Interaction of diltiazem with an intracellularly accessible binding site on Ca(V)1.2

Abstract

Background and purpose: Diltiazem inhibits Ca(V)1.2 channels and is widely used in clinical practice to treat cardiovascular diseases. Binding determinants for diltiazem are located on segments IIIS6, IVS6 and the selectivity filter of the pore forming α₁ subunit of Ca(V)1.2. The aim of the present study was to clarify the location of the diltiazem binding site making use of its membrane-impermeable quaternary derivative d-cis-diltiazem (qDil) and mutant α₁ subunits.

Experimental approach: Ca(V)1.2 composed of α1, α2-δ and β2a subunits were expressed in tsA-201 cells and barium currents through Ca(V)1.2 channels were recorded using the patch clamp method in the whole cell configuration. qDil was synthesized and applied to the intracellular side (via the patch pipette) or to the extracellular side of the membrane (by bath perfusion).

Key results: Quaternary derivative d-cis-diltiazem inhibited Ca(V)1.2 when applied to the intracellular side of the membrane in a use-dependent manner (59 ± 4% at 300 µM) and induced only a low level of tonic (non-use-dependent) block (16 ± 2% at 300 µM) when applied to the extracellular side of the membrane. Mutations in IIIS6 and IVS6 that have previously been shown to reduce the sensitivity of Ca(V)1.2 to tertiary diltiazem also had reduced sensitivity to intracellularly applied qDil.

Conclusion and implications: The data show that use-dependent block of in Ca(V)1.2 by diltiazem occurs by interaction with a binding site accessible via a hydrophilic route from the intracellular side of the membrane.

Figure 1

Use-dependent block of Ca_V1.2 by intracellularly applied quaternary diltiazem. (A) Use-dependent inhibition of wild-type channels measured in the absence or presence of 50, 100, 300 or 500 µM quaternary diltiazem in the intracellular (pipette) solution. Data points are the mean from 4–6 experiments. (B) The IC₅₀ values [d-cis-diltiazem: 95 ± 5 µM (Hill slope n_H= 1.6 ± 0.4) and quaternary derivative of d-cis-diltiazem (qDil): 85 ± 9 µM (n_H= 1.3 ± 0.2) ] were obtained by fitting the data points to the Hill equation (as described in Methods). Channel block was estimated as peak I_Ba inhibition during trains of 20 pulses (0.2 Hz, 100 ms) applied from a holding potential of −80 mV to +20 mV in control (Table 1) and in the presence of quaternary diltiazem. (C) Superimposed I_Ba during a train of 20 pulses with 300 µM quaternary diltiazem in pipette. (D) Acceleration of current decay during the first pulse in train. Current traces were normalized and averaged. The mean peak current densities were −14.7 ± 0.9 (control) and −13.8 ± 0.9 pA·pF⁻¹ (first pulse current after 3 min of 300 µM qDil in the pipette). Histograms indicate remaining current at the end of the first pulse.

Figure 2

Extracellular quaternary diltiazem and intracellular [quaternary benzothiazepine [(cis)-1,3,4,5-tetrahydro-4-(4-methoxyphenyl)-3-methyl-6- (trifluoromethyl)-1-[2-trimethylammonio)ethyl]-2H-1-benzazepin-2-o ne] (SQ32,428) ] do not inhibit Ca_V1.2. (A) Superimposed I_Ba during a train of 20 pulses (same protocol as in Figure 1) in the absence of drug, with 300 µM quaternary diltiazem in the bath solution and with 300 µM SQ32,428 in pipette. (B) Structures of quaternary diltiazem and benzothiazepinone SQ32,428. (C) Lack of significant I_Ba inhibition by extracellularly applied quaternary derivative of d-cis-diltiazem (qDil) (300 µM) and intracellularly applied SQ32,428 (300 µM). Peak current decay with 300 µM qDil in the pipette is shown for comparison as a broken line (data from Figure 1A). Data points are the mean from 4–6 experiments. (D) Illustrates tonic I_Ba inhibition (block after 3 min in drug at rest at −80 mV) induced by 100 and 300 µM Dil, d-cis-diltiazem (Dil) and SQ32,428 applied extracellularly.

Figure 3

Mutations of the putative d-cis-diltiazem binding site affect I_Ba inhibition by intracellularly applied quaternary derivative of d-cis-diltiazem (qDil). (A) Amino acid sequence of the transmembrane segments IIIS6 and IVS6 of the Ca_V1.2 α₁ subunit. Putative diltiazem binding determinants are highlighted. (B) Peak current decay in mutants I1150A, I1153A and V1165A channels induced by 300 µM quaternary diltiazem in the pipette solution (protocol as in Figure 1). (C) Remaining currents after 20 pulses in wild-type (WT) and the indicated IIIS6 mutants. Asterisks denote that the steady-state block value for quaternary diltiazem of the indicated mutant channel is significantly different from that of WT (Student's t-test: *P < 0.05, **P < 0.01, #P= 0.057). (D) Use-dependent inhibition of I460A and M1464A channels by 300 µM of qDil in the pipette solution. (E) Remaining currents of WT Ca_V1.2 and the indicated IVS6 mutants. Asterisks indicate that the steady-state block value for quaternary diltiazem of the indicated mutant channel is significantly different from that of WT (Student's t-test: *P < 0.05, **P < 0.01). (F) Use-dependent inhibition of selectivity filters mutants F1117G, E1118Q and E1419Q by 300 µM qDil in the pipette solution. (G) Remaining currents after 20 pulses in WT and the indicated Ca_V1.2 mutants in 100 and 300 µM qDil in the pipette. Asterisks indicate where there is a significant difference between the steady-state block of the indicated mutant channel and the WT (Student's t-test: *P < 0.05, #P= 0.057). The broken lines in (B, D and F) represent peak current inhibition in wild-type (taken from Figure 1A). Channel block in (C, E and G) was estimated by subtracting ‘steady state’ inhibition after 20 pulses in drug-free solution (Table 1) from channel block induced by 100 µM or 300 µM of qDil.

Figure 4

Changes in channel gating induced by d-cis-diltiazem (Dil) and quaternary derivative of d-cis-diltiazem (qDil). Steady-state activation (A) and inactivation (B) of wild-type (WT) in the absence (control) or presence of 300 µM qDil applied from intracellular side or 300 µM of Dil applied by bath perfusion (see Table 2 for parameters of the Bolzmann distributions).

Figure 5

Recovery of wild-type Ca_V1.2 from block by intracellularly applied quaternary derivative of d-cis-diltiazem (qDil) and extracellularly applied d-cis-diltiazem (Dil). (A–B) Recovery from block by 300 µM intracellular quaternary or 300 µM extracellular tertiary diltiazem at −80 mV holding potentials. Block was elicited by a standard conditioning train of 20 pulses in the presence of qDil and recovery monitored by applying short (20 ms) test pulses at different times after the train. The mean time constants of recovery from block by Dil and qDil were 32.3 ± 5.2 (n= 7) and 37.1 ± 4.9 (n= 9) respectively.