The Journal of Membrane Biology

Wright, J.

doi: 10.1007/BF01870785pmid: N/A

Ion: solute cotransporters frequency are incapable of achieving equilibrium between the solute accumulation and the transmembrane difference of the electrochemical potential of the ion. The presence of uncoupled flows of ion and solutes (leaks) is often advanced as an explanation. Here an alternative is discussed. The net accumulation of solute may be so slow that equilibrium can never be attained at finite times (e.g., several hours). Cotransporters may exhibit strong product inhibition, and the net influx of solute approaches zero far from equilibrium. The inherent slowness of net transport under these conditions is termed catalytic inefficiency. The likelihood that galactoside: H+ cotransport inEscherichia coli, hexose: H+ cotransport inChlorella vulgaris, andd-glucose: Na+ cotransport in brush-border membranes exhibit catalytic inefficiency is examined. The existence of strong product inhibition complicates the determination of the stoichiometry of cotransport and the characterization of chemically modified or mutant cotransporters.

Bertl, Adam

doi: 10.1007/BF01870786pmid: N/A

The membrane of mechanically prepared vesicles ofChara corallina has been investigated by patch-clamp techniques. This membrane consists of tonoplast as demonstrated by the measurement of ATP-driven currents directed into the vesicles as well as by the ATP-dependent accumulation of neutral red. Addition of 1mm ATP to the bath medium induced a membrane current of about 3.2 mA·m−2 creating a voltage across the tonoplast of about −7 mV (cytoplasmic side negative). On excised tonoplast patches, currents through single K+-selective channels have been investigated under various ionic conditions. The open-channel currents saturate at large voltage displacements from the equilibrium voltage for K+ with limiting currents of about +15 and −30 pA, respectively, as measured in symmetric 250mm KCl solutions. The channel is virtually impermeable to Na+ and Cl−. However, addition of Na+ decreases the K+ currents. TheI–V relationships of the open channel as measured at various K+ concentrations with or without Na+ added are described by a 6-state model, the 12 parameters of which are determined to fit the experimental data.

Mulhern, Dally; Fishman, Peter; Spiegel, Sarah

doi: 10.1007/BF01870787pmid: 2769735

The fluorescent anionic dye, bisoxonol, and flow cytometry have been used to monitor changes in the membrane potential of rat thymocytes exposed to the B subunit of cholera toxin. The B subunit induced a rapid hyperpolarization, which was due to activation of a Ca2+-sensitive K+ channel. Reduction of extracellular Ca2+ to <1 μm by the addition of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid immediately abolished the hyperpolarization caused by the B subunit. Cells treated with quinine and tetraethylammonium lost their ability to respond to the B subunit, whereas 4-aminopyridine did not have any effect. Thus, calcium-sensitive and not voltage-gated K+ channels appeared to be responsible for the hyperpolarization. The results of ion substitution experiments indicated that extracellular Na+ was not essential for changes in membrane potential. Further studies with ouabain, amiloride and furosemide demonstrated that electrogenic Na+/K+ ATPase, Na+/H+ antiporter and Na+/K+/Cl− cotransporter, respectively, were not involved in the hyperpolarization process induced by the B subunit. Thus, crosslinking of several molecules of ganglioside GM1 on the cell surface of rat thymocytes by the pentavalent B subunit of cholera toxin modulated plasma membrane permeability to K+ by triggering the opening of Ca2+-sensitive K+ channels. A role for gangliosides in regulating ion permeability would have important implications for the function of gangliosides in various cellular phenomena.

Roper, S.; McBride, D.

doi: 10.1007/BF01870788pmid: 2475632

The presence and regional localization of voltagegated ion channels on taste cells inNecturus maculosus were studied. Lingual epithelium was dissected from the animal and placed in a modified Ussing chamber such that individual taste cells could be impaled with intracellular microelectrodes and the chemical environment of the apical and basolateral membranes of cells could be strictly controlled. That is, solutions bathing the the mucosal and serosal surfaces of the epithelium could be exchanged independently and the effects of pharmacological agents could be tested selectively on the apical or basolateral membranes of taste cells. In the presence of amphibian physiological saline, action potentials were elicited by passing brief depolarizing current pulses through the recording electrode. Action potentials provided a convenient assay of voltage-gated ion channels. As in other excitable tissues, blocking current through Na+, K+, or Ca2+ channels had predictable and consistent effects on the shape and magnitude of the action potential. A series of experiments was conducted in which the shape and duration of regenerative action potentials were monitored when the ionic composition was altered and/or pharmacological blocking agents were added to the mucosal or to the serosal chamber. We have found the following: (1) voltage-gated K+ channels (delayed rectifier) are found predominately, if not exclusively, on the chemoreceptive apical membrane; (ii) voltage-gated Na+ and Ca2+ channels are found on the apical (chemoreceptive) and basolateral (synaptic) membrane; (iii) there is a K+ leak channel on the basolateral membrane which appears to vary seasonally in its sensitivity to TEA. The nonuniform distribution of voltage-gated K+ channels and their predominance on the apical membrane may be important in taste transduction: alterations in apical K+ conductance may underlie receptor potentials ellicted by rapid stimuli.

Todd, A.; Mehlhorn, Rolf; Macey, Robert

doi: 10.1007/BF01870789pmid: 2549250

Permeabilities for a homologous series of amine and carboxylate nitroxide spin probes were measured in human red blood cells by an electron paramagnetic resonance (EPR) method. Permeabilities determined in this study are much lower than would be predicted for a sheet of bulk hydrocarbon and the polarity of the rate-limiting region is shown to be greater than bulk hydrocarbon. This suggests that the rate-limiting region for permeation of these nonelectrolytes is somewhere in the membrane periphery rather than in the center of the membrane. The red cell membrane does not discriminate between these probes on the basis of molecular volume, as might be predicted by a simple free-volume theory of membrane permeation.

Todd, A.; Mehlhorn, Rolf; Macey, Robert

doi: 10.1007/BF01870790pmid: 2549251

Permeabilities for an homologous series of amine nitroxide spin probes were measured in liposomes of varying composition by an electron paramagnetic resonance (EPR) method. Results show that the rate-limiting step in permeation is not adsorption/desorption at the aqueous/membrane interface for two probes in phosphatidylcholine/phosphatidic acid liposomes and for one probe in phosphatidylcholine/cholesterol/phosphatidic acid liposomes. Accordingly, we interpret observed selectivity patterns for the entire series of probes in liposomes and red cells in terms of the properties of the bilayer interior.

Reeves, W.; McDonald, Glenn; Mehta, Pramod; Andreoli, Thomas

doi: 10.1007/BF01870791pmid: 2769736

ADH, acting through cAMP, increases the potassium conductance of apical membranes of mouse medullary thick ascending limbs of Henle. The present studies tested whether exposure of renal medullary apical membranes in vitro to the catalytic subunit of cAMP-dependent protein kinase resulted in an increase in potassium conductance. Apical membrane vesicles prepared from rabbit outer renal medulla demonstrated bumetanide-and chloride-sensitive22Na+ uptake and barium-sensitive, voltage-dependent86Rb+-influx. When vesicles were loaded with purified catalytic subunit of cAMP-dependent protein kinase (150 mU/ml), 1mm ATP, and 50mm KCl, the barium-sensitive86Rb+ influx increased from 361±138 to 528±120pm/mg prot · 30 sec (P<0.01). This increase was inhibited completely when heat-stable protein kinase inhibitor (1 μg/ml) was also present in the vesicle solutions. The stimulation of86Rb+ uptake by protein kinase required ATP rather than ADP. It also required opening of the vesicles by hypotonic shock, presumably to allow the kinase free access to the cytoplasmic face of the membranes. We conclude that cAMP-dependent protein kinase-mediated phosphorylation of apical membranes from the renal medulla increases the potassium conductance of these membranes. This mechanism may account for the ADH-mediated increase in potassium conductance in the mouse mTALH.

Dulhunty, Angela

doi: 10.1007/BF01870792pmid: 2769737

The structure of the triad junction was examined in thin sections of mammalian fast-twitch skeletal muscle. The aims of the experiments were twofold: first, to examine relationships between the contents of the junctional gap and the terminal cisternae that could be significant in excitation-contraction coupling and, second, to look for structures in the transverse tubules that could support a calcium buffer system. Procedures known to stabilize cytoskeletal elements were used in an attempt to retain the original structure. “Feet”, “pillars” and “bridges” were often seen side by side in the same junction. In one such junction, the average center-to-center spacing between four bridges was 30.9±1.7 nm and between five foot-like structures was 29.2±1.4 nm. The subunit structure of the feet could be seen in many sections. The lumen of the terminal cisternae was filled with a tetragonal network of calsequestrin which formed parallel strands near the junctional membrane, in register with the feet. The strands overlay the area occupied by “rods” seen in freeze-fracture replicas of terminal cisterna membrane. The contents of the transverse tubules were aggregated into bands, or “tethers”, which extended across the short axis of the tubule at regular intervals of about 30 nm. The tethers consisted of flattened discs, stacked across the long axis of the tubule, aligned with the junctional feet. Lanthanum staining of the tethers indicated cationic binding sites that could buffer luminal calcium ion concentration in the vicinity of the voltage sensor for contraction. It is suggested (i) that the control of calcium concentration near the voltage sensor is necessary for normal activation, (ii) that feet, pillars and bridges are different images of a spanning structure, and (iii) that the regular alignment of tethers, feet and calsequestrin is functionally significant in excitation-contraction coupling.

Changya, Lu; Gallacher, David; Irvine, Robin; Potter, Barry; Petersen, Ole

doi: 10.1007/BF01870793pmid: 2788744

We have examined the effects of various inositol polyphosphates, alone and in combination, on the Ca2+-activated K+ current in internally perfused, single mouse lacrimal acinar cells. We used the patch-clamp technique for whole-cell current recording with a set-up allowing exchange of the pipette solution during individual experiments so that control and test periods could be directly compared in individual cells. Inositol 1,4,5-trisphosphate (Ins 1,4,5 P3) (10–100 μm) evoked a transient increase in the Ca2+-sensitive K+ current that was independent of the presence of Ca2+ in the external solution. The transient nature of the Ins 1,4,5 P3 effect was not due to rapid metabolic breakdown, as similar responses were obtained in the presence of 5mm 2,3-diphosphoglyceric acid, that blocks the hydrolysis of Ins 1,4,5 P3, as well as with the stable analoguedl-inositol 1,4,5-trisphosphorothioate (Ins 1,4,5 P(S)3) (100 μm). Ins 1,3,4 P3 (50 μm) had no effect, whereas 50 μm Ins 2,4,5 P3 evoked responses similar to those obtained by 10 μm Ins 1,4,5 P3. A sustained increase in Ca2+-dependent K+ current was only observed when inositol 1,3,4,5-tetrakisphosphate (Ins 1,3,4,5 P4) (10 μm) was added to the Ins 1,4,5 P3 (10 μm)-containing solution and this effect could be terminated by removal of external Ca2+. The effect of Ins 1,3,4,5 P4 was specifically dependent on the presence of Ins 1,4,5 P3 as it was not found when 10 μm concentrations of Ins 1,3,4 P3 or Ins 2,4,5 P3 were used. Ins 2,4,5 P3 (but not Ins 1,3,4 P3) at the higher concentration of 50 μm did, however, support the Ins 1,3,4,5 P4-evoked sustained current activation. Ins 1,3,4 P3 could not evoke sustained responses in combination with Ins 1,4,5 P3 excluding the possibility that the action of Ins 1,3,4,5 P4 could be mediated by its breakdown product Ins 1,3,4 P3. Ins 1,3,4,5 P4 also evoked a sustained response when added to an Ins 1,4,5 P(S)3-containing solution. Ins 1,3,4,5,6 P5 (50 μm) did not evoke any effect when administered on top of Ins 1,4,5 P3. In the absence of external Ca2+, addition of Ins 1,3,4,5 P4 to an Ins 1,4,5 P3-containing internal solution evoked a second transient K+ current activation. Readmitting external Ca2+ in the continued presence internally of Ins 1,4,5 P3 and Ins 1,3,4,5 P4 made the response reappear. We conclude that both Ins 1,4,5 P3 and Ins 1,3,4,5 P4 play crucial and specific roles in controlling intracellular Ca2+ homeostasis.