Temperature jump as a new technique to study the kinetics of fast transport of protons across membranes

Abstract

Application of a temperature jump (2.5 degrees C) to a suspension of liposomes, having phosphate (delta pK/delta T approximately 0.005) as the internal buffer and tris(hydroxymethyl)aminomethane (delta pK/delta T approximately 0.031) as the external buffer, created a delta pH (pHin - pHout) of positive sign in ca. 5 microseconds. Decay of this delta pH was monitored by using the fluorescent pH indicator 8-hydroxy-1,3,6-pyrenetrisulfonic acid entrapped inside the liposome. This technique is useful to study transmembrane proton movement in the time range 5 microseconds-10 s at physiological pH values. The kinetics of proton transport aided by ion carriers such as nigericin, monensin, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and valinomycin were studied by our method. The electrogenic nature of transport by CCCP and valinomycin and electroneutral ion transport by nigericin and monensin were shown. From the kinetics of proton transport aided by gramicidin, the time-averaged single-channel conductance of gramicidin channels was estimated to be (2.1 +/- 0.5) X 10(-16) S for H+ at pH 7.5.

Lipid raft components cholesterol and sphingomyelin increase H+/OH− permeability of phosphatidylcholine membranes

“…The agreement between the single channel proton conductance that we arrived at and the value reported by Krishnamoorthy [22] at pH 7.5 indicates that our proton permeability coefficients are reasonable. For pure POPC liposomes, P H+ was 2.36 × 10 −2 cm/s.…”

“…Since our assay system gives us a value which is in excellent agreement with one estimate of single channel water conductance and not more than 5-fold different from the other estimate, we believe that our proton permeability coefficients under the specific conditions employed are likely to be reasonably accurate. When 1 µM gramicidin was used instead of 10 µM, conductances of 2.67 × 10 −17 and 1.60 × 10 −16 S were obtained -also in very good agreement with the Krishnamoorthy [22] value.…”

“…Since we can estimate the number of channels as well as the number of liposomes, our measurements of proton permeability, which rely upon pH-dependent fluorescence quenching of intravesicular CF, then allowed us to calculate a single channel proton conductance for gramicidin. That number was compared with a previously determined value for proton conductance measured at a similar pH [22].…”

Rotational Dynamics of Lipid/Detergent Mixtures: A Mechanism for Membrane Protein Reconstitution into Lipid Vesicles

“…Phospholipid (soybean) dispersions were obtained by sonication of the lipid in buffered aqueous solution in nitrogen atmosphere. 9 Asolectin contains 95-98% phospholipids of various types ( Figure 1C). …”

Rapid proton release during flash‐induced oxidation of cytochrome c oxidase

“…However, in studies of uncoupler-cat~ysed proton equilibration in vesicle systems [8,9], half-lives of a few milliseconds have been found at comparable concentrations of uncouplers. Conclusions from experiments using uncouplers would require that proton equilibration across the lipid membrane is rapid compared to the process under study, and therefore more work seems necessary to define conditions for complete uncoupling on the sub-millisecond time scale.…”