ATP triggers a robust intracellular [Ca2+ ]-mediated signalling pathway in human synovial fibroblasts.

NEW FINDINGS: What is the central question of this study? What are the main [Ca2+ ]i signalling pathways activated by ATP in human synovial fibroblasts? What is the main finding and its importance? In human synovial fibroblasts ATP acts through a linked G-protein (Gq ) and phospholipase C signalling mechanism to produce IP3 , which then markedly enhances release of Ca2+ from the endoplasmic reticulum. These results provide new information for the detection of early pathophysiology of arthritis. ABSTRACT: In human articular joints, synovial fibroblasts (HSFs) have essential physiological functions that include synthesis and secretion of components of the extracellular matrix and essential articular joint lubricants, as well as release of paracrine substances such as ATP. Although the molecular and cellular processes that lead to a rheumatoid arthritis (RA) phenotype are not fully understood, HSF cells exhibit significant changes during this disease progression. The effects of ATP on HSFs were studied by monitoring changes in intracellular Ca2+ ([Ca2+ ]i ), and measuring electrophysiological properties. ATP application to HSF cell populations that had been enzymatically released from 2-D cell culture revealed that ATP (10-100 µm), or its analogues UTP or ADP, consistently produced a large transient increase in [Ca2+ ]i . These changes (i) were initiated by activation of the P2 Y purinergic receptor family, (ii) required Gq -mediated signal transduction, (iii) did not involve a transmembrane Ca2+ influx, but instead (iv) arose almost entirely from activation of endoplasmic reticulum (ER)-localized inositol 1,4,5-trisphosphate (IP3 ) receptors that triggered Ca2+ release from the ER. Corresponding single cell electrophysiological studies revealed that these ATP effects (i) were insensitive to [Ca2+ ]o removal, (ii) involved an IP3 -mediated intracellular Ca2+ release process, and (iii) strongly turned on Ca2+ -activated K+ current(s) that significantly hyperpolarized these cells. Application of histamine produced very similar effects in these HSF cells. Since ATP is a known paracrine agonist and histamine is released early in the inflammatory response, these findings may contribute to identification of early steps/defects in the initiation and progression of RA.

Cloning and characterization of a potassium-dependent sodium/calcium exchanger in Drosophila.

Sodium/calcium(-potassium) exchangers (NCX and NCKX) are critical for the rapid extrusion of calcium, which follows the stimulation of a variety of excitable cells. To further understand the mechanisms of calcium regulation in signaling, we have cloned a Drosophila sodium/calcium-potassium exchanger, Nckx30C. The overall deduced protein topology for NCKX30C is similar to that of mammalian NCKX, having five membrane-spanning domains in the NH(2) terminus separated from six at the COOH-terminal end by a large intracellular loop. We show that NCKX30C functions as a potassium-dependent sodium/calcium exchanger, and is not only expressed in adult neurons as was expected, but is also expressed during ventral nerve cord development in the embryo and in larval imaginal discs. Nckx30C is expressed in a dorsal-ventral pattern in the eye-antennal disc in a pattern that is similar to, but broader than that of wingless, suggesting that large fluxes of calcium may be occurring during imaginal disc development. Nckx30C may not only function in the removal of calcium and maintenance of calcium homeostasis during signaling in the adult, but may also play a critical role in signaling during development.

Structure-function relationships of the NCKX2 Na+/Ca2+-K+ exchanger.

K+-dependent Na+/Ca2+ exchangers (NCKX) have been shown to play important roles in physiological processes as diverse as phototransduction in rod photoreceptors, motor learning and memory in mice, and skin pigmentation in humans. Most structure-function studies on NCKX proteins have been carried out on the NCKX2 isoform, but sequence similarity suggests that the results obtained with the NCKX2 isoform are likely to apply to all NCKX1-5 members of the human SLC24 gene family. Here we review our recent work on the NCKX2 protein concerning the topological arrangement of transmembrane segments carrying out cation transport, and concerning residues important for transport function and cation binding.

Calcium translocation and storage of isolated intact cattle rod outer segments in darkness.

Bovine rod outer segments (rods), isolated with an intact plasma membrane and a stable calcium exchange and storage capacity, contain 2-3 mol endogenous calcium/mol rhodopsin. By means of 45Ca accumulation experiments and concomitant 40Ca analysis, the calcium metabolism of these organelles has been studied with the following results: 1. The majority of endogenous calcium is localized within disks. 2. In the presence of the ionophore A23187 the intradiskal binding sites can be titrated with external calcium. 3. The Scatchard plot of calcium binding of rods indicates the presence of a single set of intradiskal binding sites with a maximal capacity of 8-9 mol calcium/mol rhodopsin and an affinity constant of 55 microM to calcium. 4. Without A23187 more than 99% of the rod calcium appears in a bound state in equilibrium with a free calcium concentration of 15-25 microM. 5. External calcium exchanges with endogenous calcium in a fast (t 1/2 = 12 s) process with a uniform rate constant, whereas net calcium transport is very slow (t 1/2 greater than 2 h). 6. Intact rods contain a calcium translocation system, presumably located in the plasma membrane, which performs Ca-Ca exchange with a high unidirectional flux of 2 . 10(6) calcium ions/rod per s. 7. This translocation system can be saturated by external calcium (Km = 0.5 -1 microM) and has a low Q10 (1.08). Both the calcium translocation system and the calcium binding system appear to depend on the structural integrity of the stacked disks and are very sensitive to the experimental conditions. The relevance of these findings is discussed in relation to the proposed role of calcium ions as the intracellular transmitter in vertebrate rod photoreceptor cells.

Ion selectivity of the cation transport system of isolated intact cattle rod outer segments: evidence for a direct communication between the rod plasma membrane and the rod disk membranes.

The ion selectivity of cation transport through the plasma membrane of isolated intact cattle rod outer segments (rods) is investigated by means of 45Ca-exchange experiments and light-scattering experiments. These techniques appear to provide complementary information: the 45Ca experiments (45Ca fluxes in rods) describe electroneutral antiport, whereas the light-scattering experiments (shrinkage and swelling of rods upon hypertonic shocks with various electrolytes) reveal electrogenic uniport. Electroneutral symport of ions (salt transport) does not take place without addition of external ionophores and application of salts of weak acids. 1. Intact rods recover from a hypertonic shock in the presence of FCCP when lithium, sodium and potassium acetate are applied, but not when ammonium chloride, calcium and magnesium acetate are used. This indicates that the plasma membrane of isolated intact cattle rods is relatively permeable to net transport of Na+, Li+ and K+, and relatively impermeable to net transport of Cl-, Mg2+ and Ca2+ under conditions that do not give rise to diffusion potentials. 2. Rapid (t1/2 < 1 min) efflux of 45Ca from preloaded intact rods is observed when Na+, Ca2+, Sr2+, and under certain conditions also Ba2+, are added to the external medium. Li+, K+, Rb+, Cs+, Mg2+ and Mn2+ are ineffective in this respect as well as protons at pH 7.4. It is concluded that 45Ca efflux reflects electroneutral exchange diffusion of internal 45Ca with external Na+, Ca2+, Sr2+ and Ba2+, respectively. 3. All tested cations lower the rate of 45Ca uptake. The latter can be described by a single rate constant indicating a homogeneous rod preparation and a homogeneous endogenous Ca2+ pool. However, only those cations which stimulate 45Ca efflux from preloaded rods lower the final equilibrium of 45Ca uptake. Except for the effects of K+, Rb+ and Cs+ the reduction of the rate of 45Ca uptake by external cations appears to arise from competition for a common site on the plasms membrane. The observed affinities for this site do not correlate with actual transport (as indicated by the ability to stimulate 45Ca efflux). 4. K+ increases the affinity of the exchange diffusion system to Ca2+ from 1 microM to 0.15 microM and changes the relative affinities with respect to Ca2+ for the other cations (Na+, Mg2+, Mn2+, Sr2+, Ba2+). Furthermore, the maximal rate of Ba-Ca exchange is strongly stimulated by K+, whereas the maximal rate of Ca-Ca exchange is reduced at saturating Ca2+ concentrations. 5. The exchange diffusion transport mode can be turned off by external Na+ in a process that is not of a stochastic nature, which implies interdependence of individual transport entities and which results in an inhomogeneity of the endogenous Ca2+ pool. K+ acts as antagonist of Na+ in this effect. The revelence of these findings is discussed in relation to the generally accepted view, that a diffusable transmitter in the rod cytosol communicates the photochemical event in the disk membrane to the electrical properties of the plasma membrane...

Transfer of high-energy phosphate in bovine rod outer segments. A nucleotide buffer system.

The nucleotide requirement for the recycling of NADPH, necessary in rod outer segment cytoplasm for the reduction of the chromophore upon bleaching, has been investigated. It is found that the latter process specifically requires ATP. Using this specificity the pathways have been investigated, by which in the rod outer segment cytoplasm ATP is resynthesized from other high-energy phosphate donors. It is found that enzyme activities are present, which can rapidly reshuffle the high-energy phosphate groups between the various adenine and guanine nucleotides as well as from creatine phosphate. These phosphate transfers may occur so rapidly that light-induced changes in nucleotide concentrations could easily be a delusive criterion for the enzymatic process underlying these changes.

Light-induced calcium release in isolated intact cattle rod outer segments upon photoexcitation of rhodopsin.

By applying flash-spectrophotometry with the calcium-indicating dye arsenazo III rapid light-triggered calcium release in various cattle rod outer segment preparations was studied. It is shown that light-induced calcium signals can be unambiguously discriminated from underlying absorption changes due to photolysis of rhodopsin and apparent absorption changes resulting from lightscattering transients. The following results have been obtained: 1. Calcium-induced arsenazo III responses can be quantitatively and kinetically resolved within the time domain of the visual transduction process. 2. Photoexcitation of rhodopsin results in calcium release from intradiscal binding sites. 3. Calcium released does not appear in the cytoplasmic space unless the disc membrane is made permeable to calcium ions by an ionophore. 4. The shortest observed half-rise time of calcium release (300 ms) is possibly limited by the ionophore. 5. The stoichiometric ratio of calcium released/rhodopsin bleached is 0.5 at a free calcium concentration of 2 microM. The amount of calcium released is proportional to the precentage of rhodopsin bleaching (from 1--10%). 6. Upon disruption of the disc stack by lysis of intact rod outer segments the light-induced calcium release is greatly altered. The results are discussed in relation to previous reports on a light-induced calcium release from retinal discs and in terms of the proposed role of calcium as an intracellular transmitter in vertebrate photoreceptors.

Interplay between hydroxylamine, metarhodopsin II and GTP-binding protein in bovine photoreceptor membranes.

The decay reactions of metarhodopsin II and the dissociation of the complex between rhodopsin (in the metarhodopsin II state) and the GTP-binding protein (G-protein) (in its inactive, GDP-binding form) have been compared at various concentrations of hydroxylamine. The reactions of the chromophore were measured by absorption changes in the visible range, the complex dissociation by changes in the near-infrared scattering. An additional monitor of the complex was given by the G-protein-dependent equilibrium between metarhodopsin I and metarhodopsin II. For all measurements, fragments of isolated bovine rod outer segments in suspension were used. In the absence of hydroxylamine, the rhodopsin-G-protein complex dissociated within 20-30 min at room temperature. The presence of hydroxylamine greatly accelerated (e.g., 5-fold at 1 mM NH2OH) the dissociation. Under all conditions, the free, dissociated G-protein can reassociate to metarhodopsin II produced by subsequent bleaching. Dissociation of the metarhodopsin II-G-protein complex required the decay of photoproducts with a maximal absorbance of 380 nm, but was not affected by the simultaneous presence of metarhodopsin III or metarhodopsin III - like photoproducts with a maximal absorbance between 450 and 470 nm. Despite the acceleration of metarhodopsin II-G-protein dissociation by NH2OH, metarhodopsin II-G-protein was relatively stabilized as compared to free metarhodopsin II. The ratio of the decay rates of free metarhodopsin II and metarhodopsin III-G-protein was increased as much as 10-fold in the presence of 25 mM NH2OH. The results indicate a mutual interdependence of retinal, opsin and G-protein.

Interfacial potentials at the disk membranes of isolated intact cattle rod outer segments as a function of the occupation state of the intradiskal cation-exchange binding sites.

Two different methods have been used to determine the interfacial potential at the disk membranes of intact isolated bovine rod outer segments: (1) The photolysis products of rhodopsin are known to be dependent on pH. We have used this property in order to probe the interfacial potential at disk membranes which is considered to change the surface pH at the disk membrane seen by rhodopsin. (2) The pK value of the amphiphilic pH-indicating dye neutral red (uncharged basic form) in water is 6.6, but adsorbed to disk membranes at least 7.8. This makes the distribution of neutral red between disk membranes and bulk water dependent on the interfacial potential at the disk membrane if the pH in the bulk solution is less than 7.8. Both methods yielded comparable results on the influence of ions and ion carriers on the interfacial potential at disk membranes. In particular, we have studied the effect of different occupation states on the internal binding capacity (of rod outer segments) for divalent cations. In the presence of the ionophore A23187, addition of EDTA to a suspension of intact rod outer segments removed all endogenous divalent cations (Schnetkamp, P.P.M. (1979) Biochim. Biophys. Acta 554, 441--459) and resulted in an interfacial pH at the disk membrane surface of about 6.4, whereas the bulk pH was 7.4. Subsequent addition of 2 mM Mn2+ saturated the internal binding capacity and resulted in an apparent shift towards alkaline pH of the surface pH at the disk membrane by 1.0--1.1 pH units. This could indicate a change of the interfacial potential by 60--65 mV. The same change of ionic conditions resulted in a change of the interfacial potential by 72 mV as determined from the partitioning behaviour of neutral red. These results were independent of the presence of H+ ionophores such as carbonyl cyanide p-trifluoromethoxy-phenylhydrazone and gramicidin. We conclude that the above results can be explained by the presence of fixed net negative charges (charge density: 0.5--1.5 electronic charges/rhodopsin molecule) at the intradiskal membrane surface. That the above charge density can be attributed to the intradiskal membrane surface is inferred from the observation that the presence of A23187 was required for access of divalent cations to the membrane interface involved in both rod outer segments with an intact as well as with a leaky plasma membrane.

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.

Optical measurements of Na-Ca-K exchange currents in intact outer segments isolated from bovine retinal rods.

The properties of Na-Ca-K exchange current through the plasma membrane of intact rod outer segments (ROS) isolated from bovine retinas were studied with the optical probe neutral red. Small cellular organelles such as bovine ROS do not offer an adequate collecting area to measure Na-Ca-K exchange currents with electrophysiological techniques. This study demonstrates that Na-Ca-K exchange current in bovine ROS can be measured with the dye neutral red and dual-wavelength spectrophotometry. The binding of neutral red is sensitive to transport of cations across the plasma membrane of ROS by the effect of the translocated cations on the surface potential of the intracellular disk membranes (1985. J. Membr. Biol. 88: 249-262). Electrogenic Na+ fluxes through the ROS plasma membrane were measured with a resolution of 10(5) Na+ ions/ROS per s, equivalent to a current of approximately 0.01 pA; maximal electrogenic Na-Ca-K exchange flux in bovine ROS was equivalent to a maximal exchange current of 1-2 pA. Electrogenic Na+ fluxes were identified as Na-Ca-K exchange current based on a comparison between electrogenic Na+ flux and Na(+)-stimulated Ca2+ release with respect to flux rate, Na+ dependence, and ion selectivity. Neutral red monitored the net entry of a single positive charge carried by Na+ for each Ca2+ ion released (i.e., monitored the Na-Ca-K exchange current). Na-Ca-K exchange in the plasma membrane of bovine ROS had the following properties: (a) Inward Na-Ca-K exchange current required internal Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 0.9 microM), whereas outward Na-Ca-K exchange current required both external Ca2+ (half-maximal stimulation at a free Ca2+ concentration of 1.1 microM) and external K+. (b) Inward Na-Ca-K exchange current depended in a sigmoidal manner on the external Na+ concentration, identical to Na(+)-stimulated Ca2+ release measured with Ca(2+)-indicating dyes. (c) The neutral red method was modified to measure Ca(2+)-activated K+ fluxes (half-maximal stimulation at 2.7 microM free Ca2+) via the Na-Ca-K exchanger in support of the notion that the rod Na-Ca exchanger is in effect a Na-Ca-K exchanger. (d) Competitive interactions between Ca2+ and Na+ ions on the exchanger protein are described.

Cation selectivity of and cation binding to the cGMP-dependent channel in bovine rod outer segment membranes.

The properties of the cGMP-dependent channel present in membrane vesicles prepared from intact isolated bovine rod outer segments (ROS) were investigated with the optical probe neutral red. The binding of neutral red is sensitive to transport of cations across vesicular membranes by the effect of the translocated cations on the surface potential at the intravesicular membrane/water interface (Schnetkamp, P. P. M. J. Membr. Biol. 88: 249-262). Only 20-25% of ROS membrane vesicles exhibited cGMP-dependent cation fluxes. The cGMP-dependent channel in bovine ROS carried currents of alkali and earth alkali cations, but not of organic cations such as choline and tetramethylammonium; little discrimination among alkali cations (K greater than Na = Li greater than Cs) or among earth alkali cations (Ca greater than Mn greater than Sr greater than Ba = Mg) was observed. The cation dependence of cGMP-induced cation fluxes could be reasonably well described by a Michaelis-Menten equation with a dissociation constant for alkali cations of about 100 mM, and a dissociation constant for Ca2+ of 2 mM. cGMP-induced Na+ fluxes were blocked by Mg2+, but not by Ca2+, when the cations were applied to the cytoplasmic side of the channel. cGMP-dependent cation fluxes showed a sigmoidal dependence on the cGMP concentration with a Hill coefficient of 2.1 and a dissociation constant for cGMP of 92 microM. cGMP-induced cation fluxes showed two pharmacologically distinct components; one component was blocked by both tetracaine and L-cis diltiazem, whereas the other component was only blocked by tetracaine.

Na+- and cGMP-induced Ca2+ fluxes in frog rod photoreceptors.

We have examined the Ca2+ content and pathways of Ca2+ transport in frog rod outer segments using the Ca2+-indicating dye arsenazo III. The experiments employed suspensions of outer segments of truncated, but physiologically functional, frog rods (OS-IS), intact isolated outer segments (intact OS), and leaky outer segments (leaky OS with a plasma membrane leaky to small solutes, but with sealed disk membranes). We observed the following. Intact OS or OS-IS isolated and purified in Percoll-Ringer's solution contained an average of 2.2 mM total Ca2+, while leaky OS contained 2.0 mM total Ca2+. This suggests that most of the Ca2+ in OS-IS is contained inside OS disks. Phosphodiesterase inhibitors increased the Ca2+ content to approximately 4.2 mM in intact OS or OS-IS, whereas the Ca2+ content of leaky OS was not altered. Na-Ca exchange was the dominant pathway for Ca2+ efflux in both intact and leaky OS/OS-IS. The rate of Na-Ca exchange in intact OS/OS-IS was half-maximal between 30 and 50 mM Na+; at 50 mM Na+, this amounted to 5.8 X 10(7) Ca2+/OS X s or 0.05 mM total Ca2+/s. This is much larger than the Ca2+ component of the dark current. Other alkali cations could not replace Na+ in Na-Ca exchange in either OS-IS or leaky OS. They inhibited the rate of Na-Ca exchange (K greater than or equal to Rb greater than Cs greater than or equal to Li greater than TMA) and, as the inhibition became greater, a delay developed in the onset of Na-Ca exchange. The inhibition of Na-Ca exchange by alkali cations correlates with the prolonged duration of the photoresponse induced by these cations (Hodgkin, A. L., P. A. McNaughton, and B. J. Nunn. 1985. Journal of Physiology. 358:447-468). In addition to Na-Ca exchange, disk membranes in leaky OS showed a second pathway of Ca2+ transport activated by cyclic GMP (cGMP). The cGMP-activated pathway required the presence of alkali cations and had a maximal rate of 9.7 X 10(6) Ca2+/OS X s. cGMP caused the release of only 30% of the total Ca2+ from leaky OS. The rate of Na-Ca exchange in leaky OS amounted to 1.9 X 10(7) Ca2+/OS X s.(ABSTRACT TRUNCATED AT 400 WORDS)

A derivative of amiloride blocks both the light-regulated and cyclic GMP-regulated conductances in rod photoreceptors.

Vertebrate rod photoreceptors in the dark maintain an inward current across the outer segment membrane. The photoresponse results from a light-induced suppression of this dark current. The light-regulated current is not sensitive to either tetrodotoxin or amiloride, potent blockers of Na+ channels. Here, we report that a derivative of amiloride, 3',4'-dichlorobenzamil (DCPA), completely suppresses the dark current and light response recorded from rod photoreceptors. DCPA also blocks a cyclic GMP-activated current in excised patches of rod plasma membrane and a cGMP-induced Ca++ flux from rod disk membranes. These results are consistent with the notion that the Ca++ flux mechanism in the disk membrane and the light-regulated conductance in the plasma membrane are identical. DCPA also inhibits the Na/Ca exchange mechanism in intact rods, but at a 5-10-fold-higher concentration than is required to block the cGMP-activated flux and current. The blocking action of DCPA in 10 nM Ca++ is different from that in 1 mM Ca++, which suggests either that the conductance state of the light-regulated channel may be modified in high and low concentrations of Ca++, or that there may be two ionic channels in the rod outer segment membrane.

Stable association of G proteins with beta 2AR is independent of the state of receptor activation.

beta 2-Adrenergic receptors expressed in Sf9 cells activate endogenous Gs and adenylyl cyclase [Mouillac B., Caron M., Bonin H., Dennis M. and Bouvier M. (1992) J. Biol. Chem. 267, 21733-21737]. However, high affinity agonist binding is not detectable under these conditions suggesting an improper stoichiometry between the receptor and the G protein and possibly the effector molecule as well. In this study we demonstrate that when beta 2-adrenergic receptors were co-expressed with various mammalian G protein subunits in Sf9 cells using recombinant baculoviruses signalling properties found in native receptor systems were reconstituted. For example, when beta 2AR was co-expressed with the Gs alpha subunit, maximal receptor-mediated adenylyl cyclase stimulation was greatly enhanced (60 +/- 9.0 versus 150 +/- 52 pmol cAMP/min/mg protein) and high affinity, GppNHp-sensitive, agonist binding was detected. When G beta gamma subunits were co-expressed with Gs alpha and the beta 2AR, receptor-stimulated GTPase activity was also demonstrated, in contrast to when the receptor was expressed alone, and this activity was higher than when beta 2AR was co-expressed with Gs alpha alone. Other properties of the receptor, including receptor desensitization and response to inverse agonists were unaltered. Using antisera against an epitope-tagged beta 2AR, both Gs alpha and beta gamma subunits could be co-immunoprecipitated with the beta 2AR under conditions where subunit dissociation would be expected given current models of G protein function. A desensitization-defective beta 2AR (S261, 262, 345, 346A) and a mutant which is constitutively desensitized (C341G) could also co-immunoprecipitate G protein subunits. These results will be discussed in terms of a revised view of G protein-mediated signalling which may help address issues of specificity in receptor/G protein coupling.

Calcium homeostasis in vertebrate retinal rod outer segments.

The outer segments of vertebrate retinal rod photoreceptors (ROS) exhibit dynamic Ca2+ fluxes. In darkness, Ca2+ continuously enters via the light-sensitive, cGMP-gated channels and this requires the presence of a powerful Ca2+ extrusion mechanism in the ROS plasma membrane. Our laboratory has characterized a Na/Ca+K exchanger in the ROS plasma membrane, which utilizes both inward Na+ gradient and outward K+ gradient to extrude Ca2+. Here, I review our work on the functional properties of the Na/Ca+K exchanger including the stoichiometry, ion binding sites and regulation of Ca2+ transport via Na/Ca+K exchange. Inactivation of the Ca2+ extrusion mode of the Na/Ca+K exchanger will be discussed as a mechanism to prevent lowering of cytosolic free Ca2+ to undesirably low values of < 1 nM that are expected from the coupling stoichiometry of the Na/Ca+K exchanger and that are expected to occur when Ca2+ influx via the cGMP-gated channels is interrupted during saturation of rod photoreceptors in bright light. This review also reexamines the contribution of internal Ca2+ stores (i.e. disks) to Ca2+ homeostasis in ROS.

Calcium metabolism in vertebrate photoreceptors.

So far all attempts to demonstrate a rapid, light-stimulated release of calcium from disks into the cytosol at a sufficiently high stoichiometry have failed. Either the release stoichiometry was too small or the velocity too slow to account for the amplification in visual transduction. The multitude of failures demonstrate that regulation of intracellular calcium is a very delicate process and the idea of a robust calcium channel in the disk membrane that is opened by rhodopsin itself is certainly an oversimplification. The strongest evidence in favour of the "calcium transmitter hypothesis" is the large calcium efflux from rods in a retina. However as long as the source of the calcium efflux inside the rod cells is unknown conclusions about the role of this calcium efflux are premature. Unfortunately, measurements of intracellular calcium, such as those by Brown and coworkers (93,94) in their pioneering work on photoreceptors in the ventral eye of Limulus, have not yet been feasible in vertebrates.

Voltage-dependent gating and block of the cyclic-GMP-dependent current in bovine rod outer segments.

The properties of the cyclic-GMP-activated conductance in the plasma membrane of bovine rod outer segments were studied in excised membranes. Multiple-channel and single-channel currents were recorded by the patch-clamp technique in symmetrical NaCl solutions which were free of divalent cations. The current-voltage relationship for the current, recorded when a large population of channels was activated, exhibited outward rectification. Rectification decreased as the concentration of cyclic-GMP was increased, and the concentration of cyclic-GMP required for half maximal activation of the channel decreased with depolarization. At a concentration of 1-3 microM cyclic-GMP, single-channel activity could be observed from these excised patches. The conductance of the open channel was 6 pS and was independent of the membrane potential. These results are consistent with the interpretation that under these conditions, the mechanism responsible for the outward rectification is due to an increase in the probability of an open channel as the membrane is depolarized. The cyclic-GMP-activated current could be blocked by L-cis-diltiazem. Block was voltage and time dependent. The time constant for the onset of block and its steady state level increased with depolarization. The extent of block by diltiazem was not enhanced as the cyclic-GMP concentration was increased, suggesting that the channel is not required to be open for block to occur. Complete block was never attained even for high concentrations of diltiazem. However, the diltiazem-resistant component of the cyclic-GMP-activated current could be blocked by tetracaine.

cDNA cloning of the human retinal rod Na-Ca + K exchanger: comparison with a revised bovine sequence.

PURPOSE: To clone the complementary DNA of the human retinal rod Na-Ca + K exchanger. METHODS: A human retinal cDNA library was screened initially with a radiolabeled probe representing the entire bovine rod Na-Ca + K exchanger cDNA and subsequently with probes from polymerase chain reaction fragments of the human retinal rod Na-Ca + K exchanger obtained after the initial screen. Twelve positive clones were used to obtain the entire coding sequence of the human retinal rod Na-Ca + K exchanger. RESULTS: The cDNA of the human retinal rod Na-Ca + K exchanger codes for a protein of 1081 amino acids, which shows 64.3% overall identity with the bovine retinal rod Na-Ca + K exchanger at the amino acid level. The two sets of putative transmembrane-spanning domains and their short connecting loops showed the highest degree of identity (94%-95%), whereas the extracellular loop at the N terminus showed a 59% identity. The large cytosolic loop that bisects the two sets of transmembrane-spanning domains contained two large deletions in the human exchanger; the first deletion contains 18 amino acids, whereas the second deletion involves a series of repeats that are dominated by acidic amino acid residues observed in the bovine, but not in the human, sequence. The authors observed that the bovine sequence contains a ninth repeat in addition to the eight repeats of the published sequence. CONCLUSIONS: The authors cloned the cDNA of the human retinal rod Na-Ca + K exchanger as a first step in examining the possibility that this gene could be the locus of disease-causing mutations.

The stoichiometry of Na-Ca+K exchange in rod outer segments isolated from bovine retinas.

Ca2+ extrusion in the outer segments of retinal rods (ROS) is mediated by a protein that couples both the inward Na+ gradient and the outward K+ gradient to Ca2+ extrusion. Na(+)-stimulated Ca2+ release from ROS requires internal K+ and is accompanied by release of internal K+, whereas a slow component of Na(+)-stimulated Ca2+ release does not require K+. In this paper we discuss our observations on the K+ transport via Na-Ca+ K exchange in bovine ROS, on the electrogenicity and stoichiometry of the ROS Na-Ca+ K exchanger, and on the mechanism on coupling Ca2+ to K+ via this protein. Finally, we discuss briefly the physiological implications of Na-Ca+ K exchange.