PUB - Publikationen an der Universität Bielefeld

In contact with lipid bilayers and Ca2+-ions, the intracellular protein human 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 predominant at high values [AN] greater than or equal to (K) over bar= 5 nM at 296 K. At 50 mM Ca2+, symmetrical on both membrane sides, AN added at the cis 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 trimer T, which at [AN] > K changes its membrane organization to a higher oligomer, probably to the side-by-side double-trimer T,. The smaller type (II) channel 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 the voltage U-m= U-ext + U-AN, where U-AN = 0.02 +/- 0.002 V is the electrostatic 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 voltage-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 according to a functional of a (.) U-m(2). The Nernst term Gamma(i) refers to asymmetrical 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 open-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)]epsilon(0)(epsilon(w)-epsilon(m))/(2 kTd) we determine the mean pore radius of the complex in its fully open state as (r) over bar(p) = 0.86 +/- 0.05 mm. The adsorbed annexin V (Ca2+) monomer appears to electrostatically facilitate the electric pore formation at the contact interface between the protein and the lipid phase. The complex rapidly flickers and thus limits the ion transport in a voltage-dependent manner. (C) 2000 Elsevier Science B.V. All rights reserved.