Interaction of verapamil with lipid membranes and P-glycoprotein: connecting thermodynamics and membrane structure with functional activity

Abstract

Verapamil and amlodipine are calcium ion influx inhibitors of wide clinical use. They are partially charged at neutral pH and exhibit amphiphilic properties. The noncharged species can easily cross the lipid membrane. We have measured with solid-state NMR the structural changes induced by verapamil upon incorporation into phospholipid bilayers and have compared them with earlier data on amlodipine and nimodipine. Verapamil and amlodipine produce a rotation of the phosphocholine headgroup away from the membrane surface and a disordering of the fatty acid chains. We have determined the thermodynamics of verapamil partitioning into neutral and negatively charged membranes with isothermal titration calorimetry. Verapamil undergoes a pK-shift of DeltapK(a) = 1.2 units in neutral lipid membranes and the percentage of the noncharged species increases from 5% to 45%. Verapamil partitioning is increased for negatively charged membranes and the binding isotherms are strongly affected by the salt concentration. The electrostatic screening can be explained with the Gouy-Chapman theory. Using a functional phosphate assay we have measured the affinity of verapamil, amlodipine, and nimodipine for P-glycoprotein, and have calculated the free energy of drug binding from the aqueous phase to the active center of P-glycoprotein in the lipid phase. By combining the latter results with the lipid partitioning data it was possible, for the first time, to determine the true affinity of the three drugs for the P-glycoprotein active center if the reaction takes place exclusively in the lipid matrix.

FIGURE 1

Chemical structures and conformational models of three calcium channel antagonists: verapamil, nimodipine, and amlodipine. The three-dimensional structures were obtained by searching the most amphiphilic, energy-minimized conformation with the minimal cross-sectional area, A_D. Oxygen and nitrogen molecules are shown in red and blue, respectively. Hydrogen-bond acceptors, constituting the binding modules for P-glycoprotein, are connected with dotted yellow lines. Pgp does not accept secondary amino groups (−NHR) or −NO₂ groups (for details, see (7)).

FIGURE 2

Deuterium and phosphorus-31 NMR spectra of POPC liposomes deuterated at the α-position of the choline headgroup (−POCD₂CH₂N⁺). Approximately twenty-five milligrams of POPC was suspended in 50 μL buffer (25 mM, MES 0.1 M NaCl, pH 5.5, deuterium-depleted water) containing different concentrations of verapamil. The two top spectra correspond to pure POPC membranes without verapamil. The spectra below are characterized by increasing drug concentrations. The verapamil/POPC molar ratio from top to bottom is: 0, 0.02, 0.04, 0.07, 0.11, and 0.14. Virtually all verapamil is incorporated into the membrane. (Number of FIDs: ²H NMR spectra 8 K, ³¹P NMR 2 K.)

FIGURE 3

Variation of the deuterium NMR quadrupole splittings of POPC membranes with the verapamil/lipid molar ratio. (A) Phosphocholine headgroup segments: (▪) α-CD₂ POPC (−POCD₂CH₂N); (•) β-CD₂-POPC (−POCH₂CD₂N⁺). (B) POPC deuterated at the cis-double bond of the oleic acyl chain: (▪) C-9′ deuteron, (•) C-10′ deuteron. Measurements at 22°C in buffer (MES 25 mM + 0.1 M NaCl, pH 5.5).

FIGURE 4

²H-NMR spectra of POPC membranes deuterated at the cis-double bond of the sn-2-oleic acyl chain and suspended in buffer with various verapamil concentrations. Approximately twenty-five milligrams of lipid suspended in 50 μL buffer (MES 25 mM + 0.1 M NaCl, pH 5.5) were used. The verapamil/lipid molar ratios from bottom-to-top are 0.003, 0.021, 0.037, and 0.048. The smooth lines are the simulated deuterium NMR spectra. The lower panel shows the loss in signal intensity of the C-9′ and C-10′ deuteron as a function of the verapamil/lipid molar ratio referenced to the pure POPC spectrum. The C-9′ deuteron with a 13 kHz splitting shows a much steeper intensity loss than the C-10′ deuteron with a 2 kHz splitting. (4 K free induction decays for all spectra.)

FIGURE 5

Titration of a 100 μM verapamil solution in 50 mM HEPES, pH 7.4 with 30 nm unilamellar lipid vesicles in the same buffer. Lipid composition is POPC/POPG (75:25 mol/mol). The injection of the lipid vesicles was in 5 μL steps. Measuring temperature 37°C. (A) Heat flow and (B) cumulative heat of reaction as a function of injection number.

FIGURE 6

Reaction enthalpies of verapamil binding to phospholipid vesicles (30 nm) of different lipid composition. Variation of the binding enthalpy, , with the salt concentration. (▪) POPC/POPG 75:25 mol/mol, (•) pure POPC, and (▵) POPC/DOTAP 95:5 mol/mol.

FIGURE 7

Verapamil binding isotherms for POPC/POPG membranes (75:25 mol/mol) at three different salt concentrations. All measurements made at pH 7.4 and 37°C. 50 mM HEPES + 50 mM NaCl; 50 mM HEPES; and 25 mM HEPES. The solid lines were calculated with the partition constants, K_p, given in Table 1 and the Gouy-Chapman theory. A rapid translocation of the neutral form of verapamil across the membrane was assumed.

FIGURE 8

Binding of verapamil to POPC SUVs and mixed POPC/DOTAP (94.2:15.8 mol/mol) SUVs at various salt concentrations. (○) Pure POPC SUVs; (□) POPC/DOTAP SUVs. All measurements in 50 mM Tris or HEPES buffer + various concentrations of NaCl at 37°C: The solid lines are theoretical binding isotherms calculated with the partition coefficients listed in Table 1.

FIGURE 9

Variation of the verapamil binding enthalpies with the buffer dissociation enthalpies. POPC vesicles with 30 nm diameter. Measurements made in Tris (ΔH_Diss = 11.51 kcal/mol), HEPES (ΔH_Diss = 4.9 kcal/mol), and phosphate (ΔH_Diss = 1.22 kcal/mol) at 37°C (36).

FIGURE 10

Pgp activation profiles obtained by a phosphate release assay with inside-out vesicles prepared from NIH-MDR-G185 cells. (▪) Verapamil; (○) amlodipine.