Assaying P-Type ATPases Reconstituted in Liposomes

Reconstitution of P-type ATPases in unilamellar liposomes is a useful technique to study functional properties of these active ion transporters. Experiments with such liposomes provide an easy access to substrate-binding affinities of the ion pumps as well as to the lipid and temperature dependence of the pump current. Here, we describe two reconstitution methods by dialysis and the use of potential-sensitive fluorescence dyes to study transport properties of two P-type ATPases, the Na,K-ATPase from rabbit kidney and the K(+)-transporting KdpFABC complex from E. coli. Several techniques are introduced how the measured fluorescence signals may be analyzed to gain information on properties of the ion pumps.