Ca2+ buffer sites in intact bovine rod outer segments: Introduction to a novel optical probe to measure ionic permeabilities in suspensions of small particles

Summary The nature of the Ca²⁺ buffer sites in intact rod outer segments isolated from bovine retinas (ROS) was investigated. The predominant Ca²⁺ buffer in intact ROS was found to be negatively charged groups confined to the surface of the disk membranes. Accordingly, Ca²⁺ buffering in ROS was strongly influenced by the electrostatic surface potential. The concentration of Ca²⁺ buffer sites was about 30mm, 80% of which were located at the membrane surface in the intradiskal space. A comparison with observations in model systems suggests that phosphatidylserine is the major Ca²⁺ buffer site in ROS. Protons and alkali cations could replace Ca²⁺ as mobile counterions for the fixed negatively charged groups. At physiological ionic strength, the total number of these diffusible, but osmotically inactive, counterions was as large as the number of osmotically active cations in ROS. The surface potential is dependent on the concentration of cations in ROS and can be measured with the optical dye neutral red. Addition of cations to the external solution led to the release of the internally bound dye as the cations crossed the outer membrane. The chemical and spectral properties of the dye enable its use as a real-time indicator of cation transport across the outer envelope of small particles in suspension. In this study, the dye method is illustrated by the use of well-defined ionophores in intact ROS and in liposomes. In the companion paper this method is used to describe the cation permeabilities native to ROS.     

Silver ions induce a rapid Ca2+ release from isolated intact bovine rod outer segments by a cooperative mechanism

Summary Micromolar concentrations of silver ion activate large Ca²⁺ fluxes across the plasma membrane of intact rod outer segments isolated from bovine retinas (intact ROS). The rate of Ag⁺-induced Ca²⁺ efflux from intact ROS depended on the Ag⁺ concentration in a sigmoidal manner suggesting a cooperative mechanism with a Hill coefficient between 2 and 3. At a concentration of 50 m Ag⁺ the rate of Ca²⁺ efflux was 7×10⁶ Ca²⁺/outer segment/sec; this represents a change in total intracellular Ca²⁺ by 0.7mm/outer segment/sec. Addition of the nonselective ionophore gramicidin in the absence of external alkali cations greatly reduced the Ag⁺-induced Ca²⁺ efflux from intact ROS, apparently by enabling internal alkali cations to leak out. Adding back alkali cations to the external medium restored Ag⁺-induced Ca²⁺ efflux when gramicidin was present. In the presence of gramicidin, Ag⁺-induced Ca²⁺ efflux from intact ROS was blocked by 50 m tetracaine orl-cis diltiazem, whereas without gramicidin both blockers were ineffective. Bothl-cis diltiazem and tetracaine are blockers of one kinetic component of cGMP-induced Ca²⁺ flux across ROS disk membranes. The ion selectivity of the Ag⁺-induced pathway proved to be broad with little discrimination between the alkali cations Li⁺, Na⁺, K⁺, and Cs⁺ or between Ca²⁺ and Mg²⁺. The properties of the Ag⁺-induced pathway(s) suggest that it may reflect the cGMP-dependent conductance opened in the absence of cGMP by silver ions.     

The isolation of stable cattle rod outer segments with an intact plasma membrane

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter.Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH.The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

Effect of potassium ions and membrane potential on the Na-Ca-K exchanger in isolated intact bovine rod outer segments

Two recent studies reported that Na-Ca exchange in the outer segments of tiger salamander rod photoreceptors (Cervetto, L., Lagnado, L., Perry, R. J., Robinson, D. W., and McNaughton, P. A. (1989) Nature 337, 740-743) and of bovine rod photoreceptors (Schnetkamp, P. P. M., Basu, D. K., and Szerencsei, R. T. (1989) Am. J. Physiol. 257, C153-157) requires and transports K+ in a 4Na/(1Ca+1K) stoichiometry. In this study, we have examined the effects of K+ ions and membrane potential on the kinetics of Na-Ca and Ca-Ca exchange in rod outer segments isolated from bovine retinas. The objective was to establish the ion selectivity and voltage dependence of the different cation binding sites on the Na-Ca-K exchange protein. Potassium ions activated Na-Ca exchange when present on the Ca2+ side, although the extent of activation decreased with decreasing Na+ concentration. Potassium ions inhibited Na-Ca exchange when present on the Na+ side; inhibition arose from competition between Na+ and K+ for a common single cation-binding site. Activation of Na-Ca exchange by K+ displayed a different ion selectivity than that observed for inhibition of Na-Ca exchange by K+. The results are interpreted in terms of a three-site model for the rod Na-Ca-K exchanger. The rate of forward Na-Ca exchange decreased by 1.75-fold for a 60 mV depolarization of the plasma membrane but only at lower Na+ concentrations. The rate of Ca-Ca exchange was not affected by changes in membrane potential.     

The isolation of stable cattle rod outer segments with an intact plasma membrane.

A procedure is described to purify and stabilize cattle rod outer segments with an intact plasma membrane. Three criteria are applied to assess the integrity of the latter. Upon photolysis in these rod outer segments: (1) exogenous ATP cannot phosphorylate rhodopsin located in the disk membrane. (2) Endogenous cofactors (NADPH, NADPH-regenerating system) are still available in the rod cytosol and consequently retinol is the final photoproduct of photolysis of rhodopsin. (3) The rod cytosol can maintain a pH different from that of the medium, since the later stages of rhodopsin photolysis are independent of the medium pH. The stability and homogeneity of the preparation appear to be much better than those of freshly isolated frog rod outer segments, which have been used most frequently so far for experiments on the physiology of rod outer segments. In addition, these cattle rod outer segments remain intact during various manipulations and therefore considerably extend the experimental possibilities when intact rod outer segments are required.     

ATP-dependent calcium uptake activity associated with a disk membrane fraction isolated from bovine retinal rod outer segments

Ca2+ sequestration and release from disks of rod outer segments may play a critical role in visual transduction. An ATP-dependent Ca2+ uptake activity has been identified in association with purified disks of bovine rod outer segments. A crude preparation of osmotically active disks was obtained from rod outer segments by hypoosmotic shock and subsequent flotation on a 5% Ficoll 400 solution. These "crude" disks were further purified by separation into two distinct components by centrifugation in a linear Ficoll gradient. Disks comprised the major component; at least 60% of the protein was rhodopsin. This fraction also contained a Ca2+ uptake activity stimulated approximately 4-fold by ATP. The initial rate was approximately 0.21 nmol of Ca2+ (mg of protein)-1 min-1. Most of the ATP-dependent accumulation of 45Ca2+ was released by the calcium ionophore A23187. The uptake activity was sensitive to vanadate (Ki approximately 20 microM) and insensitive to the mitochondrial Ca2+ uptake inhibitor ruthenium red (10 microM). The ATP-dependent Ca2+ uptake exhibited Michaelis-Menten activation kinetics with respect to [Ca2+] (Km approximately 6 microM). The osmotic properties of the sealed disk membranes were exploited to determine whether the association of Ca2+ transport activity with the disks was merely coincidental. The sedimentation properties of these disks, upon centrifugation on a second Ficoll linear density gradient, varied with the osmolarity of the gradient solution. In several separate gradient solutions of differing osmotic and ionic strengths, the Ca2+ uptake activity always comigrated with the disks. These results indicate that the ATP-dependent Ca2+ uptake activity was physically associated with sealed native disk membranes. The characteristics of the Ca2+ uptake activity suggest that it may play a major role in the regulation of cytosolic Ca2+ levels in rod cells in vivo.     

Ionic blockage of the light-regulated sodium channels in isolated rod outer segments.

We have investigated, with osmotic techniques, the light-regulated Na+ channels in rod outer segments (ROS) and ROS fragments freshly isolated from the frog retina. Values of Na+ permeability (PNa) similar to those observed electrophysiologically in the retina were observed using the osmotic technique (continuous flow) described by Korenbrot and Cone. In the other osmotic techniques that we explored, PNa was greatly diminished, if not completely suppressed; however, we found with these techniques that antioxidant conditions (N2 atmosphere or EDTA) significantly increased PNa, suggesting that the Na+ channels are highly sensitivive to membrane oxidation. Using the continuous flow technique, we investigated the H+ and Ca++ dependence of the Na+ channels and found that both of these ions, at micromolar activities, can block the channels. Raising the external H+ activity decreases PNa (reversibly) in a single "sigmoidal" response with an apparent pKa of 5.8. Similarly, in the presence of the ionophores X537A or A23187 which allow equilibration of Ca++ across membranes, the Na+ channels are blocked when the external Ca++ activity is increased from 10(-7) to 10(-5) M. This high sensitivity to both H+ and Ca++ ions suggests that high field strength anionic sites may exist in or near the Na+ channels and that the channels are blocked when these sites bind H+ or Ca++ ions.     

Characterization of a truncated form of arrestin isolated from bovine rod outer segments.

The inactivation of photolyzed rhodopsin requires phosphorylation of the receptor and binding of a 48-kDa regulatory protein, arrestin. By binding to phosphorylated photolyzed rhodopsin, arrestin inhibits G protein (Gt) activation and blocks premature dephosphorylation, thereby preventing the reentry of photolyzed rhodopsin into the phototransduction pathway. In this study, we isolated a 44-kDa form of arrestin, called p44, from fresh bovine rod outer segments and characterized its structure and function. A partial primary structure of p44 was established by a combination of mass spectrometry and automated Edman degradation of proteolytic peptides. The amino acid sequence was found to be identical with arrestin, except that the C-terminal 35 residues (positions 370-404) are replaced by a single alanine. p44 appeared to be generated by alternative mRNA splicing, because intron 15 interrupts within the nucleotide codon for 369Ser in the arrestin gene. Functionally, p44 binds avidly to photolyzed or phosphorylated and photolyzed rhodopsin. As a consequence of its relatively high affinity for bleached rhodopsin, p44 blocks Gt activation. The binding characteristics of p44 set it apart from tryptic forms of arrestin (truncated at the N- and C-termini), which require phosphorylation of rhodopsin for tight binding. We propose that p44 is a novel splice variant of arrestin that could be involved in the regulation of Gt activation.     

Identification of the sodium-calcium exchanger as the major ricin-binding glycoprotein of bovine rod outer segments and its localization to the plasma membrane

After neuraminidase treatment the Na+/Ca2+ exchanger of bovine rod outer segments was found to specifically bind Ricinus communis agglutinin. SDS gel electrophoresis and Western blotting of ricin-binding proteins purified from rod outer segment membranes by lectin affinity chromatography revealed the existence of two major polypeptides of Mr 215K and 103K, the former of which was found to specifically react with PMe 1B3, a monoclonal antibody specific for the 230-kDa non-neuraminidase-treated Na+/Ca2+ exchanger. Reconstitution of the ricin affinity-purified exchanger into calcium-containing liposomes revealed that neuraminidase treatment had no significant effect on the kinetics of Na+/Ca2+ exchange activation by sodium. We further investigated the density of the Na+/Ca2+ exchanger in disk and plasma membrane preparations using Western blotting, radioimmunoassays, immunoelectron microscopy, and reconstitution procedures. The results indicate that the Na+/Ca2+ exchanger is localized in the rod photoreceptor plasma membrane and is absent or present in extremely low concentrations in disk membranes, as we have previously shown to be the case for the cGMP-gated cation channel. Previous reports describing the existence of Na+/Ca2+ exchange activity in rod outer segment disk membrane preparations may be due to the fusion of plasma membrane components and/or the presence of contaminating plasma membrane vesicles.     

Ionic permeability of the mitochondrial outer membrane

The ionic permeability of the outer mitochondrial membrane (OMM) was studied with the patch clamp technique. Electrical recording of intact mitochondria (hence of the outer membrane (OM)), derived from mouse liver, showed the presence of currents corresponding to low conductances (< 50 pS), as well as of four distinct conductances of 99 pS,152 pS, 220 pS and 307 pS (in 150 mM KCl). The latter were voltage gated, being open preferentially at positive (pipette) potentials. Very similar currents were found by patch clamping liposomes containing the isolated OM derived from rat brain mitochondria. Here a conductance of approximately 530 pS, resembling in its electrical characteristics a conductance already attributed to mitochondrial contact sites (Moran et al. 1990), was also detected. Immunoblot assays of mitochondria and of the isolated OM with antibodies against the outer membrane voltage-dependent anion channel (VDAC) (Colombini 1979), showed the presence of the anion channel in each case. However, the typical electrical behaviour displayed by such a channel in planar bilayers could not be detected under our experimental conditions. From this study, the permeability of the OMM appears different from what has been reported hitherto, yet is more in line with that multifarious and dynamic structure which apparently should belong to it, at least within the framework of mitochondrial biogenesis (Pfanner and Neupert 1990).     

Cation channels in the plasma membrane of retinal rod outer segments activated by cGMP

Properties of cGMP-activated cation channels were investigated on isolated patches of the ROS plasma membrane using the "patch clamp" technique. The channels were shown to be characterized by ideal cation selectivity under physiological conditions and are nearly equally permeable for cations of alkaline metals. At the same time they are permeable for some bivalence cations (PNa approximately PCa). Other channel properties are described and their comparative analysis is given. It suggests that cGMP-activated cation channels represent a new type of cation channels.     

A simple and rapid procedure for the isolation of intact bovine rod outer segments (ROS)

A method is described which allows the rapid isolation and purification of intact rod outer segments (ROS) from cattle eyes. It requires very fresh retinal material and can be completed within less than 2 h of the death of the animals. Cattle eyes are dissected in the usual manner, the retinae are isolated and the ROS are separated from the rest of the retina by gentle vortexing and filtration through a nylon mesh. The resulting crude ROS suspension is purified on a discontinuous sucrose density gradient. Two fractions are obtained, the major one consisting of mostly intact ROS, the minor one of RIS-ROS, i.e. of ROS which are still connected to part of their inner segment. The ROS are washed once and can be stored on ice for several days without loosing their intact plasma membrane. They can be transformed to leaky ROS by a quick freeze/thawing cycle or, if one wants unobstructed access to the interdiskal space, they can be subjected to a mild lysis treatment. The resulting ROS material is characterised using light microscopy, electron microscopy, light scattering, gel electrophoresis and absorption spectroscopy. It contains unusually low levels of 48k-protein and very high levels of G-protein. The latter cannot be washed out in the presence of GTP-gamma-S, even in the case of leaky ROS.     

Evidence that a light-accelerated abrupt change in membrane characteristics of bovine rod outer segment fragments takes place at acidic pH

Changes in the turbidity of suspensions of bovine rod outer segment fragments induced by rhodopsin bleaching were measured in the presence of various concentrations of divalent cations at acidic pH (4.7–5.4). Unlike the situation at neutral pH, the turbidity of the suspensions increased drastically by bleaching at acidic pH. It was found that the extent of turbidity change became maximum at a particular concentration of divalent cations (i.e., 5 mM CaCl₂, 5 mM MgCl₂, or 5 mM mixed divalent cations). However, the turbidity increment in the presence of 5 mM MgCl₂ was greatly enhanced by the addition of a minute amount of CaCl₂. These results evidently show that the membrane characteristic is abruptly changed by bleaching at acidic pH in particular. It is also suggested that there are two kinds of binding sites for Ca ions: one is a Ca²⁺ specific site, and the other is a nonspecific site to which Mg²⁺ can also bind.     

Lipoperoxidation of rod outer segments of bovine retina is inhibited by soluble binding proteins for fatty acids

In the present study it was investigated if soluble-binding proteins for fatty acids (FABPs) present in neural retina show protection from in vitro lipoperoxidation of rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe⁺⁺ system, at 37°C during 90-120 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of soluble binding proteins for fatty acids. The fatty acid composition of rod outer segment membranes was substantially modified when subjected to non-enzymatic lipoperoxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3) and arachidonic acid (20:4 n-6). As a result of this, the unsaturation index, a parameter based on the maximal rate of oxidation of specific fatty acids was higher in the native and control membranes when compared with peroxidized ones. A similar decrease of chemiluminescence was observed with the addition of increasing concentrations of native or delipidated FABP retinal containing fractions to rod outer segment membranes. These results indicate that soluble proteins with fatty acid binding properties may act as antioxidant protecting rod outer segment membranes from deleterious effect.     

Differential membrane protein phosphorylation in bovine retinal rod outer segment disk membranes as a function of disk age

The outer segment portion of photoreceptor rod cells is composed of a stacked array of disk membranes. Newly formed disks are found at the base of the rod outer segment (ROS) and are relatively high in membrane cholesterol. Older disks are found at the apical tip of the ROS and are low in membrane cholesterol. Disk membranes were separated based on their membrane cholesterol content and the extent of membrane protein phosphorylation determined. Light induced phosphorylation of ROS disk membrane proteins was investigated using magic angle spinning³¹P NMR. When intact rod outer segment preparations were stimulated by light, in the presence of endogenously available kinases, membrane proteins located in disks at the base of the ROS were more heavily phosphorylated than those at the tip. SDS-gel electrophoresis of the phosphorylated disk membranes subpopulations identified a phosphoprotein species with a molecular weight of approximately 68–72 kDa that was more heavily phosphorylated in newly formed disks than in old disks. The identity of this phosphoprotein is presently under investigation. When the phosphorylation reaction was carried out in isolated disk membrane preparations with exogenously added co-factors and kinases, there was no preferential protein phosphorylation. Taken collectively, these results suggest that within the ROS there is a protein phosphorylation gradient that maybe indicative of co-factor or kinase heterogeneity.     

Ionic channels in the plasma membrane ofSchizosaccharomyces pombe: Evidence from patch-clamp measurements

Patch-clamp studies of the yeastSchizosaccharomyces pombe reveal that the plasma membrane contains a voltage-gated channel mildly selective for potassium over sodium, lithium, and chloride. The channel exhibits several conductances with a maximum of 153 pS. The channel gates in the region of physiologically relevant voltages, being closed at hyperpolarizing and open at depolarizing voltages. It is not inhibited by tetraethylammonium, quinine, or quinidine applied from the cytoplasmic side of the membrane; similarly, ATP and stretch have no effect. The frequency of its occurrence in patches implies that about 35 channels of this kind are present in the plasma membrane of a single cell.