Interaction of Verapamil with Lipid Membranes and P-Glycoprotein: Connecting Thermodynamics and Membrane Structure with Functional Activity  M. Meier, X.Li Blatter, A. Seelig, J. Seelig  Biophysical Journal  Volume 91, Issue 8, Pages 2943-2955 (October 2006) DOI: 10.1529/biophysj.106.089581 Copyright © 2006 The Biophysical Society Terms and Conditions

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, AD. 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)). Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

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 50μL buffer (25mM, MES 0.1M 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 8K, 31P NMR 2K.) Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

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+0.1M NaCl, pH 5.5). Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

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 50μL buffer (MES 25 mM+0.1M 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 13kHz splitting shows a much steeper intensity loss than the C-10′ deuteron with a 2kHz splitting. (4K free induction decays for all spectra.) Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 5 Titration of a 100μM verapamil solution in 50mM HEPES, pH 7.4 with 30nm unilamellar lipid vesicles in the same buffer. Lipid composition is POPC/POPG (75:25mol/mol). The injection of the lipid vesicles was in 5μL steps. Measuring temperature 37°C. (A) Heat flow and (B) cumulative heat of reaction as a function of injection number. Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 6 Reaction enthalpies of verapamil binding to phospholipid vesicles (30nm) of different lipid composition. Variation of the binding enthalpy, ΔHD0, with the salt concentration. (■) POPC/POPG 75:25mol/mol, (●) pure POPC, and (▵) POPC/DOTAP 95:5mol/mol. Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 7 Verapamil binding isotherms for POPC/POPG membranes (75:25mol/mol) at three different salt concentrations. All measurements made at pH 7.4 and 37°C. 50mM HEPES+50mM NaCl; 50mM HEPES; and 25mM HEPES. The solid lines were calculated with the partition constants, Kp, given in Table 1 and the Gouy-Chapman theory. A rapid translocation of the neutral form of verapamil across the membrane was assumed. Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 8 Binding of verapamil to POPC SUVs and mixed POPC/DOTAP (94.2:15.8mol/mol) SUVs at various salt concentrations. (○) Pure POPC SUVs; (□) POPC/DOTAP SUVs. All measurements in 50mM 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. Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 9 Variation of the verapamil binding enthalpies with the buffer dissociation enthalpies. POPC vesicles with 30nm diameter. Measurements made in Tris (ΔHDiss=11.51kcal/mol), HEPES (ΔHDiss=4.9kcal/mol), and phosphate (ΔHDiss=1.22kcal/mol) at 37°C (36). Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions

Figure 10 Pgp activation profiles obtained by a phosphate release assay with inside-out vesicles prepared from NIH-MDR-G185 cells. (■) Verapamil; (○) amlodipine. Biophysical Journal 2006 91, 2943-2955DOI: (10.1529/biophysj.106.089581) Copyright © 2006 The Biophysical Society Terms and Conditions