Calcicludine binding to the outer pore of L-type calcium channels is allosterically coupled to dihydropyridine binding

Abstract

How dihydropyridines modulate L-type voltage-gated Ca2+ channels is not known. Dihydropyridines bind cooperatively with Ca2+ binding to the selectivity filter, suggesting that they alter channel activity by promoting structural rearrangements in the pore. We used radioligand binding and patch-clamp electrophysiology to demonstrate that calcicludine, a toxin from the venom of the green mamba snake, binds in the outer vestibule of the pore and, like Ca2+, is a positive modulator of dihydropyridine binding. Data were fit using an allosteric scheme where dissociation constants for dihydropyridine and calcicludine binding, KDHP and KCaC, are linked via the coupling factor, alpha. Nine acidic amino acids located within the S5-Pore-helix segment of repeat III were sequentially changed to alanine in groups of three, resulting in the mutant channels, Mut-A, Mut-B, and Mut-C. Mut-A, whose substitutions are proximal to IIIS5, exhibits a 4.5-fold reduction in dihydropyridine binding and is insensitive to calcicludine binding. Block of Mut-A currents by calcicludine is indistinguishable from wild-type, indicating that KCaC is unchanged and that the coupling between dihydropyridine and calcicludine binding (i.e., alpha) is disrupted. Mut-B and Mut-C possess KDHP values that resemble that of the wild type. Mut-C, the most C-terminal of the mutant channels, is insensitive to calcicludine binding and block. KCaC values for the Mut-C single mutants, E1122A, D1127A, and D1129A, increase from 0.3 (wild type) to 1.14, 2.00, and 20.5 microM, respectively. Together, these findings suggest that dihydropyridine antagonist and calcicludine binding to L-type Ca2+ channels promote similar structural changes in the pore that stabilize the channel in a nonconducting, blocked state.

FIGURE 1. Calcicludine is a positive allosteric modulator of DHP binding. (A) Primary sequence of calcicludine

Calcicludine is a 60 amino acid peptide containing three disulfide bonds, 12 positively charged lysine or arginine residues (underlined) and two negatively charged glutamate residues. (B) Membranes from cells expressing wild-type Ca_V1.2 Ca²⁺ channels were incubated with ~350 pM [³H]PN200-110 and increasing concentrations of calcicludine. Each experiment was fit using Scheme 1 (see Equation 4, Materials and Methods) and normalized such that the occupancy in zero calcicludine is equal to 1.0. Note that [³H]PN200-110 binding increases as calcicludine is raised from 3 nM to 3 μM. Binding data are means ± SEM, and calcicludine concentrations where DHP binding differs significantly from that with no toxin (determined by ANOVA) are indicated with asterisks (*; P<0.05; n=6). Error bars smaller than symbols do not appear in figures. (C) Allosteric binding model for DHP and calcicludine binding. See Materials and Methods and text for details.

FIGURE 2. Acidic residues proximal to the extracellular portion of IIIS5 are important binding determinants for [³H]PN200-110

(A) Amino acid sequence of Ca_V1.2 beginning at the C-terminus of IIIS5 through the pore-loop. Peptide segments corresponding to regions spanned by Mut-A, Mut-B and Mut-C are indicated by lines above sequence. Glu-1122, Asp-1127 and Asp-1129 are indicated with arrows. The Pore Helix and P-Loop are indicated by lines below the sequence. (B) Saturation binding experiment using membranes derived form cells expressing wild-type Ca_V1.2 channels was performed as described in the Materials and Methods. The signal-to-noise can be assessed by comparing the relative levels of specific (open circles) and non-specific (solid circles) binding. (inset) Scatchard transformation of data from Panel B indicates that the cells express a single population of high affinity receptor sites. (C) Similar analyses were performed on independent membrane preparations derived from cells expressing Wild-type (n = 6), Mut-A (n = 3), Mut-B (n = 3) and Mut-C (n = 3) channels (see Table 1).

FIGURE 3. Three acidic amino acid residues, Glu-1122, Asp-1127 and Asp-1129, positioned proximal to the pore helix in domain III are important for calcicludine binding to Ca_V1.2 channels

Wild-type, Mut-A, Mut-B and Mut-C (A) and Wild-type, E1122A, D1127A and D1129A (B) Ca²⁺ channels were incubated with [³H]PN200-110 and the indicated concentrations of calcicludine as described in the Materials and Methods. Data were fit using Scheme 1 (see Equation 4, Materials and Methods) and normalized such that the occupancy in zero calcicludine is equal to 1.0. The method for fitting binding data from the individual mutants, E1122A, D1127A and D1129A, is described in the text and Legend for Table 1. DHP binding to Mut-A and Mut-C membranes does not increase in the presence of calcicludine, but block of Mut-A and not Mut-C currents by calcicludine is similar to that of wild-type (Fig. 5; see text for details). Data are means ± SEM, and significant differences between binding parameters of wild-type and mutant channels were evaluated using a 2-tailed Students-t test P<0.05 (*). Error bars smaller than symbols do not appear in figures. See Table 1 for data summary and indications of statistical significance. (A) Wild-type (n = 6), Mut-A (n = 3), Mut-B (n = 3), Mut-C (n = 3); (B) Wild-type (n = 6), E1122A (n = 3), D1127A (n = 3), D1129A (n = 3).

FIGURE 4. Mut-C channels are insensitive to by calcicludine

(A, D) Peak amplitudes of Ba²⁺ currents evoked by 300 msec step depolarizations from −100 to −10 mV every 20 seconds from cells expressing wild-type (A) and Mut-C (D) Ca²⁺ channels in the presence of 0, 100, 500 and 5000 nM calcicludine. Note that Mut-C currents are insensitive to block by calcicludine. (B, E) Sample traces resulting from voltage steps from −100 to −10 mV in the absence and presence of 500 nM calcicludine. (C, F) Current-voltage relations in the absence and presence of calcicludine were generated by depolarizing cells from a holding potential of −100 mV to 300 msec step depolarizations to potentials ranging from −60 to +80 mV. Peak currents were plotted against corresponding test voltages to give the current-voltage relationship (I-V). These data were fit using the equation, I=G(V_m−V_rev)/(1+exp[(V_h−V_m)/k]), where G is the maximal slope conductance, V_rev is the reversal potential, V_m is the membrane potential, V_h is the half activation potential and k is the slope factor. I-V measurements were made in each cell in the absence and presence of calcicludine, and were normalized by dividing the peak current at each step potential by the peak of the fit through I-V data acquired in the absence of calcicludine. Current voltage relations indicate that the gross gating properties of Mut-C (F) are similar to that of wild-type (D).

FIGURE 5. Glu-1122, Asp-1127 and Asp-1129 confer the channel’s sensitivity to block by calcicludine

Currents through Mut-C, but not Mut-A or Mut-B, are insensitive to block by calcicludine. (A) The fraction of wild-type, Mut-A, Mut-B and Mut-C current remaining after the application of the indicated concentrations of calcicludine (I/I_{no drug}) is plotted. Lines are Logistic fits through wild-type (open circles) and Mut-C (solid squares). Only 63% of the maximal Ba²⁺ currents through wild-type channels is blocked by saturating concentrations of calcicludine. Data for Mut-A and Mut-B are fit nicely with the same logistic function used to fit wild-type data. Data are summarized in Panel C and Table 1. Wild-type (n = 19), Mut-A (n = 4), Mut-B (n = 3), Mut-C (n = 7). (B) Ba²⁺ currents through E1122A, D1127A and D1129A channels exhibit a reduced sensitivity to block by calcicludine. The fraction of Ba²⁺ current remaining (I/I_{no drug}) after the addition of 0, 100, 500 and 5000 nM calcicludine to wild-type and mutant Ca²⁺ channels is plotted. Best fits through the data were made assuming a Hill coefficient of 1.0 and, as was observed for wild-type (Panel A), that a maximum of 63% of the total current is blocked at saturating concentrations of calcicludine. Wild-type (n = 19), E1122A (n = 6), D1127A (n = 6), D1129A (n = 4). (C) Summary of data depicted in Panels A and B (also, see Table 1). An IC₅₀ value for Mut-C could not be determined (n.d.). IC₅₀ values from these experiments were used to generate fits through binding data shown in Fig. 3B (see text and Legend for table 1 for details). The statistical significance of the observed differences between the blocking parameters of wild-type and mutant channels was evaluated using a 2-tailed Students-t test. Patch-clamp data are means ± SEM and significance was set at P<0.05 (*). Error bars smaller than symbols do not appear in figures.

Scheme 1