The Journal of Membrane Biology

Okazaki, Y.; Tazawa, M.

doi: 10.1007/BF01869213pmid: 1691298

Ohki, S.; Arnold, K.

doi: 10.1007/BF01869214pmid: 2325117

Membrane fusion induced by ions and its associated membrane property, surface dielectric constant, were studied with the use of acidic and neutral phospholipid vesicles. The fusion of vesicles was monitored by utilizing two fluorescence fusion assays: fluorescence content mixing method and fluorescence labelled membrane component dilution method. For the surface dielectric constant measurements, a fluorescence method was used which detected the environmental effect on the membrane surface upon the addition of various fusogenic cations. Also, the effects of poly-(ethylene glycol) on both fusion and surface dielectric properties were examined. It was found that the extent of fusion correlated well with the degree of lowering in the dielectric constant of the surface membrane, which corresponds to the increase in hydrophobicity of the membrane surface. This agrees with the previously obtained experimental results that the increase in interfacial tension of the membrane, which also corresponds to the increase in surface hydrophobicity, correlates with the extent of membrane fusion.

Blatt, Michael; Beilby, Mary; Tester, Mark

doi: 10.1007/BF01869215pmid: 2157844

It is generally agreed that solute transport across theChara plasma membrane is energized by a proton electrochemical gradient maintained by an H+-extruding ATPase. Nonetheless, as deduced from steady-state current-voltage (I-V) measurements, the kinetic and thermodynamic constraints on H+-ATPase function remain in dispute. Uncertainties necessarily surround long-term effects of the relatively nonspecific antagonists used in the past; but a second, and potentially more serious problem has sprung from the custom of subtracting, across the voltage spectrum, currents recorded following pump inhibition from currents measured in the control. This practice must fail to yield the trueI-V profile for the pump when treatments alter the thermodynamic pressure on transport.

Sjodin, R.; Mahmoud, A.; Montes, J.

doi: 10.1007/BF01869216pmid: 2109079

Giant axons from the marine annelidMyxicola infundibulum were internally dialyzed with solutions containing22Na ions as tracers of Na efflux. In experiments performed in Li-substituted seawater, Na efflux that is dependent on external Ca ion concentration, [Ca2+] o , was measured using dialysis to maintain [Na+] i at 100mm, which enhances the [Ca2+] o -dependent Na efflux component, (i.e., reverse-mode Na/Ca exchange). When dialysis fluid contained EGTA (1mm) to buffer the internal Ca concentration, [Ca2+] i , to desired levels, Na efflux lost its normal sensitivity to external calcium. The inhibition was not simply due to the Ca-chelating action of EGTA to produce insufficient [Ca2+] i to activate Na/Ca exchange. The addition of EGTA inhibited Ca o -dependent Na efflux even when a large enough excess of [Ca2+] i was present to saturate the EGTA and still produce elevated values of [Ca2+] i . Control experiments showed that these high values of [Ca2+] i resulted in normal Na/Ca exchange in the absence of EGTA. It is concluded that the presence of EGTA itself interferes with the manifestation of reverse-mode Na/Ca exchange inMyxicola giant axons.

Bosma, Martha; Moody, William

doi: 10.1007/BF01869217pmid: 2157845

Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidianCiona intestinalis to investigate the properties of two voltage-dependent Ca2+ currents found in this cell. The peak of the low threshold current (channel I) occurred at −20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca2+ and Mg2+ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during changel I openings were recorded when micromolar concentrations of Ca2+ or Mg2+ were added to the patch pipette solutions. Slopes of the blocking rate constantvs. concentration gave binding constants of 6.4×106 m −1 sec−1 for Mg2+ and 4.5×108 m −1 sec−1 for Ca2+. The Ca2+ block was somewhat relieved at negative potentials, whereas the Mg2+ block was not, suggesting that Ca2+, but not Mg2+, can exit from the binding site toward the cell interior.

Hijden, H.; Grell, E.; Pont, J.; Bamberg, E.

doi: 10.1007/BF01869218pmid: 2157846

Membrane fragments containing the H+K−-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na+K+-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J. 4:3079–3085). Since H+K+-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K+. By separation of the H+ and K+ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K+ or at low concentrations of K+. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K+ transport during dephosphorylation is electrogenic, too, in contrast to the Na+K+-ATPase where the K+ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H+K+-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.

Kikuchi, Kazuhiro; Kikuchi, Toshiko; Ghishan, Fayez

doi: 10.1007/BF01869219pmid: 2157847

The developmental maturation of Na+−H+ antiporter was determined using a well-validated brush-border membrane vesicles (BBMV's) technique. Na+ uptake represented transport into an osmotically sensitive intravesicular space as evidenced by an osmolality study at equilibrium. An outwardly directed pH gradient (pH inside/pH outside=5.2/7.5) significantly stimulated Na+ uptake compared with no pH gradient conditions at all age groups; however, the magnitude of stimulation was significantly different between the age groups. Moreover, the imposition of greater pH gradient across the vesicles resulted in marked stimulation of Na+ uptake which increased with advancing age. Na+ uptake represented an electroneutral process.