Biomedical subjects

M D Bownds

Publications and source records attributed to M D Bownds.

At least 19 recordsLinked to original sources

PubMed2000

The gain of rod phototransduction: reconciliation of biochemical and electrophysiological measurements.

We have resolved a central and long-standing paradox in understanding the amplification of rod phototransduction by making direct measurements of the gains of the underlying enzymatic amplifiers. We find that under optimized conditions a single photoisomerized rhodopsin activates transducin molecules and phosphodiesterase (PDE) catalytic subunits at rates of 120-150/s, much lower than indirect estimates from light-scattering experiments. Further, we measure the Michaelis constant, Km, of the rod PDE activated by transducin to be 10 microM, at least 10-fold lower than published estimates. Thus, the gain of cGMP hydrolysis (determined by kcat/Km) is at least 10-fold higher than reported in the literature. Accordingly, our results now provide a quantitative account of the overall gain of the rod cascade in terms of directly measured factors.

Animals↗

PubMed1996

Phosphorylation of non-bleached rhodopsin in intact retinas and living frogs.

The photoresponse in retinal photoreceptors begins when a molecule of rhodopsin is excited by a photon of light. Photoexcited rhodopsin activates an enzymatic cascade including the G-protein transducin and cyclic GMP phosphodiesterase. As a result, cytoplasmic cyclic GMP concentration is decreased and the photoresponse is initiated. This process is terminated when rhodopsin is phosphorylated by rhodopsin kinase and subsequently blocked by a protein called arrestin. It has been noted by several investigators that light can cause phosphorylation of not only photoexcited but also non-excited rhodopsin in rod photoreceptors. A goal of this study was to determine how much non-bleached rhodopsin is phosphorylated. To determine how the structural integrity of the photoreceptor influences the extent of non-bleached rhodopsin phosphorylation, we studied the reaction in electropermeabilized rod outer segments, in rod outer segments still attached to isolated retinas and in living frogs. In the first two preparations, we found that the maximum extent of non-bleached rhodopsin phosphorylation was approximately 1% of the total rhodopsin pool. In living frogs, the maximal amount of non-bleached rhodopsin phosphorylation was approximately 2% of the total rhodopsin pool and occurred after prolonged illumination by the relatively dim light intensity of 20 lux. These data appear to exclude models for light adaptation that postulate high levels of phosphorylation of non-bleached rhodopsin in rod photoreceptors.

Animals↗

PubMed1995

Rhodopsin kinase inhibition by recoverin. Function of recoverin myristoylation.

Recoverin is a Ca(2+)-binding protein that may play a role in vertebrate photoreceptor light adaptation by imparting Ca2+ sensitivity to rhodopsin kinase. It is heterogeneously acylated (mostly myristoylated) at its amino-terminal glycine. Recent studies have shown that recoverin myristolyation is necessary for its Ca(2+)-dependent membrane association and cooperative Ca2+ binding. We have addressed several issues concerning the role of recoverin myristoylation with respect to inhibition of rhodopsin kinase. We find that 1) myristoylation of recoverin is not necessary for inhibition of rhodopsin kinase, 2) myristoylation of recoverin induces a cooperative Ca(2+)-dependence for rhodopsin kinase inhibition, and 3) each Ca(2+)-binding site on the nonmyristoylated recoverin partially inhibits rhodopsin kinase. The available data suggest that the functions of recoverin myristoylation in the living rod are to induce a sharp Ca2+ dependence of rhodopsin kinase inhibition and to bring this dependence into the rod's physiological Ca2+ concentration range.

Animals↗

PubMed1995

Inhibition of rhodopsin kinase by recoverin. Further evidence for a negative feedback system in phototransduction.

Recoverin is a 23-kDa Ca(2+)-binding protein found predominantly in vertebrate photoreceptor cells. Recent electrophysiological and biochemical studies suggest that recoverin may regulate the photoresponse by inhibiting rhodopsin phosphorylation. We find in both cell homogenates and reconstituted systems that the inhibition of rhodopsin phosphorylation by recoverin occurs over a significantly higher free Ca2+ range than previously reported. Half-maximal inhibition occurs at 1.5-3 microM free Ca2+ and is cooperative with a Hill coefficient of approximately 2. Measurements of transducin activation demonstrate that this inhibition prolongs the lifetime of catalytically active rhodopsin. Ca(2+)-recoverin directly inhibits rhodopsin kinase activity, and Ca(2+)-dependent binding of recoverin to rod outer segment membranes is not required for its action. Extrapolation of the in vitro data to in vivo conditions based on simple mass action calculations places the Ca(2+)-recoverin regulation within the physiological free Ca2+ range in intact rod outer segment. The data are consistent with a model in which the fall in free Ca2+ that accompanies rod excitation exerts negative feedback by relieving inhibition of rhodopsin phosphorylation.

Animals↗

PubMed1995

An effector site that stimulates G-protein GTPase in photoreceptors.

Heterotrimeric G-proteins mediate between receptors and effectors, acting as molecular clocks. G-protein interactions with activated receptors catalyze the replacement of GDP bound to the alpha-subunit with GTP. alpha-Subunits then modulate the activity of downstream effectors until the bound GTP is hydrolyzed. In several signal transduction pathways, including the cGMP cascade of photoreceptor cells, the relatively slow GTPase activity of heterotrimeric G-proteins can be significantly accelerated when they are complexed with corresponding effectors. In the phototransduction cascade the GTPase activity of photoreceptor G-protein, transducin, is substantially accelerated in a complex with its effector, cGMP phosphodiesterase. Here we characterize the stimulation of transducin GTPase by a set of 23 mutant phosphodiesterase gamma-subunits (PDE gamma) containing single alanine substitutions within a stretch of the 25 C-terminal amino acid residues known to be primarily responsible for the GTPase regulation. The substitution of tryptophan at position 70 completely abolished the acceleration of GTP hydrolysis by transducin in a complex with this mutant. This mutation also resulted in a reduction of PDE gamma affinity for transducin, but did not affect PDE gamma interactions with the phosphodiesterase catalytic subunits. Single substitutions of 7 other hydrophobic amino acids resulted in a 50-70% reduction in the ability of PDE gamma to stimulate transducin GTPase, while substitutions of charged and polar amino acids had little or no effect. These observations suggest that the role of PDE gamma in activation of the transducin GTPase rate may be based on multiple hydrophobic interactions between these molecules.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1994

Regulation of transducin GTPase activity in bovine rod outer segments.

The photoreceptor G-protein, transducin, belongs to the class of heterotrimeric GTP-binding proteins that transfer information from activated seven-span membrane receptors to effector enzymes or ion channels. Like other G-proteins, transducin acts as a molecular clock. It is activated by photoexcited rhodopsin which catalyzes the exchange of transducin-bound GDP for GTP and then stays active until bound GTP is hydrolyzed by an intrinsic GTPase activity. Our previous study on the components of the amphibian phototransduction cascade (Arshavsky, V. Y., and Bownds, M. D. (1992) Nature 357, 416-417) has shown that transducin GTPase can be significantly accelerated by the target enzyme, cGMP phosphodiesterase (PDE), and more specifically its gamma-subunit (PDE gamma). Here we report that an analogous mechanism is present in bovine photoreceptors. Addition of recombinant PDE gamma to the test photoreceptor membranes which retain transducin but are depleted of endogenous PDE causes a significant acceleration of transducin GTPase activity. A similar effect was observed with the PDE holoenzyme, but not with the complex of PDE alpha- and beta-subunits prepared by a limited proteolysis of PDE with trypsin. The activating effect of PDE gamma is increased as test membrane concentration increases, exceeding 20-fold at rhodopsin concentrations over 80 microM and approaching the rate of the photoresponse turnoff. This suggests either that photoreceptor membranes contain a further factor which is essential for PDE-dependent regulation of transducin-bound GTP hydrolysis or that components of the phototransduction cascade interact in a cooperative manner. We also report that the GTPase-activating epitope is located within the C-terminal third of PDE gamma: the peptide corresponding to the 25 C-terminal amino acid residues of PDE gamma can accelerate transducin GTPase almost as well as the full-length PDE gamma. A part of the GTPase activating epitope is located within the 3 C-terminal amino acid residues: the truncation PDE gamma mutant lacking these residues accelerates transducin GTPase considerably less than the whole length PDE gamma.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1994

cGMP binding sites on photoreceptor phosphodiesterase: role in feedback regulation of visual transduction.

A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transduction lowers the cGMP binding affinity for noncatalytic sites on PDE and accelerates the dissociation of cGMP from these sites. These sites can exist in three states: high affinity (Kd = 60 nM) for the nonactivated PDE, intermediate affinity (Kd approximately 180 nM) when the enzyme is activated in a complex with transducin, and low affinity (Kd > 1 microM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by transducin causes a 10-fold increase in the rate of cGMP dissociation from one of the two noncatalytic sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP dissociation rate from both sites > 50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of light-adapted photoreceptors.

Animals↗

PubMed1994

Rod outer segment structure influences the apparent kinetic parameters of cyclic GMP phosphodiesterase.

Cyclic GMP hydrolysis by the phosphodiesterase (PDE) of retinal rod outer segments (ROS) is a key amplification step in phototransduction. Definitive estimates of the turnover number, kcat, and of the Km are crucial to quantifying the amplification contributed by the PDE. Published estimates for these kinetic parameters vary widely; moreover, light-dependent changes in the Km of PDE have been reported. The experiments and analyses reported here account for most observed variations in apparent Km, and they lead to definitive estimates of the intrinsic kinetic parameters in amphibian rods. We first obtained a new and highly accurate estimate of the ratio of holo-PDE to rhodopsin in the amphibian ROS, 1:270. We then estimated the apparent kinetic parameters of light-activated PDE of suspensions of disrupted frog ROS whose structural integrity was systematically varied. In the most severely disrupted ROS preparation, we found Km = 95 microM and kcat = 4,400 cGMP.s-1. In suspensions of disc-stack fragments of greater integrity, the apparent Km increased to approximately 600 microM, though kcat remained unchanged. In contrast, the Km for cAMP was not shifted in the disc stack preparations. A theoretical analysis shows that the elevated apparent Km of suspensions of disc stacks can be explained as a consequence of diffusion with hydrolysis in the disc stack, which causes active PDEs nearer the center of the stack to be exposed to a lower concentration of cyclic GMP than PDEs at the disc stack rim. The analysis predicts our observation that the apparent Km for cGMP is elevated with no accompanying decrease in kcat. The analysis also predicts the lack of a Km shift for cAMP and the previously reported light dependence of the apparent Km for cGMP. We conclude that the intrinsic kinetic parameters of the PDE do not vary with light or structural integrity, and are those of the most severely disrupted disc stacks.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Noncatalytic cGMP-binding sites of amphibian rod cGMP phosphodiesterase control interaction with its inhibitory gamma-subunits. A putative regulatory mechanism of the rod photoresponse.

The cGMP phosphodiesterase (PDE) of retinal rods plays a central role in phototransduction. Illumination leads to its activation by a rod G-protein (Gt, transducin), thus causing a decrease in intracellular cGMP concentration, closure of plasma membrane cationic channels gated by cGMP, and development of the photoresponse. The PDE holoenzyme is an alpha beta gamma 2 tetramer. The alpha- and beta-subunits each contain one catalytic and one, or possibly two, noncatalytic cGMP-binding sites. Two identical gamma-subunits serve as protein inhibitors of the enzyme. Their inhibition is removed when they bind to Gt-GTP during PDE activation. Here we report that the noncatalytic cGMP-binding sites regulate the binding of PDE alpha beta with PDE gamma and as a result determine the mechanism of PDE activation by Gt. If the noncatalytic sites are empty, Gt-GTP physically removes PDE gamma from PDE alpha beta upon activation. Alternatively, if the noncatalytic sites are occupied by cGMP, Gt-GTP releases PDE gamma inhibitory action but remains bound in a complex with the PDE heterotetramer. The kinetic parameters of activated PDE in these two cases are indistinguishable. This mechanism appears to have two implications for the physiology of photoreceptor cells. First, the tight binding of PDE gamma with PDE alpha beta when the noncatalytic sites are occupied by cGMP may be responsible for the low level of basal PDE activity observed in dark-adapted cells. Second, occupancy of the noncatalytic sites ultimately controls the rate of PDE inactivation (cf. Arshavsky, V. Yu., and Bownds, M. D. (1992) Nature 357, 416-417), for the GTPase activity that terminates PDE activity is slower when these sites are occupied and Gt stays in a complex with PDE holoenzyme. In contrast GTPase acceleration is maximal when the noncatalytic sites are empty and Gt-PDE gamma dissociates from PDE alpha beta. Because cGMP levels are known to decrease upon illumination over a concentration range corresponding to the binding constants of the noncatalytic sites, the binding might be involved in determining the lifetime of activated PDE, after a single flash and/or during dark adaptation.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1992

Regulation of deactivation of photoreceptor G protein by its target enzyme and cGMP.

The photoreceptor G protein, transducin, is one of the class of heterotrimeric G proteins that mediates between membrane receptors and intracellular enzymes or ion channels. Light-activated rhodopsin catalyses the exchange of GDP for GTP on multiple transducin molecules. Activated transducin then stimulates cyclic GMP phosphodiesterase by releasing an inhibitory action of the phosphodiesterase gamma-subunits. This leads to a decrease in cGMP levels in the rod, and closure of plasma membrane cationic channels gated by cGMP. In this and other systems, turn-off of the response requires the GTP bound to G protein to be hydrolysed by an intrinsic GTPase activity. Here we report that the interaction of transducin with cGMP phosphodiesterase, specifically with its gamma-subunits, accelerates GTPase activity by several fold. Thus the gamma-subunits of the phosphodiesterase serve a function analogous to the GTPase-activating proteins that regulate the class of small GTP-binding proteins. The acceleration can be partially suppressed by cGMP, most probably through the non-catalytic cGMP-binding sites of phosphodiesterase alpha and beta-subunits. This cGMP regulation may function in light-adaptation of the photo-response as a negative feedback that decreases the lifetime of activated cGMP phosphodiesterase as light causes decreases in cytoplasmic cGMP.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMed1991

cGMP suppresses GTPase activity of a portion of transducin equimolar to phosphodiesterase in frog rod outer segments. Light-induced cGMP decreases as a putative feedback mechanism of the photoresponse.

In rod photoreceptor cells, the light response is triggered by an enzymatic cascade that causes cGMP levels to fall: excited rhodopsin (Rho*)----rod G-protein (transducin, Gt)----cGMP-phosphodiesterase (PDE). This results in the closure of plasma membrane channels that are gated by cGMP. PDE activation by Gt occurs when GDP bound to the alpha-subunit of Gt (Gt alpha) is exchanged with free GTP. The interaction of Gt alpha-GTP with the gamma-subunits of PDE releases their inhibitory action and causes cGMP hydrolysis. Inactivation is thought to be caused by subsequent hydrolysis of Gt alpha-GTP by an intrinsic Gt-GTPase activity. Here we report that there are two portions of Gt in frog rod outer segments (ROS) expressing different rates of GTP hydrolysis: 19.5 +/- 3 mmol of Gt/mol of Rho, equivalent to that amount which participates in PDE activation, hydrolyzing GTP at a rate of approximately 0.6 turnover/s ("fast") and the remaining Gt (80.5 +/- 3 mmol/mol Rho) hydrolyzing GTP at a rate of 0.058 +/- 0.009 turnover/s. Fast GTPase activity is abolished in the presence of cGMP. This effect occurs over the physiological range of cGMP concentration changes in ROS, half-saturating at approximately 2 microM and saturating at 5 microM cGMP. cGMP-dependent suppression of GTPase is specific for cGMP; cAMP in millimolar concentration does not affect GTPase, while the poorly hydrolyzable cGMP analogue, 8-bromo-cGMP, mimics the effect. GTPase regulation by cGMP is not affected by Ca2+ over the concentration range 5-500 nM, which spans the physiological changes in cytoplasmic Ca2+ in rod cells. We suggest that the fast cGMP-sensitive GTPase activity is a property of the Gt that activates PDE. In this model, cGMP serves not only as a messenger of excitation but also modulates GTPase activity, thereby mediating negative feedback regulation of the pathway via PDE turnoff: a light-dependent decrease in cGMP accelerates the hydrolysis of GTP bound to Gt, resulting in the rapid inactivation of PDE.

Animals↗

PubMed1991

Dim background light and Cerenkov radiation from 32P block reversal of rhodopsin phosphorylation in intact frog retinal rods.

The phosphorylation of photoexcited rhodopsin (Rho*) is thought to inactivate this receptor by inhibiting its interaction with the GTP-binding protein transducin (Gt). Here we report that the time course of phosphorylation-dephosphorylation after bright illumination of intact rod outer and inner segments (ROS-RIS) incubated in 33Pi can be altered if the ROS-RIS are first exposed to levels of dim illumination that cause light adaptation in these ROS-RIS. The dephosphorylation of greater than 10(7) phosphorylated rhodopsin molecules/ROS following a bright flash can be blocked by prior dim continuous illumination (generating 10(3) Rho*/ROS/s) that cumulatively bleaches approximately 10(5) rhodopsin molecules/ROS. The phenomenon has not been previously noted because these low levels of light are emitted as a result of Cerenkov radiation from the 32P isotope that is usually employed to monitor rhodopsin phosphorylation. The inhibition of rhodopsin dephosphorylation by dim conditioning illumination is observed in intact ROS-RIS but is lost when ROS-RIS are electropermeabilized or fragmented.

Animals↗

PubMed1990

Transducin activation in electropermeabilized frog rod outer segments is highly amplified, and a portion equivalent to phosphodiesterase remains membrane-bound.

An electropermeabilized preparation of frog retinal rod outer segments (ROS) has been developed to examine the light sensitivity and amplification of visual transduction reactions in a minimally disturbed environment. Electropermeabilized ROS are indistinguishable from whole and osmotically intact ROS in the light microscope and retain 3-fold more protein than mechanically disrupted ROS. They differ from mechanically fragmented ROS in several respects. Illumination results in more amplified activation of the GTP-binding protein transducin (Gt) than previously observed: bleaching as little as approximately 1 rhodopsin molecule (Rho*) in every 10 disks within a single ROS activates 37,000 molecules of Gt per Rho*, equivalent to 70% of the light-activatable Gt present on a single disk face. This amplification is maintained over approximately 1 decade of light intensity but drops sharply as disk faces begin to absorb a second photon. Lower amplification is observed in fragmented ROS and derives from the fact that physical disruption of ROS causes Gt to bind GTP and elute from the membrane, thus decreasing the amount remaining and available for light activation. Illumination of electropermeabilized ROS in the presence of GTP or of the nonhydrolyzable substrate guanosine 5'-(gamma-thio)triphosphate (GTP gamma S) causes redistribution of Gt: an amount (approximately 20 mmol/mol Rho) equivalent to the amount of inhibitory gamma subunit of phosphodiesterase (PDE) remains internal and bound to nucleotide, and the remaining activated Gt diffuses out in a manner graded with light intensity. This suggests that PDE activation by Gt alpha may not require dissociation of Gt alpha bound to the gamma subunit of PDE in a form than can elute from ROS. Two further differences between electropermeabilized and mechanically disrupted ROS are noted: the addition of ATP to electropermeabilized ROS does not affect the light sensitivity or kinetics of the GTP binding reaction, and a specificity for light-induced GTP versus GDP binding is observed.

Animals↗

PubMed1990

Light activation of one rhodopsin molecule causes the phosphorylation of hundreds of others. A reaction observed in electropermeabilized frog rod outer segments exposed to dim illumination.

A rhodopsin phosphorylation reaction that occurs with high-gain is observed if measurements are made in electropermeabilized frog rod outer segments (ROS) stimulated by a dim flash of light in the operating range of the photoreceptor. Flashes of light exciting 1000 or fewer of the 3 x 10(9) rhodopsins present/ROS results in the incorporation of 1400 phosphates from ATP into the rhodopsin pool for each excited rhodopsin (Rho*). This amplification decreases with increasing light intensity, falling most sharply after each disk has absorbed one photon. The high-gain reaction is lost if the ROS are broken into vesicles by shearing, leaving a low-gain rhodopsin phosphorylation characterized in previous studies using brighter illumination. The high-gain but not the low-gain phosphorylation appears to be regulated by G-protein and by calcium levels in the range over which intracellular calcium changes when rod photoreceptors are illuminated. Kinetic measurements made on the phosphorylation observed at higher light intensities shows that it initially occurs rapidly enough for a role in terminating the photoresponse. The high-gain phosphorylation observed at lower light intensities may play a global role in regulating light-adaptation of the rod photoreceptor, and its existence suggests that a search for a similar high-gain modification in systems using the homologous beta-adrenergic or muscarinic acetylcholine receptors might be rewarding.

Adenosine Triphosphate↗

PubMed1989

Cyclic GMP levels and membrane current during onset, recovery, and light adaptation of the photoresponse of detached frog photoreceptors.

We have used a preparation of isolated, intact rod photoreceptors to correlate the effects of flash illumination on the intracellular cyclic GMP content and the membrane current. We find that the recovery of cyclic GMP levels after brief flash illumination requires approximately twice as much time as the recovery of the membrane current. In contrast, the subsecond kinetics of the cyclic GMP response to light are faster than the kinetics of membrane current suppression. Both cyclic GMP and the membrane current show graded responses to a wide range of flash intensities; however, in a low Ca2+-Ringer's solution, dim flashes can trigger a decrease in cyclic GMP concentration with no corresponding decrease in membrane current. These results suggest that either other factors can regulate the membrane current, or that measurements of total cellular cyclic GMP do not accurately reflect dynamic changes in cyclic GMP concentration in the vicinity of the light-regulated channel. Changes in cyclic GMP concentration in the presence of background illumination exhibit adaptational behavior similar to that observed in a light-adapted photoresponse: acceleration in the response kinetics and a decrease in response amplitude. That this result is observed in rods depleted of internal Ca2+ suggests a Ca2+-independent mechanism by which background illumination can accelerate the cyclic GMP response.

Acclimatization↗

PubMed1989

Stimulation of protein phosphorylations in frog rod outer segments by protein kinase activators. Suppression of light-induced changes in membrane current and cGMP by protein kinase C activators.

Addition of protein kinase C activators to electropermeabilized frog rod photoreceptors enhances the phosphorylation of proteins with molecular masses of 54, 24, 19, 17, 12, and 11 kDa. The latter two correspond to components I and II, which are also phosphorylated by cyclic nucleotide-dependent protein kinase. Stimulation of phosphorylation by the protein kinase C activator oleoylacetylglycerol (OAG) is half-maximal at 7.7 microM OAG and is reduced by the protein kinase C inhibitor H-7. In contrast with earlier observations, no effects of calcium, calmodulin, or insulin on protein phosphorylations are observed. We find evidence for only three protein kinases in rod outer segments: a protein kinase C-like activity, cAMP-dependent protein kinase, and rhodopsin kinase. With the exception of components I and II, the substrate proteins for each kinase are distinct. Treatment of intact rods with OAG decreases the amplitude of the photoresponse and dark levels of cGMP up to 40%, as well as depressing the light-stimulated decrease in cGMP levels. These effects are observed between 0.1 and 1 microM OAG. The data suggest that OAG-sensitive reactions may modulate pathways that support the light response.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1989

Calcium regulates some, but not all, aspects of light adaptation in rod photoreceptors.

The role of calcium as a regulator of light adaptation in rod photoreceptors was examined by manipulation of the intracellular Ca2+ concentration through the use of the calcium ionophore A23187 and external Ca2+ buffers. These studies utilized suspensions of isolated and purified frog rod outer segments that retain their mitochondria-rich inner segments (OS-IS). Three criteria of the dark- and light-adapted flash response were characterized as a function of the Ca2+ concentration: (a) the time to peak, (b) the rate of recovery, and (c) the response amplitude or sensitivity. For all Ca2+ concentrations examined, the time to peak of the flash response was accelerated in the presence of background illumination, suggesting that mechanisms controlling this aspect of adaptation are independent of the Ca2+ concentration. The recovery kinetics of the flash response appeared to depend on the Ca2+ concentration. In 1 mM Ca2+-Ringer's and 300 nM Ca2+-Ringer's + A23187, background illumination enhanced the recovery rate of the response; however, in 10 and 100 nM Ca2+-Ringer's + A23187, the recovery rates were the same for dark- and light-adapted responses. This result implies that a critical level of Ca2+ may be necessary for background illumination to accelerate the recovery of the flash response. The sensitivity of the flash response in darkness (SDF) was dependent on the Ca2+ concentration. In 1 mM Ca2+-Ringer's SDF was 0.481 pA per bleached rhodopsin (Rh*); a background of four Rh*/s decreased SDF by half (Io). At 300 nM Ca2+ + A23187, SDF was reduced to 0.0307 pA/Rh* and Io increased to 60 Rh*/s. At 100 nM Ca2+ + A23187, SDF was reduced further to 0.0025 pA/Rh* and Io increased to 220 Rh*/s. In 10 nM Ca2+ + A23187, SDF was lowered to 0.00045 pA/Rh* and Io raised to 760 RhI/s. Using these values of SDF and Io for each respective Ca2+ concentration, the dependence of the flash sensitivity on background intensity could be described by the Weber-Fechner relation. Under low Ca2+ conditions + A23187, bright background illumination could desensitize the flash response. These results are consistent with the idea that the concentration of Ca2+ may set the absolute magnitude of response sensitivity in darkness, and that there exist mechanisms capable of adapting the photoresponse in the absence of significant changes in cytoplasmic Ca2+ concentration.

Adaptation, Ocular↗

PubMed1987

Identification of frog photoreceptor plasma and disk membrane proteins by radioiodination.

Several functions have been identified for the plasma membrane of the rod outer segment, including control of light-dependent changes in sodium conductance and a sodium-calcium exchange mechanism. However, little is known about its constituent proteins. Intact rod outer segments substantially free of contaminants were prepared in the dark and purified on a density gradient of Percoll. Surface proteins were then labeled by lactoperoxidase-catalyzed radioiodination, and intact rod outer segments were reisolated. Membrane proteins were identified by polyacrylamide gel electrophoresis and autoradiography. The surface proteins labeled included rhodopsin, the major membrane protein, and 12 other proteins. Several control experiments indicated that the labeled proteins are integral membrane proteins and that label is limited to the plasma membrane. To compare the protein composition of plasma membrane with that of the internal disk membrane, purified rod outer segments were lysed by hypotonic disruption or freeze-thawing, and plasma plus disk membranes were radioiodinated. In these membrane preparations, rhodopsin was the major iodinated constituent, with 12 other proteins also labeled. Autoradiographic evidence indicated some differences in protein composition between disk and plasma membranes. A quantitative comparison of the two samples showed that labeling of two proteins, 24 kilodaltons (kDa) and 13 kDa, was enriched in the plasma membrane, while labeling of a 220-kDa protein was enriched in the disk membrane. These plasma membrane proteins may be associated with important functions such as the light-sensitive conductance and the sodium-calcium exchanger.

Animals↗