Shevchenko T., Livshits V.A. and Kalamkarov G.R. Influence of sylfhydryl group modification on arrestin interaction with phosphorylated rhodopsin. Biologicheskie membrani (USSR), 6: 1248-1255, 1989.
   Abstract.  3.4±0.2 mol of SH-groups per mol of protein were found in the molecule of arrestin.  These SH-goupd can be modified by 5,5'-dithiobis-2-nitrobenzoic acid and 1-oxyl-2,2,6,6-tetramethyl-3-(piperidinomethyl)-4-chloromercuri-D^5-dehydropiperidine under both native and denaturation conditions. The rate of the SH group modification in arrestine is much slower than in the transducin a-subunit. Modification of SH groups in arrestin, unlike those of transducin, entails no alteration of its binding with bleached phosphorylated rhodopsin.  The differences may be due to replacement of Cys³⁴⁷ in transducin by Ala³⁸⁸ in arrestin.  Modification of 4 SH groups in phosphorylated rhodopsin with spin labels is not essential for its interaction with arrestin.
6. Pogozheva I.D., Shevchenko T., Livshits V.A. and Kalamkarov G.R. Interaction of rhodopsin and arrestin:the role of rhodopsin phosphorylation.  Biologicheskie membrani (USSR), 6: 1237-1247, 1989.
   Abstract. The binding of arrestin with several forms of rhodopsin photolysis intermediates has been investigated. The dissociation constants are 0.7-2 mM for unphosphorylated and phosphorylated rhodopsin in their 11-cis retinal states, 11,7±0.2 mM and 0,7±0.2 mM for metarhodopsin II and phosphorylated metarhodopsin II, 6.3±2.5 mM and 5.3±1.5 mM for opsin and its phosphorylated form. The binding sites of the phosphorylated and unphosphorylated rhodopsin differ in their affinity for arrestin, as well as in the pH and ionic strength dependences of the extent of arrestin binding. Using the spin label technique itwas shown the the degree of immobilization of arrestin in these binding sites is also different. Only binding of arrestin to phosphorylated metarhodopsin II results in the inhibition of cGMP phosphodiesterase activity in the rod outer segments. The arrestin affinity strongly decreases after papainolysis of the phosphorylated rhodopsin. It was shown that metarhodopsin II phosphorylation results not only in the lower K_d values of arrestin binding, but also in a higher K_d of metarhodopsin-transducin-GDP complex.
7. Pogozheva I.D., Kuznetsov V.A., Fedorovich I.B. and Ostrovskii M.A. ESR Saturation transfer study of photoinduced changes in the hydrophilic regions of rhodopsin. Biologicheskie membrani (USSR), 2: 880-896, 1985.
   Abstract. Rhodopsin aggregation and conformational mobility of its polar regions near Cys¹⁴⁰ and Cys³¹⁶ have been studied using spin labels strongly and weakly immobilized on the protein. Photoinduced changes in ESR spectra of both labels were observed and shown to correspond to metarhodopsin II formation.  The magnitude of these changes is directly proportional to the rhodopsin photobleaching degree and decreases with the pH rise and with the protein solubilization by digitonin. No photoinduced association of rhodopsin was found under experimentsl conditions. The observed increase in label's mobility is caused by structural rearrangements of their binding sites, the changes being more pronounced near Cys¹⁴⁰. The conformational mobility if the hydriphilic regions decreases again on decay of metarhodopsin II.
8. Pogozheva I.D., Kuznetsov V.A., Fedorovich I.B. and Ostrovskii M.A. Conformational mobility and interactions of rhodopsin domains. Biologicheskie membrani (USSR), 2: 897-905, 1985.
   Abstract.  To get an insight into the structure of the rhodospin hydrophilic regions and the character of its changes in the metarhodopsin-II formation, the rotational mobilities of spin labels attached to Cys¹⁴⁰ and Cys³¹⁶ protein residues have been studies as function of rhodopsin cleavage degree by papain in the dark and under illumination.  The proteolytic removal of C-terminus Cys³²²-Ala³⁴⁸ and of loop Glu²³⁷-Ala²⁴¹ was shown to decrease the mobility of strongly  immobilized labels and to eliminate sterical hindrances for rotation of weakly immobilized labels.  Photoinduced conformational changes in both label's binding sites are conserved under such structural perturbation of rhodopsin hydrophylic regions, and their magnitudes increase as the label mobilities in these sites decrese on proteolysis.  The results obtained suggest that C-terminus of rhodopsin is packed on the protein surface so that it contacts with the Cys¹⁴⁰ region, whereas conformational rearrangements on the metarhodopsin II formation correspond to loosening of protein hydrophilic regions and to changes in spatial localization of cleaved parts of rhodopsin.  Transmission of conformational changes from retinal to the protein intracellular surface is provided by noncovalent interactions in hydrophobic protein regions.
9. Pogozheva I.D., Kuznetsov V.A., Livshits V.A. and Kuznetsov A.N. Influence of a magnetic field on the aggregation of rhodopsin molecules during photooxidation of photoreceptor membranes. Biofizika (USSR). 28(2):336-337, 1983.
10. Pogozheva I.D., Kuznetsov V.A., Fedorovich I.B., Livshits V.A., Ostrovskii MA. Manoshkina N.B. Aggregation of rhodopsin molecules during damaging exposure of photoreceptor membranes to light. Biofizika (USSR). 26(4):692-700, 1981.
    Abstract. Injuring light induced structural changes in rod outer segment (ROS) membranes are studied using "ST EST spectroscopy" for spin labelled rhodopsin, ESR of lipid spin label and SDS gel-electrophoresis. Free SH-group content of rhodopsin and lipid peroxidation level were simultaneously determined as well. A decrease of rotational mobility of rhodopsin in ROS induced by prolonged illumination is shown to result from irreversible protein aggregation caused by disulfide bond formation between "hydrophobic" SH-groups of rhodopsin. Some decrease of lipid microviscosity and degree of order are found, in contrast to considerable rise in microviscosity due to Fe2+-ascorbate induced lipid peroxidation of ROS membranes. Lipid oxidation is found to accelerate protein aggregation which in its turn influences the state of lipid bilayer.
11. Pogozheva I.D., Fedorovich I.B., Ostrovskii M.A. and Emanuel' N.M. Photodamage of rhodopsin molecule. Oxidation of SH-group. Biofizika.(USSR). 26(3):398-403, 1981.
    Abstract. Illumination of rod outer segments with bright visible light results in the oxidation of both protein and lipid components of the photoreceptor membrane. The oxidation degree depends on the intensity and time of illumination. The inhibitors of free radical processes completely inhibit lipid oxidation and somewhat decrease protein oxidation. Photooxidation systems of lipids and rhodopsin also react differently to oxygen content in the incubation medium. Retinal is the photosensitizer of the oxidation of the photoreceptor membrane components.
12. Pogozheva I.D., Kuznetsov V.A., Livshits V.A., Fedorovich I.B. and Ostrovskii MA. Reversible pH-dependent aggregation of rhodopsin molecules in photoreceptor membranes. Doklady Akademii Nauk SSSR (Proc. Acad. Sci. USSR).  260(5):1254-1258, 1981.
    Abstract.   The mobility of spin labels  strongly immobilized on frog and bovine rhodopsin molecules in photoreceptor membrane reversibly decreases at pH<5 and pH near 7.0-7.4.  Thes could be related with the aggregation of spin-labeled rhodopsin molecules.  In the same conditions the spin-spin interactions of weakly immobilized labels increase at label:rhodopsin mol ratio 0.75-1 and do not change at the ratio 0.15, that also suggest the aggregation of  rhodospin molecules.  The dimer fromation has been observed on SDS electrophoresis of photoreceptor membrane after their  fixation with glutaraldehyde (1 mM, 15 min, 20 degrees C, pH 7-7.4).  The treatment of photoreceptor membrane by 1.5% digitonin prevents the aggregation.  The aggregation increases at the ionic srength lower and higher, than 0.2.