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

In contact with lipid bilayers and Ca2+-ions, the intracellular protein human annexin V (wild-type), Mr=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]≥K̄=5 nM at 296 K. At 50 mM Ca2+, symmetrical on both membrane sides, AN added at the cis side, the conductance is gCa(I)=22±2 pS and at symmetrical 0.1 M K+-conditions: gK(I)=32±3 pS, associated with two mean open-times τ̄1(I)=0.68±0.2 ms and τ̄2(I)=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]>K̄ changes its membrane organization to a higher oligomer, probably to the side-by-side double-trimer T2. The smaller type (II) channel events are predominant at low [AN]≤K̄ and refer to the (electroporative) adsorption complex of the monomer. The conductances gi(II) for symmetrical concentrations depend non-linearly on the voltage Um=Uext+UAN, where UAN=0.02±0.002 V is the electrostatic contribution of the Ca2+–AN complex and Uext the externally applied voltage. There is only one mean open-time τ̄o(II) which is voltage-dependent according to a functional of b·Um2 where b=113.9±15 V−2, yielding an activation Gibbs free energy of Ga=RT·b·Um2. The conformational flicker probability fi(II) in gi(II)=gi0(II)·Γi·fi(II) is non-linearly voltage-dependent according to a functional of a·Um2. The Nernst term Γi refers to asymmetrical ion concentrations. From a=50 V−2, independent of the ion type, we obtain fi0(II)=0.03±0.002 and the conductances for the fully open-channel states: gCa0(II)=69±3 pS (0.05 M Ca2+) and gK0(II)=131±5 pS (1.2 M K+). From the electroporation term a =πrp2ε0εw−εm/2 kT d we determine the mean pore radius of the complex in its fully open state as r̄p=0.86±0.05 nm. 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.