P-glycoprotein senses its substrates and the lateral membrane packing density:: Consequences for the catalytic cycle

P-glycoprotein (ABCB1) prevents absorption (e.g., blood-brain barrier) or enhances excretion (e.g., kidney) by moving substrates from the cytosolic to the extracellular membrane leaflet at the expense of ATP hydrolysis. It translocates various drugs and functions in membranes exhibiting different lateral packing densities. To gain more functional insight, we measured the temperature dependence of the P-glycoprotein ATPase activity in NIH-MDRI-G185 cell membranes in the absence and presence of three drugs (promazine, verapamil, and PSC833), exhibiting significantly different transporter affinities. Activation enthalpies (Delta H(double dagger)) and entropies (T Delta S(double dagger)) were derived from Eyring plots. In the absence of drugs, the activation enthalpy and the free energy of activation for P-glycoprotein ATPase activity was determined as Delta H(double dagger) = 92.6 +/- 4.2 kJ/mol and Delta G(double dagger) =73.1 +/- 7.2 kJ/mol, respectively. Increasing the drug concentration reduced the activation enthalpy, whereby the drug with the highest transporter affinity had the strongest effect (Delta Delta H(double dagger) = -21 %). The free energy of activation decreased for activating (Delta Delta G(double dagger)=similar to-3.8%) and increased for inhibitory compounds (Delta Delta G(double dagger)=similar to+0.7%). The drug-specific changes of the free energy of activation are thus barely above thermal energy. A comparison with literature data revealed that a decrease of the lateral membrane packing density reduces the enthalpic and the entropic contribution to the free energy of activation. Although the P-glycoprotein ATPase activity increases only slightly with decreasing lateral membrane packing density, the mode of action changes from strongly entropy-driven at high, to essentially enthalpy-driven at low packing densities. This Suggests that the transporter and the membrane form a functional entity.