Electroporative fast pore-flickering of the annexin V-lipid surface complex, a novel gating concept for ion transport

In contact with lipid bilayers and Ca2+-ions, the intracellular protein hum an annexin V (wild-type), M-r= 35 800, forms two types of cation-selective channels for the transport of Ca2+-, K+-, Na+- and Mg2+-ions, depending on the protein concentration [AN]. Type (I) channel events are large and predo minant at high values [AN] greater than or equal to (K) over bar= 5 nM at 2 96 K. At 50 mM Ca2+, symmetrical on both membrane sides, AN added at the ci s side, the conductance is g(Ca)(I)= 22 +/- 2 pS and at symmetrical 0.1 M K +-conditions: g(K)(I) = 32 +/- 3 pS, associated with two mean open-times <( tau)over bar>(1)(I)= 0.68 +/- 0.2 ms and <(tau)over bar>(2)(1)= 31 +/- 2 ms . Monoclonal anti-AN antibodies added to the trans-side first increase the mean open-times and then abolish the channel activity, suggesting that type (I) channels refer to a membrane spanning protein complex, probably a trim er T, which at [AN] > K changes its membrane organization to a higher oligo mer, probably to the side-by-side double-trimer T,. The smaller type (II) c hannel events are predominant at low [AN]less than or equal to (K) over bar and refer to the (electroporative) adsorption complex of the monomer. The conductances gi(II) for symmetrical concentrations depend non-linearly on t he voltage U-m= U-ext + U-AN, where U-AN = 0.02 +/- 0.002 V is the electros tatic contribution of the Ca2+-AN complex and U-ext the externally applied voltage. There is only one mean open-time <(tau)over bar>(0)(II) which is v oltage-dependent according to a functional of b (.) U-m(2) where b = 113.9 +/- 15 V-2, yielding an activation Gibbs free energy of G(a) = RT (.) B (.) U-m(2). The conformational flicker probability f(i)(II) in g(i)(II) = g(i) (0)(II) (.) Gamma(i) (.) f(i)(II) is non-linearly voltage-dependent accordi ng to a functional of a (.) U-m(2). The Nernst term Gamma(i) refers to asym metrical ion concentrations. From a = 50 V-2, independent of the ion type, we obtain f(i)(0)(II)=0.03 +/- 0.002 and the conductances for the fully ope n-channel states: g(Ca)(0)(II)= 69 +/- 3 pS (0.05 M Ca2+) and g(K)(0)(II)= 131 +/- 5 pS (1.2 M K+). From the electroporation term a = pi[r(p)(2)]epsil on(0)(epsilon(w)-epsilon(m))/(2 kTd) we determine the mean pore radius of t he complex in its fully open state as (r) over bar(p) = 0.86 +/- 0.05 mm. T he adsorbed annexin V (Ca2+) monomer appears to electrostatically facilitat e the electric pore formation at the contact interface between the protein and the lipid phase. The complex rapidly flickers and thus limits the ion t ransport in a voltage-dependent manner. (C) 2000 Elsevier Science B.V. All rights reserved.