Enhancement of rates of H+, Na+ and K+ transport across phospholipid vesicular membrane by the combined action of carbonyl cyanide m-chlorophenylhydrazone and valinomycin: temperature-jump studies

Abstract

Enhancement of delta pH relaxation rate by the combined action of valinomycin (VAL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) has been studied under a variety of concentration conditions in soyabean phospholipid (SBPL) vesicles after creating a pH gradient across the vesicular membrane delta pH by temperature jump. After taking note of the changes by VAL and CCCP induced membrane disorder (using nigericin and monensin mediated delta pH decay as probes) the following could be inferred about the mechanism of enhancement of delta pH decay rate: (i) in solutions containing KCl, the rate limiting species have been identified to be (a) Val-K(+)-CCCP-, at low [Val]0 and [CCCP]0 (with translocation rate constant k2 approximatley 3.2 x 10(3) s-1); (b) CCCPH, at high [Val]0 (with translocation rate constant k1 approximately 2 x 10(5) s-1); (c) the neutral valinomycin species Val, at high [CCCP]0. (ii) In solutions containing NaCl, in our concentration range, the rate limiting species are Val-Na(+)-CCCP-. (iii) The apparent dissociation constant K*M of Val-M+ decreases with pH in SBPL vesicles but is independent of pH in vesicles prepared from PC + 6% PA. (iv) The differences in the ionic strength dependencies of kinetic data shows that the environments of Na+ and K+ binding sites on VAL are different. (v) In vesicle solutions containing 100 mM MCl, the cation selectivity of VAL (towards K+ in preference to Na+) is reduced when CCCP- is already bound to it in the membrane. The CCCP- dissociation constant of Val-M(+)-CCCP- is smaller with M+ = Na+ (approximatley 0.22 mM at 100 mM NaCl) when compared to that with M+ = K+ (approximately 2 mM at 100 mM KCl). Attributing these differences to the differences in electrostatic interaction between CCCP- and M+ in Val-M(+)-CCCP-, we can say that CCCP- binds closer to the Na+ binding site than to the K+ binding site on VAL.

New insights into the P‐glycoprotein‐mediated effluxes of rhodamines

“…At these concentrations neither valinomycin nor FCCP inhibits the P‐gp‐mediated efflux of drug [22]. It has previously been observed that FCCP and valinomycin in combination can precipitate in the presence of potassium [23,24]. However, under our experimental conditions, the FCCP concentration was 100‐fold higher than that of valinomycin.…”

Synergy in K+/H+ exchange across phospholipid vesicle membranes with combinations of valinomycin and chlorophenolsThe T-jump experiments described in this work were carried out during James Hudson's visit to Tata Institute of Fundamental Research, Mumbai 400 005, India during 2000–2001.

“…[8][9][10] The magnitude of t depends on the size of the vesicles. 12 By monitoring the optical densities of vesicle suspensions at 700 nm the vesicle sizes were regulated such that the variation of t was within 20%. Reproducibility of relaxation traces (amplitudes and t) even after subjecting the same sample to several Tjumps confirmed that the integrity of vesicles was not affected and vesicles did not fuse due to T-jump in our experiments.…”

“…If the rate limiting step of DpH decay is the translocation of a rate limiting species across the membrane with rate constant k r1 and if b i is the internal buffer capacity of vesicles. 8,10,12 1=t ¼ ðln 10=b i Þk r1 ½rate limiting species m ð1Þ…”

“…The above-mentioned basic requirements for fast DpH decay can be fulfilled in various ways. It has been shown in the literature that the following also facilitate DpH decay: (a) gramicidin-A channels through which both H + and M + are transported; 9 (b) synergy associated with a combination of two species: a protonophore and a M + carrier 12,13 (such as by the mechanism of Fig. 1); (c) synergy from a combination of two species: a protonophore and gramicidin-A channel; 14 y The T-jump experiments described in this work were carried out during James Hudson's visit to Tata Institute of Fundamental Research, Mumbai 400 005, India during 2000-2001.…”

Evidence for Dimer Participation and Evidence Against Channel Mechanism in A23187-Mediated Monovalent Metal Ion Transport Across Phospholipid Vesicular Membrane

“…If the translocation of the M ϩ carriers Cal 2 MM or Cal 2 MH is the rate-limiting step it must be possible to increase the translocation rates (and 1/) by disturbing the membrane order such as by adding valinomycin at sufficiently high concentrations (Prabhananda and Kombrabail, 1995). The ⌬pH relaxation traces shown in Fig.…”