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.     

Regulation of intracellular free Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. II. Thermodynamic competence of transmembrane Na+ and K+ gradients and inactivation of Na(+)-dependent Ca2+ extrusion.

Regulation of cytosolic free Ca2+ in the physiologically relevant submicromolar range was measured in isolated intact bovine rod outer segments (ROS) with the intracellular Ca(2+)-indicating dye fluo-3. Changes in free Ca2+ were compared with changes in total Ca2+ measured with 45Ca fluxes and a good qualitative correlation was observed. Ca2+ homeostasis in isolated bovine ROS was exclusively mediated via the Na-Ca-K exchanger. Free cytosolic Ca2+ concentration was lowered by an increase in the inward Na+ gradient, was raised by an increase in external K+, and was raised by depolarization of the plasma membrane. The simplest stoichiometry consistent with these qualitative observations is 4Na:(1Ca + 1K). The individual K:Ca, Na:Ca, and K:Na coupling ratios were deduced from quantitative changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ and K+ gradients. The observed changes in free Ca2+ did not agree with changes in free Ca2+ calculated on the basis of the above fixed stoichiometry which may reflect the flexibility in the Ca:K coupling ratio observed before in flux experiments (Schnetkamp, P. P. M., Szerencsei, R. T., and Basu, D. K. (1991) J. Biol. Chem. 266, 198-206). The most dramatic discrepancy was observed for the Na:Ca coupling ratio: the expected very large changes in cytosolic free Ca2+ upon changes in the transmembrane Na+ gradient were not observed. Rapid Na(+)-induced Ca2+ extrusion was unable to lower cytosolic free Ca2+ below 100 nM, even under nonequilibrium conditions and despite the observation that Ca2+ influx via reverse Na-Ca-K exchange readily occurred at a free external Ca2+ concentration of 20 nM. We conclude that the Na(+)-dependent extrusion mode of the Na-Ca-K exchanger occurs in a brief (20-s) burst of high maximal velocity transport followed by a nearly complete inactivation of transport. The importance of our findings for Ca2+ homeostasis in functioning rod photoreceptors is discussed.     

Regulation of free cytosolic Ca2+ concentration in the outer segments of bovine retinal rods by Na-Ca-K exchange measured with fluo-3. I. Efficiency of transport and interactions between cations.

Regulation of free cytosolic Ca2+ concentration in the rod outer segments (ROS) isolated from bovine retinas was examined with the fluorescent Ca(2+)-indicating dye fluo-3. In situ calibration of cytosolic fluo-3 was done in the presence of the Ca2+ ionophore A23187 and yielded a dissociation constant of 500 nM for the Ca(2+)-fluo-3 complex. Ca2+ influx in Ca(2+)-depleted ROS was completely abolished when internal Na+ was removed suggesting that Ca2+ influx exclusively occurred via Na-Ca-K exchange. The most striking observation was that Na-Ca-K exchange could mediate a rapid increase in cytosolic free Ca2+ over the most of the usable indicating range of fluo-3 (from 10 nM to 2 microM), even when exposed to free external Ca2+ concentrations as low as 10 nM. From a comparison between changes in free Ca2+ and changes in total Ca2+, we conclude that physiologically occurring changes in cytosolic free Ca2+ are mediated by exchange fluxes less than 1% of the maximal Na-Ca-K exchange flux. The Na-Ca-K exchanger could mediate both K(+)-dependent and K(+)-independent Ca2+ influx; Li+ caused a complete inhibition of K(+)-independent Ca2+ influx, but had no effect on K(+)-dependent Ca2+ influx. We examined the complex interactions of alkali cations with Ca2+ influx and discuss the results in terms of a three-site model for the Na-Ca-K exchanger (Schnetkamp, P. P. M. and Szerencsei, R. T. (1991) J. Biol. Chem. 266, 189-197). Ca2+ competed with one Mg2+ ion or two Na+ ions for binding to a common site. High K+ concentration greatly diminished the ability of Na+ and Mg2+ to compete with Ca2+ for this common site on the exchanger protein. As a result, high internal K+ induced a conformation of the exchange protein that kinetically favoured Ca2+ extrusion.     

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.     

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.     

Sodium ions selectively eliminate the fast component of guanosine cyclic 3',5'-phosphate induced Ca2+ release from bovine rod outer segment disks

Guanosine cyclic 3',5'-phosphate (cGMP) induced Ca2+ release from bovine rod outer segment (ROS) disks showed two kinetic components that could be distinguished in three ways: (1) The slow component (half-rise time of about 30 s) was blocked by 1-cis diltiazem [cf. Koch, K. W., & Kaupp, U. B. (1985) J. Biol. Chem. 260, 6788-6800], whereas the fast component (half-rise time of less than 1 s) was not affected by 1-cis diltiazem. (2) The slow component required the presence of alkali cations, whereas the fast component did not. (3) Preincubation with Na+ (50 mM) selectively eliminated the fast component, whereas the slow component was not affected. The action of Na+ appeared to be caused by Na-Ca exchange removing Ca2+ from a pool that can also be accessed by cGMP. The slow component of cGMP-induced Ca2+ release was not affected by Na+ and, hence, appears to reside in disks that do not contain a functional Na-Ca exchanger. The local anesthetic tetracaine blocked both the slow and fast component of cGMP-induced Ca2+ release from bovine ROS disks.     

Transport of K+ by Na(+)-Ca2+, K+ exchanger in isolated rods of lizard retina.

Transport of K+ by the photoreceptor Na(+)-Ca2+, K+ exchanger was investigated in isolated rod outer segments (OS) by recording membrane current under whole-cell voltage-clamp conditions. Known amounts of K+ were imported in the OS through the Ca(2+)-activated K+ channels while perfusing with high extracellular concentration of K+, [K+]o. These channels were detected in the recordings from the OS, which probably retained a small portion of the rest of the cell. The activation of forward exchange (Na+ imported per Ca2+ and K+ extruded) by intracellular K+, Ki+, was described by first-order kinetics with a Michaelis constant, Kapp(Ki+), of about 2 mM and a maximal current, Imax, of about -60 pA. [Na+]i larger than 100 mM had little effect on Kapp(Ki+) and Imax, indicating that Nai+ did not compete with Ki+ for exchange sites under physiological conditions, and that Na+ release at the exchanger intracellular side was not a rate-limiting step for the exchange process. Exchanger stoichiometry resulted in one K+ ion extruded per one positive charge imported. Exchange current was detected only if Ca2+ and K+ were present on the same membrane side, and Na+ was simultaneously present on the opposite side. Nonelectrogenic modes of ion exchange were tested taking advantage of the hindered diffusion found for Cai2+ and Ki+. Experiments were carried out so that the occurrence of a putative nonelectrogenic ion exchange, supposedly induced by the preapplication of certain extracellular ion(s), would have resulted in the transient presence of both Cai2+ and Ki+. The lack of electrogenic forward exchange in a subsequent switch to high Nao+, excluded the presence of previous nonelectrogenic transport.     

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)     

Na+, K+, and Cl- transport in resting pancreatic acinar cells

To understand the role of Na+, K+, and Cl- transporters in fluid and electrolyte secretion by pancreatic acinar cells, we studied the relationship between them in resting and stimulated cells. Measurements of [Cl-]i in resting cells showed that in HCO3(-)-buffered medium [Cl- ]i and Cl- fluxes are dominated by the Cl-/HCO3- exchanger. In the absence of HCO3-, [Cl-]i is regulated by NaCl and NaK2Cl cotransport systems. Measurements of [Na+]i showed that the Na(+)-coupled Cl- transporters contributed to the regulation of [Na+]i, but the major Na+ influx pathway in resting pancreatic acinar cells is the Na+/H+ exchanger. 86Rb influx measurements revealed that > 95% of K+ influx is mediated by the Na+ pump and the NaK2Cl cotransporter. In resting cells, the two transporters appear to be coupled through [K+]i in that inhibition of either transporter had small effect on 86Rb uptake, but inhibition of both transporters largely prevented 86Rb uptake. Another form of coupling occurs between the Na+ influx transporters and the Na+ pump. Thus, inhibition of NaK2Cl cotransport increased Na+ influx by the Na+/H+ exchanger to fuel the Na+ pump. Similarly, inhibition of Na+/H+ exchange increased the activity of the NaK2Cl cotransporter. The combined measurements of [Na+]i and 86Rb influx indicate that the Na+/H+ exchanger contributes twice more than the NaK2Cl cotransporter and three times more than the NaCl cotransporter and a tetraethylammonium-sensitive channel to Na+ influx in resting cells. These findings were used to develop a model for the relationship between the transporters in resting pancreatic acinar cells.     

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 1. Mechanism of inhibition by amiloride analogues

The mechanism by which terminal guanidino nitrogen substituted analogues of amiloride inhibit Na-Ca exchange in purified cardiac sarcolemmal membrane vesicles has been investigated. These inhibitors block both Nai-dependent Ca2+ uptake and Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange monitored in K+ is noncompetitive vs Ca2+ but competitive vs Na+. Substitution of sucrose for K+ results in mixed kinetics of inhibition vs Ca2+, suggesting a complex interaction between inhibitor and carrier under this condition. Amiloride derivatives also block two other modes of carrier action: Na-Na exchange is inhibited in a competitive fashion with Na+ and kinetics of Ca-Ca exchange inhibition are mixed vs Ca2+ in either sucrose or K+. However, Ca-Ca exchange inhibition can be alleviated by increasing K+ concentration. Dixon analyses of Na-Ca exchange block with mixtures of inhibitors suggest that these agents are interacting at more than one site. In addition, Hill plots of inhibition are biphasic with Hill coefficients of 1 and 2 at low and high inhibitor concentrations, respectively. These results indicate that amiloride derivatives are mechanism-based inhibitors that interact at two classes of substrate-binding sites on the carrier; at low concentration they bind preferentially to a site that is exclusive for Na+, while at higher concentration they also interact at a site that is common for Na+, Ca2+, and K+.     

The sodium-calcium exchanger of bovine rod photoreceptors: K(+)-dependence of the purified and reconstituted protein

The K(+)-dependence of the rod photoreceptor sodium-calcium exchanger was investigated using the Ca2(+)-sensitive dye arsenazo III after reconstitution of the purified protein into proteoliposomes. The uptake of Ca2+ by Na(+)-loaded liposomes was found to be greatly enhanced by the presence of external K+ (EC50 approximately 1 mM) in a Michaelis-Menten manner, suggesting that one K+ ion is involved in the transport of one Ca2+ ion. We also found a minimal degree of Ca2+ uptake in the total absence of K+. Other alkali cations, notably Rb+ and, to a lesser extent, Cs+, were also able to stimulate Na(+)-Ca2+ exchange. We also investigated the K(+)-dependence of the photoreceptor Na(+)-Ca2+ exchanger by determining the effects of electrochemical K+ gradients on the Na(+)-activated Ca2+ efflux from proteoliposomes. We found that, under conditions of membrane voltage clamp with FCCP, inwardly directed electrochemical K+ gradients (i.e., K0+ greater than Ki+) inhibited, whereas an outwardly directed electrochemical K+ gradient (i.e., Ki+ greater than K0+) enhanced, Na(+)-dependent Ca2+ efflux, consistent with the notion that K+ is cotransported in the same direction as Ca2+. The investigation of the reconstituted exchanger at physiological (i.e. Ki+ = 110 mM, K0+ = 2.5 mM) potassium concentrations revealed that the Na(+)-dependence of Ca2(+)-efflux was highly cooperative (n = 3.01 from Hill plots), indicating that at least three, but possibly four, Na+ ions are exchanged for one Ca2+ ion. Under these conditions the reconstituted exchanger showed a Km for Na+ of 26.1 mM, and a turnover number of 115 Ca2+.s-1 per exchanger molecule. Our results with the purified and reconstituted sodium-calcium exchanger from rod photoreceptors are therefore consistent with previous reports (Cervetto, L., Lagnado, L., Perry, R.J., Robinson, D.W. and McNaughton, P.A. (1989) Nature 337, 740-743; Schnetkamp, P.P.M., Basu, D.K. and Szerencsei, R.T. (1989) Am. J. Physiol. 257, C153-C157) that the sodium-calcium exchanger of rod photoreceptors cotransports K+ under physiological conditions with a stoichiometry of 4 Na+:1 Ca2+, 1K+.     

On the structural basis for ionic selectivity among Na+, K+, and Ca2+ in the voltage-gated sodium channel.

Voltage-sensitive sodium channels and calcium channels are homologous proteins with distinctly different selectivity for permeation of inorganic cations. This difference in function is specified by amino acid residues located within P-region segments that link presumed transmembrane elements S5 and S6 in each of four repetitive Domains I, II, III, and IV. By analyzing the selective permeability of Na+, K+, and Ca2+ in various mutants of the mu 1 rat muscle sodium channel, the results in this paper support the concept that a conserved motif of four residues contributed by each of the Domains I-IV, termed the DEKA locus in sodium channels and the EEEE locus in calcium channels, determines the ionic selectivity of these channels. Furthermore, the results indicate that the Lys residue in Domain III of the sodium channel is the critical determinant that specifies both the impermeability of Ca2+ and the selective permeability of Na+ over K+. We propose that the alkylammonium ion of the Lys(III) residue acts as an endogenous cation within the ion binding site/selectivity filter of the sodium channel to tune the kinetics and affinity of inorganic cation binding within the pore in a manner analogous to ion-ion interactions that occur in the process of multi-ion channel conduction.     

Diphtheria toxin-induced channels in Vero cells selective for monovalent cations

Ion fluxes associated with translocation of diphtheria toxin across the surface membrane of Vero cells were studied. When cells with surface-bound toxin were exposed to low pH to induce toxin entry, the cells became permeable to Na+, K+, H+, choline+, and glucosamine+. There was no increased permeability to Cl-, SO4(-2), glucose, or sucrose, whereas the uptake of 45Ca2+ was slightly increased. The influx of Ca2+, which appears to be different from that of monovalent cations, was reduced by several inhibitors of anion transport and by verapamil, Mn2+, Co2+, and Ca2+, but not by Mg2+. The toxin-induced fluxes of N+, K+, and protons were inhibited by Cd2+. Cd2+ also protected the cells against intoxication by diphtheria toxin, suggesting that the open cation-selective channel is required for toxin translocation. The involvement of the toxin receptor is discussed.     

Inhibition of sodium-calcium exchange in cardiac sarcolemmal membrane vesicles. 2. Mechanism of inhibition by bepridil

Bepridil, an antiarrhythmic agent, inhibits Na-Ca exchange in cardiac sarcolemmal membrane vesicles (Ki = 30 microM) by a novel mechanism, different from that determined for amiloride analogues [Slaughter, R. S., Garcia, M. L., Cragoe, E. J., Jr., Reeves, J. P., & Karczorowski, G. J. (1988) Biochemistry (preceding paper in this issue)]. Bepridil causes partial inhibition of Nai-dependent Ca2+ uptake but complete block of Nao-dependent Ca2+ efflux. Inhibition of Na-Ca exchange is noncompetitive vs Ca2+ but competitive vs Na+ in both K+ and sucrose. Bepridil also blocks Ca-Ca exchange, with or without K+ present. However, K+ has two effects on inhibition: it reduces the potency of bepridil and causes inhibition to become partial. Inhibition of Ca-Ca exchange displays noncompetitive kinetics vs Ca2+ in either sucrose or K+. Dixon analyses of Na-Ca exchange inhibition caused by mixtures of bepridil and amiloride analogues demonstrate that these compounds produce a competitive interaction at a common carrier site that is evident only at low concentrations of amiloride inhibitors. Hill plots of bepridil inhibition of Na-Ca and Ca-Ca exchange display unitary Hill coefficients. These results indicate that bepridil interacts at only one substrate-binding site, the site selective for Na+, where amiloride analogues also preferentially interact. However, unlike amiloride, bepridil does not interact at the common Na+, Ca2+-binding site of the carrier.(ABSTRACT TRUNCATED AT 250 WORDS)     

In squid nerve fibers monovalent activating cations are not cotransported during Na+/Ca2+ exchange

Squid axons display a high activity of Na+/Ca2+ exchange which is largely increased by the presence of external K+, Li+, Rb+ and NH+4. In this work we have investigated whether this effect is associated with the cotransport of the monovalent cation along with Ca2+ ions. 86Rb+ influx and efflux have been measured in dialyzed squid axons during the activation (presence of Ca2+i) of Ca2+o/Na+i and Ca2+i/Ca2+o exchanges, while 86Rb+ uptake was determined in squid optic nerve membrane vesicles under equilibrium Ca2+/Ca2+ exchange conditions. Our results show that although K+o significantly increases Na+i-dependent Ca2+ influx (reverse Na+/Ca2+ exchange) and Rb+i stimulates Ca2+o-dependent Ca2+ efflux (Ca2+/Ca2+ exchange), no sizable transport of rubidium ions is coupled to calcium movement through the exchanger. Moreover, in the isolated membrane preparation no 86Rb+ uptake was associated with Ca2+/Ca2+ exchange. We conclude that in squid axons although monovalent cations activate the Na+/Ca2+ exchange they are not cotransported.     

Electrophysiological characterization of ionic transport by the retinal exchanger expressed in human embryonic kidney cells.

The retinal Na+:Ca2+, K+exchanger cDNA was transiently expressed in human embryonic kidney (HEK 293) cells by transfection with plasmid DNA. The correct targeting of the expressed protein to the plasma membrane was confirmed by immunocytochemistry. The reverse exchange offrent (Ca2+ imported per Na+ extruded) was measured in whole-cell voltage-clamp experiments after intracellular perfusion with Na+ (Na+i, 128 mM) and extracellular perfusion with Ca2+ (Ca2o+, 1 mM) and Ko+ (20 mM). As expected, the exchange current was suppressed by removing Ca2o+. Surprisingly, however, it was also abolished by increasing Na+o to almost abolish the Na+ gradient, and it was almost unaffected by the removal of Ko+. Apparently, then, at variance with the exchanger in the rod outer segment, the retinal exchanger expressed in 293 cells acts essentially as a Na+:Ca2+ exchanger and does not require K+ for its electrogenic activity.     

Phenylalkylamine-sensitive calcium channels in osteoblast-like osteosarcoma cells. Characterization by ligand binding and single channel recordings

(-)-[3H]Desmethoxyverapamil ((-)-DMV) binds saturably to homogenates of the osteoblast-like cell lines UMR 106 and ROS 17/2.8 with KD values of 45 and 61 nM and Bmax values of 6.0 and 5 pmol/mg protein, respectively. Binding is stereoselective with (-)-DMV 8-10 times more potent than (+)-DMV. None of the dihydropyridine or benzothiazepine Ca2+ antagonists examined affect (-)-[3H]DMV binding. Monovalent cations such as Li+, Na+, and K+ inhibit (-)[3H]DMV binding in the 100-400 mM range. Divalent cations such as Ba2+, Sr2+, Ca2+, and Mg2+ are effective binding inhibitors in the 2-5 mM range. ROS 17/2.8 cells express a channel on the apical plasma membrane which conducts Ba2+ and Ca2+. With 110 mM BaCl2 or CaCl2 as charge carriers the single channel conductance is 3-5 picosiemens. In cell-excised patches the channel selects for Ba2+ over Na+ 3.3:1. In the absence of divalent ions the channel conducts Na+ ions with a single channel conductance of 13 picosiemens. This Na+ conductance decreases with physiological levels of Ca2+. The channel appears related to the (-)-[3H]DMV binding site, since its conductance is blocked by verapamil in a dose-dependent manner. Moreover, DMV blocks the channel stereoselectively with relative potencies of the isomers corresponding to their affinities for the binding site. The dihydropyridine drugs BAY K 8644 or (+)-202-791 do not affect channel opening. These binding and biophysical data indicate that osteoblast cells have a phenylalkylamine receptor associated with a Ca2+ channel.     

Na+/Ca2+ exchangers: three mammalian gene families control Ca2+ transport

Mammalian Na+/Ca2+ exchangers are members of three branches of a much larger family of transport proteins [the CaCA (Ca2+/cation antiporter) superfamily] whose main role is to provide control of Ca2+ flux across the plasma membranes or intracellular compartments. Since cytosolic levels of Ca2+ are much lower than those found extracellularly or in sequestered stores, the major function of Na+/Ca2+ exchangers is to extrude Ca2+ from the cytoplasm. The exchangers are, however, fully reversible and thus, under special conditions of subcellular localization and compartmentalized ion gradients, Na+/Ca2+ exchangers may allow Ca2+ entry and may play more specialized roles in Ca2+ movement between compartments. The NCX (Na+/Ca2+ exchanger) [SLC (solute carrier) 8] branch of Na+/Ca2+ exchangers comprises three members: NCX1 has been most extensively studied, and is broadly expressed with particular abundance in heart, brain and kidney, NCX2 is expressed in brain, and NCX3 is expressed in brain and skeletal muscle. The NCX proteins subserve a variety of roles, depending upon the site of expression. These include cardiac excitation-contraction coupling, neuronal signalling and Ca2+ reabsorption in the kidney. The NCKX (Na2+/Ca2+-K+ exchanger) (SLC24) branch of Na+/Ca2+ exchangers transport K+ and Ca2+ in exchange for Na+, and comprises five members: NCKX1 is expressed in retinal rod photoreceptors, NCKX2 is expressed in cone photoreceptors and in neurons throughout the brain, NCKX3 and NCKX4 are abundant in brain, but have a broader tissue distribution, and NCKX5 is expressed in skin, retinal epithelium and brain. The NCKX proteins probably play a particularly prominent role in regulating Ca2+ flux in environments which experience wide and frequent fluctuations in Na+ concentration. Until recently, the range of functions that NCKX proteins play was generally underappreciated. This situation is now changing rapidly as evidence emerges for roles including photoreceptor adaptation, synaptic plasticity and skin pigmentation. The CCX (Ca2+/cation exchanger) branch has only one mammalian member, NCKX6 or NCLX (Na+/Ca2+-Li+ exchanger), whose physiological function remains unclear, despite a broad pattern of expression.     

Na+, K+ and Ca2+ antagonize the glutamate- and glycine-induced decrease of [3H]MK-801 binding observed in the presence of Mg2+ at low pH.

NMDA receptors are glutamate-regulated ion channels that are of great importance for many physiological and pathophysiological conditions in the mammalian central nervous system. We have previously shown that, at low pH, glutamate decreases binding of the open-channel blocker [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten, 5,10-imine ([3H]MK-801) to NMDA receptors in the presence of 1 mM Mg2+ but not in Krebs buffer. Here, we investigated which cations that block the glutamate-induced decrease in Krebs buffer, using [3H]MK-801 binding assays in membrane preparations from the rat cerebral cortex. At pH 6.0, Na+, K+, and Ca2+ antagonized the glutamate-induced decrease with cross-over values, which is a measure of the antagonist potencies of the cations, of 81, 71, and 26 mM, respectively, in the absence of added glycine. Thus, in Krebs buffer only the concentration of Na+ (126 mM) is sufficiently high to block the glutamate-induced decrease observed at low pH. In the presence of 1 mM Mg2+ and 10 mM Ca2+ at pH 7.4, the cross-over values for Na+, K+, and Ca2+ were 264, 139, and 122 mM, respectively, in the absence of added glycine. This is the same rank order of potency as observed at pH 6.0, suggesting that the less H+-sensitive and the less Ca2+-sensitive, glutamate-induced decreases in [3H]MK-801 binding represent the same entity. The glycine site antagonists 7-chlorokynurenate (10 microM) and 7-chloro-4-hydroxy-3-(3-phenoxy)phenyl-2(H)-quinoline (L-701,324; 1 microM) antagonized the glutamate-induced decrease in [3H]MK-801 binding observed in presence of Mg2+ at pH 6.0, suggesting that glycine is required together with glutamate to induce the decrease observed at low pH. These results suggest that in addition to a previously described high-affinity binding site for H+ and Ca2+ there exist a low-affinity binding site for H+, Ca2+, Na+, and K+ on NMDA receptors. The latter site may under physiological conditions be blocked by Na+ or K+, depending on the extra/intracellular localization of the modulatory site. Both the high-affinity and low-affinity cation sites mediate antagonistic effects on the glutamate- and glycine-induced decrease of the affinity of the [3H]MK-801 binding site, which may correspond to similar changes in the affinity of the voltage-sensitive Mg2+-block site inside the NMDA receptor channel pore, which in turn may affect current and Ca2+ influx through activated NMDA receptor channels.     

Visualizing the mapped ion pathway through the Na,K-ATPase pump

The Na+,K+-ATPase pump achieves thermodynamically uphill exchange of cytoplasmic Na+ ions for extracellular K+ ions by using ATP-mediated phosphorylation, followed by autodephosphorylation, to power conformational changes that allow ion access to the pump's binding sites from only one side of the membrane at a time. Formally, the pump behaves like an ion channel with two tightly coupled gates that are constrained to open and close alternately. The marine agent palytoxin disrupts this coupling, allowing both gates to sometimes be open, so temporarily transforming a pump into an ion channel. We made a cysteine scan of Na+,K+-ATPase transmembrane (TM) segments TM1 to TM6, and used recordings of Na+ current flow through palytoxin-bound pump-channels to monitor accessibility of introduced cysteine residues via their reaction with hydrophilic methanethiosulfonate (MTS) reagents. To visualize the open-channel pathway, the reactive positions were mapped onto a homology model of Na+,K+-ATPase based on the structure of the related sarcoplasmic- and endoplasmic-reticulum (SERCA) Ca2+-ATPase in a BeF3--trapped state1,2, in which the extra-cytoplasmic gate is wide open (although the cytoplasmic access pathway is firmly shut). The results revealed a single unbroken chain of reactive positions that traverses the pump from the extracellular surface to the cytoplasm, comprises residues from TM1, TM2, TM4, and TM6, and passes through the equivalent of cation binding site II in SERCA, but not through site I. Cavity search analysis of the homology model validated its use for mapping the data by yielding a calculated extra-cytoplasmic pathway surrounded by MTS-reactive residues. As predicted by previous experimental results, that calculated extra-cytoplasmic pathway abruptly broadens above residue T806, at the outermost end of TM6 which forms the floor of the extracellular-facing vestibule. These findings provide a structural basis for further understanding cation translocation by the Na+,K+-ATPase and by other P-type pumps like the Ca2+- and H+,K+-ATPases.