Probing the architecture of an L-type calcium channel with a charged phenylalkylamine: evidence for a widely open pore and drug trapping

Abstract

Voltage-gated calcium channels are in a closed conformation at rest and open temporarily when the membrane is depolarized. To gain insight into the molecular architecture of Ca(v)1.2, we probed the closed and open conformations with the charged phenylalkylamine (-)devapamil ((-)qD888). To elucidate the access pathway of (-)D888 to its binding pocket from the intracellular side, we used mutations replacing a highly conserved Ile-781 by threonine/proline in the pore-lining segment IIS6 of Ca(v)1.2 (1). The shifted channel gating of these mutants (by 30-40 mV in the hyperpolarizing direction) enabled us to evoke currents with identical kinetics at different potentials and thus investigate the effect of the membrane potentials on the drug access per se. We show here that under these conditions the development of channel block by (-)qD888 is not affected by the transmembrane voltage. Recovery from block at rest was, however, accelerated at more hyperpolarized voltages. These findings support the conclusion that Ca(v)1.2 must be opening widely to enable free access of the charged (-)D888 molecule to its binding site, whereas drug dissociation from the closed channel conformation is restricted by bulky channel gates. The functional data indicating a location of a trapped (-)D888 molecule close to the central pore region are supported by a homology model illustrating that the closed Ca(v)1.2 is able to accommodate a large cation such as (-)D888.

FIGURE 1. Similar kinetics of I_Ba through wild-type Ca_v1.2 and mutants I781T and I781P at different test potentials

A, I_Ba currents are evoked by 100 ms test pulses to 20 mV (wild-type), −10 mV (I781T), and −20 mV (I781P) from −80 mV. B, normalized current-voltage relationships of the wild-type (n = 9, open circles), I781T (n = 6, filled circles), and I781P (n = 6, filled squares) mutant channels. Potentials of half-maximal activation (V_0.5,act) are −1.1 ± 0.8 mV, −29.9 ± 0.6 mV, and −44.1 ± 0.7 mV for wild-type, I781T, and I781P mutant channels, respectively. C, average voltage dependencies of steady-state inactivation for wild-type (n = 3, open circles), I781T (n = 3, filled circles), and I781P (n = 3, filled squares) mutant channels. Solid lines represent fits to Boltzmann functions. Potentials of half-maximal inactivation (V_0.5,inact) are −20.0 ± 0.8 mV, −52.1 ± 0.6 mV, and −59.3 ± 1.0 for wild-type, I781T, and I781P mutant channels respectively.

FIGURE 2. Similar inhibition of wild-type, I781T, and I781P mutant channels by (−)qD888

A, B, and C, use-dependent block of wild-type (A), I781T (B), and I781P (C) mutant channels by different concentrations of the calcium channel blocker (−)qD888. Use-dependent inhibition of wild-type and mutant channels was measured in the absence (○) or presence of 10 μm (■), 30 μm (▲), 100 μm (▼), or 300 μm () (−)qD888 in the pipette (intracellular) solution. Channel block was estimated as peak I_Ba inhibition during trains of 30 pulses (0.2 Hz, 100 ms) applied from a holding potential of −80 mV to +20 mV (wild-type), −10 mV (I781T) or −20 mV (I781P). D, concentration dependence of peak I_Ba inhibition by (−)qD888 in wild-type (○), I781T (●), and I781P (■) mutant channels. Channel block was estimated as the difference between “steady state” normalized current in control and in presence of (−)qD888. Data points are the mean from 3–6 experiments. The IC₅₀ values were obtained by fitting the data points to the Hill equation (as described under “Experimental Procedures”) yielding 43.7 ± 3.4 μm (wild-type), 41.0 ± 2.1 μm (I781T), and 34.3 ± 8.2 μm (I781P).

FIGURE 3. Voltage-dependent recovery from block by 100 μm intracellular (−)qD888

A, B, and C, I_Ba recovery from block by 100μm intracellular (−)qD888 of wild-type (A), I781T (B), and I781P (C) mutant channels at holding potentials of −110 (○), −100 (■), −90 (▲), or −80 mV (). Inset, block was elicited by a standard conditioning train of 30 pulses (see also Fig. 2) and recovery measured applying test pulses at different time after the conditioning train. Data points were fitted by mono-exponential functions; yielding time constants: τ₋₁₁₀ = 16 ± 3 s, τ₋₁₀₀ = 24 ± 4 s, τ₋₉₀ = 40 ± 6 s, and τ₋₈₀ = 78 ± 7 s for wild-type; τ₋₁₁₀ = 13 ± 3 s, τ₋₁₀₀ = 27 ± 4 s, τ₋₉₀ = 31 ± 5 s, and τ₋₈₀ = 52 ± 6 s for I781T mutant; τ₋₁₁₀ = 19 ± 3 s, τ₋₁₀₀ = 25 ± 5 s, τ₋₉₀ = 28 ± 4 s, and τ₋₈₀ = 71 ± 10 s for I781P mutant. D, semi-logarithmic plot of the recovery time constants versus holding potentials. Regression lines yield slopes of 0.022 mV⁻¹ (wild-type), 0.018 mV⁻¹ (I781T), and 0.017 mV⁻¹ (I781P).

FIGURE 4. Voltage-dependent recovery from block by 10 μm (−)D888

A and B, I_Ba recovery from block (10 μm (−)D888) of wild-type (A) and I781T (B) channels at holding potentials of −110 (○), −100 (■), −90 (▲), or −80 mV () (same protocol as described in the legend to Fig. 3). Data points were fitted by mono-exponential functions yielding time constants: τ₋₁₁₀ = 11 ± 1 s,τ₋₁₀₀ = 18 ± 2 s,τ₋₉₀ = 31 ± 4 s, and τ₋₈₀ = 57 ± 10 s for wild-type; τ₋₁₁₀ = 10 ± 1 s, τ₋₁₀₀ = 24 ± 4 s, τ₋₉₀ = 29 ± 3 s, and τ₋₈₀ = 53 ± 7 s for I781T mutant. C, semi-logarithmic plot of the recovery time constants versus holding potentials. Regression yields slopes of 0.023 mV⁻¹ (wild-type) and 0.022 mV⁻¹ (I781T).

FIGURE 5. Illustration of the drug binding hypothesis in a homology model of Ca_v1.2 in the open (A) and closed (B) channel conformations

Nine determinants of PAA sensitivity in segments IIIS6, IVS6, and the selectivity filter are indicated (amino acid numbering according to Dilmac et al. (16)). A, the model illustrates free access of the permanently charged (−)qD888 molecule (yellow) via a widely open inner channel mouth. B, the figure illustrates one possible conformation of (−)qD888 in the closed Ca_v1.2. The model supports the hypothesis that this compound fits into the cavity of the closed channel pore. The position of the charged molecule within the closed channel conformation (δ ranging from 0.44 to 0.57) was deduced from the voltage dependence of I_Ba recovery (Figs. 3, 4, and supplemental data). The precise location and orientation of the drug within the binding pocket remain unknown. Our data support the hypothesis of drug binding to the central pore region (supplemental data). C, bottom surface view of open pore. D, bottom surface view of closed channel pore accommodating (−)qD888.