Selective ion binding and membrane activity of synthetic cyclopeptides.

Four cyclic peptides related to the membrane-active complexones PV, cyclo-(L-Pro-Lval-D-Pro-D-Val)3, and valinomycin were synthesized: (1) cyclo-(L-Pro-L-Ala-D-Val)3 or PVPA, (2) cyclo-(L-Ala-L-Val-D-Pro-D-Val)3 or PVAV, (3) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2-L-Pro-D-Val or PV-10, (4) cyclo-(L-Pro-L-Val-D-Pro-D-Val)2 or PV-8. In a two-phase extraction assay the affinity of PV and PVPA for alkali picrates was about three orders or magnitude greater than that of valinomycin. It was about equal to valinomycin for PVAV and much lower for PV-10 and PV-8. PV, PVPA and PVAV showed a selectivity sequence similar to that of valinomycin, namely K+ approximately Rb+ greater than Cs+ greater than Na+ greater than Li+. In the series PV, PV-10, PV-, the preference for K+ over Na+ was 700, 5 and less than 1, respectively. Thus, it was possible to reverse the selectivity of PV for K+ over Na+ by reducing the ring size from 12 to 8 amino acid residues. In sheep red cell lipid bilayer membranes PVPA increased the membrane conductance significantly in the presence of either KCl or NaCl but it was less potent than PV. PV-10, PV-8 and PVAV on the other hand were ineffective in this assay. The inactivity of PVAV as a potassium carrier in membrane was in contrast to its high affinity for potassium picrate in two-phase assays. Such a behaviour may be observed of a compound that has too low an aqueous cation binding constant to use the solution-complexation mechanism of PV (Davis et al. (1976) Biochemistry 15, 768--774 and Pinkerton et al. (1969) Biochem. Biophys. Res. Commun. 35, 512--518) and too slow binding and release kinetics to use the interfacial-complexation mechanism of valinomycin.

Mechanism of ion transport through lipid bilayer-membranes mediated by peptide cyclo-(D-Val-L-Pro-L-Val-D-Pro).

The cyclic dodecapeptide PV, cyclo-(D-Val-L-Pro-L-Val-D-Pro)3, a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes. This paper reports the results of two types of relaxation experiments, namely relaxation of the membrane current after a voltage jump and decay of the membrane voltage after a charge pulse in lipid bilayer membranes exposed to PV. From the relaxation data, the rate constant for the translocation of the ion carrier complex across the membrane, as well as the partition coefficient of the complex between water and membrane solution interface were computed and found to be about one order of magnitude less than the comparable values for valinomycin (Val). Furthermore, the dependence of the initial membrane conductivity on ion concentration was used to evaluate the equilibrium constant, K, of complexation between PV and some monovalent cations in water. The values of K yield the following selectivity sequence of PV: Na+ less than NH4+ less than K+ less than Cs+ less than Rb+. These and earlier results are consistent with the idea that PV promotes cation movement across membranes by the solution complexation mechanism which involves complexation between ion and carrier in the aqueous phase and transport of the carrier across the membrane. In the particular form of the solution complexation mechanism operating here, the PV present in the PV-cation complex carrying charge across the membrane derives from the side from which the current is flowing (cis-mechanism). As shown previously, valinomycin, in contrast to PV, acts by an interfacial complexation mechanism in which the Val in the Val-cation complex derives from the side toward which current is flowing (trans-mechanism). The comparison of the kinetic properties of these two closely related compounds yields interesting insights into the relationship between chemical structure and function of ion carriers.

Effect of peptide PV on the ionic permeability of lipid bilayer membranes.

THIS PAPER REPORTS THE EFFECTS OF PEPTIDE PV (PRIMARY STRUCTURE: cyclo-(D-val-L-pro-L-val-D-pro)(delta)) on the electrical properties of sheep red cell lipid bilayers. The membrane conductance (G(m)) induced by PV in either Na(+) or K(+) medium is proportional to the concentration of PV in the aqueous phase. The PV concentration required to produce a comparable increase in G(m) in K(+) medium is about 10(4) times greater than for its analogue, valinomycin (val). Although the selectivity sequence for PV and val is similar, K(+) greater, similar Rb(+) > Cs(+) > NH(4) (+) > TI(+) > Na(+) > Li(+); the ratio of GGm in K(+) to that in Na(+) is about 10 for PV compared to > 10(3) for val. When equal concentrations of PV are added to both sides of a bilayer, the membrane current approaches a maximum value independent of voltage when the membrane potential exceeds 100 mV. When PV is added to only one side of a bilayer separating identical salt solutions of either Na(+) or K(+) salts, rectification occurs such that the positive current flows more easily away rather than toward the side containing the carrier. Under these conditions, a large, stable, zero-current potential (VVm) is also observed, with the side containing PV being negative. The magnitude of this VVm is about 90 mV and relatively independent of PV concentration when the latter is larger than 2 Times; 10(-5) M. From a model which assumes that V(m) equals the equilibrium potential for the PV-cation complexes (MS(+)) and that the reaction between PV and cations is at equilibrium on the two membrane surfaces, we compute the permeability of the membrane to free PV to be about 10(-5) cm s(-1), which is about 10(-7) times the permeability of similar membranes to free val. This interpretation is supported by the fact that the observed values of V(m) are in agreement with the calculated equilibrium potential for MS(+) over a wide range of ratios of concentrations of total PV in the two bathing solutions, if the unstirred layers are taken into account in computing the MS(+) concentrations at the membrane surfaces.

Ion transport mediated by the valinomycin analogue cyclo(L-Lac-L-Val-D-Pro-D-Val)3 in lipid bilayer membranes.

Cyclo(L-Lac-L-Val-D-Pro-D-Val)3 (PV-Lac) a structural analogue of the ion-carrier valinomycin, increases the cation permeability of lipid bilayer membranes by forming a 1:1 ion-carrier complex. The selectively sequence for PV-Lac is identical to that of valinomycin; i.e., Rb+ greater than K+ greater than Cs+ greater than or equal to NH+4 greater than Na+ greater than Li+. The steady-state zero-voltage conductance, G(0), is a saturating function of KCl concentration. A similar behavior was found for Rb+, Cs+, and NH+4. However, the ion concentration at which G(0) reaches a plateau strongly depends on membrane composition. The current-voltage curves present saturating characteristics, except at low ion concentrations of Rb+, K+, or Cs+. The ion concentration at which the saturating characteristics appear depends on membrane composition. These and other results presented in this paper agree with a model that assumes complexation between carrier and ion at the membrane-water interface. Current relaxation after voltage-jump studies were also performed for PV-Lac. Both the time constant and the amplitude of the current after a voltage jump strongly depend on ion concentration and membrane composition. These results, together with the stationary conductance data, were used to evaluate the rate constants of the PV-Lac-mediated K+ transport. In glycerolmonooleate they are: association rate constant, 2 x 10(6) M-1 s-1; dissociation rate constant, 4 x 10(5) s-1; translocation rate constant for complex, 5 x 10(4) s-1; and the rate of translocation of the free carrier (ks), 55 s-1. ks is much smaller for PV-Lac than for valinomycin and thus limits the efficiency with which the carrier is able to translocate cations across the membrane.

Kinetics of ion transport in lipid membranes induced by lysine-valinomycin and derivatives.

Lysine-valinomycine and two N epsilon-acyl derivatives are compared with respect to their potency to transport Rb+ ions across thin lipid membranes. Lysine-valinomycin acts as a neutral ion carrier only above a pH of about 7 of the aqueous solutions, while at lower pH the molecules seem to be positively charged due to a protonation of the epsilon-NH2 group of the lysine residue. A kinetic analysis based on voltage jump relaxation experiments and on the nonlinearity of the current-voltage characteristics showed that the conductance increment delta per carrier molecule for uncharged lysine-valinomycin is similar to that of natural valinomycin. The attachment of a rather bulky side group such as the dansyl or para-nitrobenzyloxycarbonyl group reduced delta by approximately one order of magnitude. Some of the relaxation data of the valinomycin analogues were influenced by an unspecific relaxation of the pure lipid membrane. This structural relaxation represents a limitation to the possibility of analyzing specific transport systems in thin lipid membranes by the voltage jump or charge pulse techniques. It is shown that the time dependence of this structural relaxation--which was first published by Sargent (1975)--is at variance with a three capacitor equivalent circuit of the membrane, which was suggested by Coster and Smith (1974) on the basis of a.c. measurements. A modified equivalent circuit has been found to represent a satisfactory analogue for the current relaxation in the presence of valinomycin. It turned out, however, that such an equivalent circuit provides little insight into the molecular mechanism of transport.

Synthesis of lysine-valinomycin by solid-phase segment condensation.

In order to obtain a readily derivatized analog of the ionophore antibiotic valinomycin, [1-lysine] valinomycin (Lys-VAL) was synthesized. The compound was built up on a polystyrene support by stepwise segment condensation and was cyclized in solution. The segments used were didepsipeptides protected by the t-butyloxycarbonyl and p-nitrobenzyloxycarbonyl groups. Derivatives prepared by acylation of the epsilon-amino group of Lys-VAL included [14C]acetyl-Lys-VAL, dansyl-Lys-VAL, palmitoyl-Lys-VAL and dithiodiglycoyl-bis-Lys-VAL. These derivatives had a high potassium binding capacity but were in general much less active than VAL in mediating ion transport in membranes.

Conformational studies of peptide cyclo-(D-Val-L-Pro-L-Val-D-Pro]3, a cation-binding analogue of valinomycin.

The solution conformation of cyclo-[D-Val-L-Pro-L-Val-D-Pro]3 (PV) and its alkali-metal ion complexes was investigated by proton nuclear magnetic resonance spectroscopy. It is concluded that the cation complexes of PV have S6 symmetry and are essentially isostructural with the K complex of valinomycin. In contrast to valinomycin, the Li- and Na-PV complexes are stable in methanol and have dissociation rate constants that are several orders of magnitude slower than the corresponding valinomycin complexes. Also in contrast to valinomycin, free PV exists in two different conformational states which interconvert at very slow rates (less than 1 s-1). One of these conformers has S6 symmetry and is structurally similar to that of the cation complexes. The other species, which has lower symmetry than S6, is the more stable conformer. Depending upon concentration and solvent polarity, the latter represents between 50 and 75% of the total mixture. It is proposed that PV may have a higher affinity for cations than valinomycin because of its higher potential energy in the uncomplexed state.

Synthesis of a 19-residue peptide with alamethicin-like activity.

This paper describes the chemical synthesis of a compound with voltage-gating characteristics similar to those observed in nerve membranes. For alamethicin (ALA), a natural antibiotic that induces such properties in lipid bilayer membranes, there are two proposed structures, one a cyclic and the other an open chain peptide. The open chain sequence (ALA-o) proposed by Martin and Williams [(1976) Biochem. J. 153, 181-190] was synthesized by stepwise solid-phase condensation of four fragments prepared by solid-phase synthesis. The product, purified to homogeneity, was not identical with the main component of natural ALA. Nevertheless, in lipid bilayer membranes the exponential dependence of conductance on voltage and the dependence of conductance on a high power of the peptide concentration were qualitatively similar for ALA-o and for natural ALA. Like ALA, ALA-o showed the characteristics of a channel-former, although the single-channel conductances were less well defined for the synthetic compound. This work establishes that a cyclic structure is not a necessary condition for a peptide to induce voltage-dependent conductances in membranes and that ALA-o possesses all the structural elements required for such an activity.

Optical and electrical studies on dansyllysine-valinomycin in thin lipid membranes.

Dansyllysine-valinomycin, a fluorescent analogue of the ionophore valinomycin was synthesized and incorporated into black lipid membranes. Its concentration inside the membrane was measured fluorometrically and was also determined from electrical relaxation experiments, which were analyzed on the basis of a previously proposed carrier model. The results of both methods agreed within less than one order of magnitude. This appears satisfactory in view of the sources of error inherent in both procedures. A conductance increment per carrier molecule of about 3 - 10(-17) omega-1 was obtained for dansyllysine-valinomycin in diphytanoyllecithin membranes at 25 degrees C and 1 M RbCl in the aqueous phases. This is about 400 times smaller compared to unmodified valinomycin in monoolein membranes. The difference is mainly caused by the change in the membrane properties and to a smaller extent by the structural modification of the carrier.