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.     

Stimulation of the salt receptor of the blowfly. III. The alkali halides

Application of solutions of each of the alkali halides to the tip of a labellar sensillum of the blowfly elicited a repetitive neural discharge from the salt receptor. The records were qualitatively similar to those for NaCl. For each of the alkali chlorides and sodium halides, the shapes of the curves of the response of the salt receptor as a function of concentration were similar to that for NaCl. The alkali halides exhibited a regular pattern of relative stimulating effectiveness for the salt receptor. The effectiveness of the anions increased monotonically with atomic number. The effectiveness of the cations was greatest for potassium and declined as the atomic number was increased or decreased. This hierarchy for stimulating effectiveness was maintained at all tested molarities. The response to a mixture of two salts appeared to be an average of those to the single salts at concentrations equal to the total concentration of the mixture. Cross-adaptation was observed between the alkali halides. The results indicate that an explanation of stimulation of the salt receptor must apply to all the salts tested and that both the anion and the cation affect a salt's stimulating effectiveness.     

Fresh-water fish chemoreceptors responsive to dilute solutions of electrolytes

The existence of the palatal chemoreceptors responding specifically to dilute solutions of salts with monovalent cations was demonstrated in carp. The distilled water effect (a response produced by the application of distilled water after chemoreceptors had been rinsed out with hypertonic salt solutions) was assigned to the activity of the same receptor. Intensity of the response to dilute solutions of salts depended on the valency of the anion: the larger the valency, the greater the response. Positively charged sites of the receptor responsive to dilute salt solutions were suggested by previous treatments with acid and alkali, and dye salts. Increase in the ionic strength of the stimulating solution by the addition of supporting electrolytes caused a depression of response. In particular, strong depression of response was caused by the addition of a supporting electrolyte with a divalent cation. Effects of polarizing current on the chemoreceptor activity were investigated. Based upon the findings in this paper, a hypothesis is presented, which explains mechanisms underlying chemoreceptor responses to dilute solutions of electrolytes in terms of an interfacial electro-kinetic process.     

The Selective Uptake of Alkali Cations by Red Beet Root Tissue

The selective uptake of alkali cations by red beet root tissuefrom solutions of chlorides has been investigated. It is shownthat when disks are transferred from distilled water to a solutionof salts, there is a rapid initial uptake of cations which isneither particularly selective, nor directly related to metabolism.On the other hand, the prolonged active accumulation of cationsexhibits strong selectivity, Na being preferred to other ions. Evidence is presented to show that the alkali cations competewith one another for the same absorption mechanism. In thisrespect the material apparently differs from that investigatedby Epstein and Hagen, in which the operation of distinct mechanismsfor some of these ions was visualized. The validity of Epsteinand Hagen's conclusion is discussed in relation to the resultspresented here.     

Large enhancement of canine taste responses to sugars by salts

The effects of changed ionic environments on the canine taste responses to sugars were examined by recording the activity of the chorda tympani nerve. a) The responses to various sugars were greatly enhanced by the presence of salts having monovalent cations such as Na+, K+, choline+, or Tris+. The responses to sugars were suppressed by high concentrations of salts. (b) The presence of 100 mM NaCl in fructose solution did not affect the maximal response and changed the Hill constant for the concentration-response relationship from 1.3 to 2.4. (c) CaCl2 greatly enhanced the response to fructose, while MgCl2 exhibited practically no effect. The presence of 20 mM CaCl2 in fructose solution changed the Hill constant from 1.2 to 2.4. (d) CaCl2 suppressed the responses to 0.5 M sugars except for fructose and sucrose and enhanced the responses to all sugars examined at 1 M. In the glucose response, the slope of the concentration-response curve was increased by the presence of CaCl2. Here the curve in the absence of CaCl2 intersected with that in the presence of CaCl2, indicating that CaCl2 suppressed the response to glucose of low concentrations and enhanced that of high concentrations. (e) The enhancement of the sugar responses by salts was not simply explained in terms of ionic permeability at the apical membranes of taste cells. The enhanced and suppressed effects of salts on the sugar responses were interpreted in terms of the cooperativity between receptor molecules for sugars.     

A model for the stimulation of taste receptor cells by salt.

A taste cell mucosal surface is regarded as a planar region containing bound anionic sites and openings to ionic channels. It is assumed that the bulk aqueous properties of the exterior phase are not continuous with the surface but terminate at a plane near the surface. The region between the (Stern) plane and the membrane is regarded as having a lower dielectric constant than bulk water. This fact admits the possibility of ion pair formation between fixed sites and mobile cations. Mobile ion pairs entering the region may also bind to a fixed anionic site. Thus, it is assumed that mobile cations and ion pairs are potential determining species at the surface. Binding cations neutralizes surface charges, whereas binding mobile ion pairs does not. This competition accounts for the observed anion effect on stimulation of tast receptors by sodium salts. The potential profile is constructed by superimposing the phase boundary potentials with an ionic diffusion potential across the membrane. The model accounts for the anion effect on receptor potential, pH effects, the reversal of polarity when cells are treated with FeCl3, and the so-called "water reponse," depolarization of the taste cell upon dilution of the stimulant solution below a critical lower limit. The proposed model does not require both bound cationic and anionic receptors, and further suggests that limited access to a Stern-like region continuous with membrane channels may generally serve to control transport of ions.     

Similarity of ion dependence of odorant responses between lipid bilayer and olfactory system

In a previous paper (Nomura and Kurihara (1987) Biochemistry 26, 6135-6140), we demonstrated that the lipid bilayer membranes exhibit membrane potential changes in response to various odorants similarly to olfactory cells. The present study demonstrated that ion dependence of the responses of the lipid membranes to odorants is related to that in the carp olfactory system in the following point. (1) The responses to odorants diminished at low concentrations of salts and recovered upon addition of salts to stimulating solutions. (2) Divalent cations were effective in supporting the responses to odorants at much lower concentrations that monovalent cations. (3) Impermeable organic cations were effective in supporting the responses. The present results suggest that the initial process of generation of the receptor potential in olfactory cells resembles that in the lipid membranes.     

Effects of impermeant medium ions on the composition of rabbit renal cortical slices

When incubated in isosmotic oxygenated medium in which chloride was completely replaced by gluconate, rabbit renal cortical slices lost chloride with sodium, potassium and water before reaching a new steady-state composition after 15-30 min. When corrected for extracellular space, there was an electroneutral loss of alkali metal cations (Na + K) with chloride, accompanied by isosmotic loss of water from the cells. The losses of chloride and water were independent of medium pH over the range of 6.4-8.2, and were the same with potassium rather than sodium as the dominant medium cation. Incubation in isosmotic sodium chloride medium restored tissue composition of slices transferred from gluconate medium. This recovery was not dependent specifically upon medium chloride, for slice water content also recovered when nitrate rather than chloride was substituted for medium gluconate. With sodium completely replaced by n-methyl d-glucamine (nmdG+), cells in slices lost far more sodium and potassium than chloride before reaching a new steady-state composition after some 30 min. However, the loss of water was as predicted from the total losses of measured inorganic ions. With sodium and chloride completely replaced by nmdG+ and gluconate, there was a greater loss of water than found with unilateral substitutions. Again, the combined loss of diffusible inorganic cations exceeded the loss of chloride but the water loss was that expected for isosmotic loss accompanying the measured losses of ions. These results reveal that both gluconate and nmdG+ behave as impermeant ions in this tissue preparation. It is suggested that, in the absence of medium sodium, sodium-hydrogen exchange is inhibited. Retained hydrogen ions are buffered on charged cellular non-diffusible solutes and the associated hydroxyl (or bicarbonate) ions are lost from the cells accompanied by the inorganic univalent cations lost in excess of chloride in nmdG+ medium.     

Effects of nystatin on intracellular contents and membrane transport of alkali cations,and cell volume in HeLa cells

Summary Nystatin (50 g/ml) had strong influence on the intracellular contents and membrane transports of monovalent ions and water in HeLa cells. The nystatin-induced changes in the intracellular ion content and cell volume were inhibited by sucrose, and Donnan and osmotic equilibria were attained. Using cells under conditions for these equilibria, the concentrations of intracellular impermeant solutes, their mean valence, the differences of their intra- and extracellular osmotic concentrations, and the circumferential tension of the cell membrane were determined. Stimulation by nystatin of the influx of one cation species, e.g. Rb, was inhibited by another cation species, e.g. Na. The stimulatory effect of nystatin on cation fluxes was reversible within 1 hr after ionophore addition, and after 1-hr treatment the intracellular contents of Na and K became proportional to their extracellular concentrations, provided that the sum of these concentrations was constant (300mm). Similar proportionality was also observed in the presence of choline, provided that the choline concentration was less than those of the alkali cations. The implications of these results in relation to the osmotic properties of cultured cells, and the experimental regulation of alkali cations in the cells, are discussed.     

Properties of cyclic nucleotide-gated channels mediating olfactory transduction. Activation, selectivity, and blockage.

Cyclic nucleotide-gated channels (cng channels) in the sensory membrane of olfactory receptor cells, activated after the odorant-induced increase of cytosolic cAMP concentration, conduct the receptor current that elicits electrical excitation of the receptor neurons. We investigated properties of cng channels from frog and rat using inside-out and outside-out membrane patches excised from isolated olfactory receptor cells. Channels were activated by cAMP and cGMP with activation constants of 2.5-4.0 microM for cAMP and 1.0-1.8 for cGMP. Hill coefficients of dose-response curves were 1.4-1.8, indicating cooperativity of ligand binding. Selectivity for monovalent alkali cations and the Na/Li mole-fraction behavior identified the channel as a nonselective cation channel, having a cation-binding site of high field strength in the pore. Cytosolic pH effects suggest the presence of an additional titratable group which, when protonated, inhibits the cAMP-induced current with an apparent pK of 5.0-5.2. The pH effects were not voltage dependent. Several blockers of Ca2+ channels also blocked olfactory cng channels. Amiloride, D 600, and diltiazem inhibited the cAMP-induced current from the cytosolic side. Inhibition constants were voltage dependent with values of, respectively, 0.1, 0.3, and 1 mM at -60 mV, and 0.03, 0.02, and 0.2 mM at +60 mV. Our results suggest functional similarity between frog and rat cng channels, as well as marked differences to cng channels from photoreceptors and other tissues.     

Amino acid and divalent ion permeability of the pores formed by the Bacillus thuringiensis toxins Cry1Aa and Cry1Ac in insect midgut brush border membrane vesicles

The pores formed by Bacillus thuringiensis insecticidal toxins have been shown to allow the diffusion of a variety of monovalent cations and anions and neutral solutes. To further characterize their ion selectivity, membrane permeability induced by Cry1Aa and Cry1Ac to amino acids (Asp, Glu, Ser, Leu, His, Lys and Arg) and to divalent cations (Mg(2+), Ca(2+) and Ba(2+)) and anions (SO(4)(2-) and phosphate) was analyzed at pH 7.5 and 10.5 with midgut brush border membrane vesicles isolated from Manduca sexta and an osmotic swelling assay. Shifting pH from 7.5 to 10.5 increases the proportion of the more negatively charged species of amino acids and phosphate ions. All amino acids diffused well across the toxin-induced pores, but, except for aspartate and glutamate, amino acid permeability was lower at the higher pH. In the presence of either toxin, membrane permeability was higher for the chloride salts of divalent cations than for the potassium salts of divalent anions. These results clearly indicate that the pores are cation-selective.     

Osmotically induced removal of water from fungal cells as determined by a spin probe technique

Effects of physical environment on plasma membrane semipermeability and osmotic induction of changes in aqueous cytoplasmic volume were studied in vegetative and spore cells of a plant pathogenic fungus, Fusarium sulphureum. A direct method, employing a spin probe molecule that partitioned between intracellular aqueous and hydrophobic phases, allowed measurement of reversible water movement out of macroconidial cells and chlamydospores exposed to solutions of high osmolarity. Equilibrium distribution of the spin probe between intracellular aqueous and lipid phases was more rapid than movement of water in and out of the cells. The extent of water removal was exponentially dependent on osmotic strength. Some cells became irreversibly permeable to divalent cations on treatment with sodium chloride above 1.5 osmolar but addition of sucrose to the suspension medium at equivalent osmolar concentrations caused water removal without adversely affecting the viability. Sucrose also protected the plasma membrane against damage during freeze-drying. Induction of plasma membrane damage by osmotic shock or freeze-drying permitted rapid permeation of nickel ions. Neither slow equilibration of intracellular components with divalent paramagnetic cations nor partial permeability of damaged plasma membranes to these ions was observed.     

The responses to choline ions induced by transition metal ions in single water fibers of the frog glossopharyngeal nerve

In single water-sensitive fibers (water fibers) of the frogglossopharyngeal nerve, application of a solution of 500 mMcholine Cl to the tongue elicited responses of varying magnitude.Some water fibers (plain choline-insensitive water fibers) barelyresponded to the solution, while some water fibers (plain choline-sensitivewater fibers) exhibited a considerable response to this solution.NiCl₂. which is barely effective in producing neural responseat concentrations below 5 mM, induced the response of plaincholine-insensitrve water fibers to choline⁺ ions. It was confirmed,in a collision test, that the Ni²⁺-induced responses to choline⁺ions were derived from water fibers. However, NiCl₂ did notaffect the magnitude of me response generated by choline⁺ ionsin plain choline-sensitive water fibers. The concentration-responsecurve for choline Cl in the presence of 1 mM NiCl₂ for plaincholine-insensitive water fibers was similar to the curves obtainedin the absence of NiCl₂ for plain choline-sensitive water fibers.Other organic salts, such as tris(hydroxymethyl)arrdnomethane-HCl,triethanotamine-HCl and tetraethylammonium Cl, elicited no responseor only a very small response from water fibers, and NiCl₂ didnot affect these responses. It is suggested that there existsa choline receptor for the response to choline⁺ ions in theapical membrane of frog taste cells and that Ni²⁺ ions exposethe sites of such choline receptors, which are deeply embeddedin the receptor membrane, to the outside medium. The effectof Ni²⁺ ions results in an increase in the number of the cholinereceptor sites available for binding of choline⁺ ions. The rankorder of effectiveness of transition metal ions in elicitingthe appearance or enhancement of the response to choline Clwas Ni²⁺ > Co²⁺ > Mn²⁺. Mg²⁺ ions had no effect on theresponse to choline⁺ ions. A similar rank order was previouslyobtained in enhancement of the responses to Ca²⁺, Mg²⁺ and Na²⁺ions (Kitada, 1994a). It seems likely that the mechanism forenhancement or elicitation of the response to choline⁺ ionsby the transition metal ions has features in common with thatfor enhancement of the responses to Ca²⁺, Mg²⁺ and Na⁺ ions.     

Affinity Shifts Induced by Cations Do Not Reliably Predict the Agonistic or Antagonistic Nature of Ligands at Brain Dopamine Receptors

The effects of Ca2+ and Na+ ions on the affinities of rat striatal dopamine D1 and D2 receptors for a wide range of agonists and antagonists were investigated. These experiments were performed at 37 degrees C, since it was found that at this physiological temperature D2 receptor affinities for dopamine and 2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene were clearly lower than at room temperature. No correlation was found between the effects of the cations on the affinities of compounds for D1 and D2 receptors and their agonistic or antagonistic nature. On the other hand, the Hill coefficients of agonists but not of antagonists were consistently and significantly below unity, with either Ca2+ or Na+ ions present and at both receptors. This suggests the existence of yet another type of heterogeneity of D1 and D2 receptor forms, to which agonists but not antagonists are sensitive. It is thus concluded that changes in D1 and D2 receptor affinities induced by cations do not predict the agonistic or antagonistic nature of a compound. However, since dopamine itself was sensitive to Na+ or Ca2+ ions, this mechanism might play a role in the regulation of receptor affinities in synaptic transmission, in addition to that exerted by guanyl nucleotides.     

Active transport of thallous ions by Streptococcus lactis.

Tl+ ions have been shown to mimic or compete with K+ in a number of membrane systems. We confirmed that in starved, valinomycin-treated cells of Streptococcus lactis 7962, Tl+ ions distributed themselves across the bacterial membrane in response to the potassium diffusion potential. In glucose-energized cells, however, Tl+ was taken up by a system specifically stimulated by sodium salts. The intracellular levels of Tl+ exceeded those attained by [3H]triphenylmethylphosphonium ion, a lipophilic cation which accumulates in response to the membrane potential. The uptake of Tl+ by (Na+ and glucose)-stimulated cells was strongly inhibited by potassium salts. These experiments suggest that metabolic energy is coupled to Tl+ transport by means of a high energy phosphate compound and that Tl+ ions are actively transported by a membrane carrier whose normal substrate is K+. The uptake of Tl+ is not a valid method for determining the streptococcal membrane potential.     

Effects of alkali metal cations on root extension in germinating tomato seedlings

Tomato seeds exhibited high germination percentages on the chloride salts of the alkali metal cations Li, Na, K, Rb, and Cs. Root extension was normal in seedlings germinated in light or dark on Li, Na, or K but was severely suppressed on Rb and Cs in both environments. Germination percentages and root extension on alkali sulphate salts were similar to those observed on alkali chloride salts. Suppression of root extension by Rb and Cs was not cultivar specific.     

Studies related to the metabolism of anabolic steroids in the horse: the metabolism of 1-dehydrotestosterone and the use of fast atom bombardment mass spectrometry in the identification of steroid conjugates

The metabolism and urinary excretion of 1,2(n)-3H-1-dehydrotestosterone were studied in cross-bred gelded horses. Approximately 40% of the dose was excreted in 24 h. The steroid metabolites were extracted by Amberlite XAD-2 resin and fractionated into glucuronides and sulphoconjugates. Unchanged 1-dehydrotestosterone was the only component identified by gas chromatography mass spectrometry after solvolysis of the sulphoconjugates. Positive and negative ion fast atom bombardment mass spectra were obtained on the purified 1-dehydrotestosterone sulphoconjugate isolated from horse urine and on the alkali metal salts of three standard steroid conjugates. Spectra obtained in the different modes were of comparable intensity. Positive ion spectra were generally more complex due to the formation of alkali metal adduct ions containing several sodium cations. The most abundant ion in the negative ion spectra corresponded to the loss of the alkali metal cation to give [M]-. Thus, the structure of a conjugate can be defined from the combination of mass spectrometric techniques.     

Diffusion of cations in salt solutions between ice walls

It is important for food, medical and energy engineering to control ice growth during cooling processes. Adding salts and antifreeze protein to water is a promising method for doing this. However, the effects of ions in solution on ice–water interface in narrow spaces have not yet been clarified. Therefore, we carried out molecular dynamics simulations of sodium chloride, potassium chloride and calcium chloride in water between ice walls. It was found that the peaks of the radial distribution function for calcium ions are highest among the cations. In addition, the diffusion coefficient of calcium ions was lower than those of the other ions. A further finding was that one water molecule on the interface and two water molecules adjacent to the interface in liquid phase were positioned in the first or second hydration shell for the calcium ion, which was located at the closest point to the interface. Such hydration shells caused the reductions in motion and diffusion coefficient of the specific calcium ion.     

The modifications of the final stages of the complement reaction by alkali metal cations.

We studied the effects of alkali metal cations on the terminal stages of complement lysis of human and sheep HK erythrocytes. Sensitized erythrocytes (EA) were reacted with limited amounts of complement for 1 hr at 37 degrees C in buffer containing 147 mM NaCl (Na buffer), which resulted in 10-40% lysis. The unlysed cells were washed with Na buffer at 0-2 degrees C and incubated for 1 hr at 37 degrees C in buffers containing 147 mM of the various alkali metal cations. Although additional lysis (25 to 65%) occurred with K, Rb, or Cs buffer, only minor degrees developed with Na or Li buffer, only minor degrees developed with Na or Li buffer. Intermediate levels occurred with 100 mM of the divalent alkali cations. Halogen ions and SCN-(147 MM), Ca++ (0.15mM), and Mg++ (0.5 mM) did not alter the effect of the alkali metal cations. Lysis occurring in K+, Rb+ or Cs+ proceeded without lag, was temperature dependent with an optimum of 43 degrees C, and had a pH optimum of 6.5. Lysis in K and Na buffers was unaffected by 10(-3) to 10(-5) M ouabain. Experiments with mixtures of cations indicated that Na+ had a mild inhibitory effect that could be totally overcome by K+, partially by Rb+, and not at all by Cs+. Li+ had a strong inhibitory effect, 6 X 10(-5) M causing 50% inhibition in buffers containing 147 mM K+, Rb+, or Cs+. By using intermediate complexes of EA and purified complement components we demonstrated that K+ enhances the lytic action of C8 on EAC1-7 as well as that of C9 on EAC1-8. It was known that Li+ facilitates lysis when acting on the entire complement reaction. We found that Li+ enhanced the lytic action of C8 on EAC1-7, with a kinetic that differed from that of the K+ effect. In addition, Li+ inhibited the enhancing effect of K+ upon lysis of EAC1-8 by C9. This occurred at concentration of Li+ similar to those which inhibited the additional lysis by K+, Rb+, and Cs+ of cells that were pretreated in Na buffer with the entire complement sequence. We propose that the major effects of alkali metal cations on complement lysis are due to their interaction with C8 and/or membrane constitutes.     

9-Amino-acridine as a probe of the electrical double layer associated with the chloroplast thylakoid membranes

Chloroplasts washed with monovalent cations are found to quench 9-amino-acridine fluorescence after resuspension in a cation-free medium. This quenching occurs in the absence of a high energy state and can be reversed by the addition of salts. The effectiveness of these salts is related to the charge carried by the cations and appears to be essentially independent of the associated anions. The order of effectiveness is polyvalent > divalent > monovalent, and virtually no variation is found within the groups of monovalent cations and divalent cations tested. Furthermore, choline and lysine are as effective as alkali metal cations, and lysyl-lysine is almost as effective as alkaline earth metal cations. These results are consistent with an effect mediated by the electrical double layer at the membrane surface rather than chemical bonding, and can be qualitatively explained in terms of the Gouy-Chapman theory.It appears that 9-amino-acridine acts as a diffusible monovalent cation which increases its fluorescence when displaced from the diffuse layer adjacent to the negatively charged membrane surface. The 9-amino-acridine fluorescence changes have been experimentally correlated with the cation-induced chlorophyll a fluorescence changes also observed with isolated chloroplasts.