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Allosteric Interactions Required for High-Affinity Binding of Dihydropyridine Antagonists to Ca_V1.1 Channels Are Modulated by Calcium in the Pore

Department of Cellular and Molecular Physiology, The Penn State Milton S. Hershey College of Medicine, Hershey, Pennsylvania (B.Z.P.); and Department of Pharmacology, University of Washington, Seattle, Washington (W.A.C.)

Received November 11, 2005; accepted May 4, 2006

	Abstract

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Dihydropyridines (DHPs) are an important class of drugs, used extensively in the treatment of angina pectoris, hypertension, and arrhythmia. The molecular mechanism by which DHPs modulate Ca²⁺ channel function is not known in detail. We have found that DHP binding is allosterically coupled to Ca²⁺ binding to the selectivity filter of the skeletal muscle Ca²⁺ channel Ca_V1.1, which initiates excitation-contraction coupling and conducts L-type Ca²⁺ currents. Increasing Ca²⁺ concentrations from approximately 10 nM to 1 mM causes the DHP receptor site to shift from a low-affinity state to a high-affinity state with an EC₅₀ for Ca²⁺ of 300 nM. Substituting each of the four negatively charged glutamate residues that form the ion selectivity filter with neutral glutamine or positively charged lysine residues results in mutant channels whose DHP binding affinities are decreased up to 10-fold and are up to 150-fold less sensitive to Ca²⁺ than wild-type channels. Analysis of mutations of amino acid residues adjacent to the selectivity filter led to identification of Phe-1013 and Tyr-1021, whose mutation causes substantial changes in DHP binding. Thermo-dynamic mutant cycle analysis of these mutants demonstrates that Phe-1013 and Tyr-1021 are energetically coupled when a single Ca²⁺ ion is bound to the channel pore. We propose that DHP binding stabilizes a nonconducting state containing a single Ca²⁺ ion in the pore through which Phe-1013 and Tyr-1021 are energetically coupled. The selectivity filter in this energetically coupled high-affinity state is blocked by bound Ca²⁺, which is responsible for the high-affinity inhibition of Ca²⁺ channels by DHP antagonists.

View larger version (60K): [in this window] [in a new window]   Fig. 1. The selectivity filter consists of four negatively charged glutamate residues that bind a single Ca2+ ion with a high affinity or two Ca2+ ions with a low affinity. A, the 1 subunit of the Ca2+ channel consists of four homologous repeats (I, II, III, and IV) that assemble to form the pore of the channel complex. Each repeat consists of six transmembrane segments, S1-S6. The loops connecting the 5th and 6th transmembrane segments from each repeat contain highly conserved glutamate residues (denoted with an E) that collectively form the selectivity filter. The selectivity filter is capable of binding a single Ca2+ ion with a high affinity or two Ca2+ ions with a low affinity. Dihydropyridine agonists and antagonists interact with amino acid residues located in transmembrane segments IIIS5, IIIS6, and IVS6 (dark cylinders). B, the pore segments from each of the four domains of the DHP-sensitive (CaV1.1-4) and -insensitive (CaV2.1-3) Ca2+ channels are aligned. The Ca2+ binding glutamate residues that form the selectivity filter are indicated with boxes.

The Ca²⁺ channel pore contains a Ca²⁺ binding site consisting of one negatively charged glutamate residue from each domain, which collectively form the selectivity filter (Fig. 1). The selectivity filter is thought to consist of a negatively charged locus capable of binding a single Ca²⁺ with high affinity or two Ca²⁺ ions with low affinity (Hille, 2001; Sather and McCleskey, 2003). In the absence of divalent cations, Ca²⁺ channels are highly permeable to monovalent cations. Under such conditions, Ca²⁺ functions as a channel blocker with an IC₅₀ in the submicromolar range. Monovalent currents are blocked by Ca²⁺ ions because monovalent cations are unable to dislodge tightly bound Ca²⁺ ions from the selectivity filter. Currents carried by Ca²⁺ ions occur when Ca²⁺ is raised to the millimolar range because repulsive forces from a second Ca²⁺ ion entering the pore increase the exit rate of the bound Ca²⁺ ion by more than 10,000-fold at physiological Ca²⁺ concentrations (Almers and McCleskey, 1984; Hess and Tsien, 1984; Yue and Marban, 1990). The original two-site barrier model nicely simulates most of the permeation properties of the channel, but it has limited value when applied to structural studies. However, the primary features of this model (i.e., binding and repulsion) almost certainly are dominant forces in a conducting pore. Binding and repulsion of ions within the selectivity filter are core features of recent models of the pore that are based more on structure than the original two-site models (Dang and McCleskey, 1998; Nonner et al., 1998; Boda et al., 2001; Corry et al., 2001; Lipkind and Fozzard, 2001).

We and others have found that high-affinity DHP binding is dependent on Ca²⁺ binding to the selectivity filter (Mitterdorfer et al., 1995; Peterson and Catterall, 1995). These findings establish an important link between DHP binding to its receptor site deep within the lipid bilayer and Ca²⁺ binding to the selectivity filter in the outer pore. This relationship offers a unique opportunity to gain a deeper understanding of the molecular basis for DHP action, ion permeation, and gating. We have systematically characterized the DHP and Ca²⁺ binding characteristics of mutant Ca²⁺ channels whose pore-forming glutamate residues have been replaced by glutamine or lysine. We analyzed these mutants using an allosteric binding model that enabled us to determine 1) the actual dissociation constants for Ca²⁺ binding to the selectivity filter at any DHP concentration, 2) the dissociation constant for DHP binding at any Ca²⁺ concentration, and 3) the magnitude of two allosteric factors that couple DHP and Ca²⁺ binding. We found that all four of the glutamate residues that form the ion selectivity filter are important for DHP and Ca²⁺ binding. Mutational analysis of several non-glutamate residues in the outer pore revealed altered DHP and Ca²⁺ sensitivity as well. Thermodynamic mutant cycle analysis (Carter et al., 1984; Hidalgo and MacKinnon, 1995) of two of these mutants, F1013G and Y1021K, indicate that Phe-1013 and Tyr-1021 are energetically coupled when a single Ca²⁺ ion is bound in the selectivity filter, suggesting that DHP binding promotes structural rearrangements in the outer pore that involve the energetic coupling of Phe-1013 and Tyr-1021 via a single bound Ca²⁺ ion. We discuss our findings in the context of current theoretical and structural models for permeation (Nonner et al., 1998; Boda et al., 2001; Corry et al., 2001; Lipkind and Fozzard, 2001; Wang et al., 2005) and propose that DHPs block monovalent and divalent currents by stabilizing a nonconducting blocked state that is structurally and functionally analogous to a channel with a single Ca²⁺ ion in its selectivity filter.

	Materials and Methods

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Preparation of Wild-Type and Mutant Ca_V1.1 Membranes. Wild-type and mutant _1S (Ca_V1.1) Ca²⁺ channels were coexpressed with the _1a and ₂ subunits as described previously (Peterson and Catterall, 1995). In brief, cDNAs encoding all the channel subunits were cotransfected into tsA-201 cells by calcium phosphate precipitation, and membranes were harvested 2 to 3 days after transfection. Cells were washed twice in buffer A (50 mM Tris, 100 µM phenylmethylsulfonyl fluoride, 100 µM benzamidine 1.0 µM pepstatin A, 1.0 µg/µl leupeptin, and 2.0 µg/ml aprotinin, pH 8.0). Cells were scraped and homogenized in the same buffer using a glass-Teflon homogenizer. The homogenate was centrifuged at 1700g for 10 min, and the resulting pellet was discarded. The supernatant was centrifuged at 100,000g for 30 min, and the resulting membrane pellet was washed and homogenized in buffer A. Membrane aliquots remained stable for several months, but they were typically used within 1 week of harvesting.

Radioligand Binding. Saturation binding assays were performed in buffer A using 20 to 100 µg of membrane protein, 0.1 to 10 nM (+)-[³H]PN200-110 (PerkinElmer Life and Analytical Sciences, Boston, MA), and the indicated concentrations of free Ca²⁺ for 120 min at 22°C. Nonspecific binding was determined by the addition of 1 µM(±)-PN200-110, thus reducing k_on for the radiolabeled ligand to an insignificant level, and bound radioligand was recovered by vacuum filtration using GF/C glass fiber filters.

View larger version (20K): [in this window] [in a new window]   Fig. 2. Ca2+ is a positive allosteric modulator of DHP binding. A, membranes prepared from cells expressing wild-type Ca2+ channels were incubated with [3H]PN200-110 at a concentration equal to its dissociation constant measured in 1 mM free Ca2+ (1 x KD1) and the indicated concentrations of free Ca2+ (see Materials and Methods). Data from one such experiment (circles) demonstrate that increasing Ca2+ from 10 nM to 1 mM results in a large increase in DHP binding with an EC50 of approximately 0.3 µM. Further increases in Ca2+ to the 100 mM range result in a large decrease in DHP binding with an IC50 in the high millimolar range. The line is a smooth fit through the data points and was generated using Scheme 1 (B). Scheme 1 describes CaV1.2 as possessing a single DHP receptor site that can exist in one of three affinity states, depending on whether zero, one, or two Ca2+ ions are bound to the selectivity filter. Scheme 1 is thermodynamically constrained such that dissociation constants for DHP and Ca2+ binding are coupled by the factors and . High-affinity DHP binding stabilizes a nonconducting, blocked state that is structurally analogous to a channel with a single Ca2+ ion in its selectivity filter (dashed box). Scheme 1 is further described in the text.

Data Analysis. DHP binding as a function of Ca²⁺ concentration was fit using an allosteric binding model (Fig. 2B, Scheme 1) described in the text with the aid of the analysis and graphics programs Excel (Microsoft, Redmond, WA) and Origin (OriginLab Corp., Northampton, MA). The statistical significance of the observed differences between the binding parameters of wild-type and mutant channels was evaluated using a two-tailed Student's t test and analysis of variance (Fig. 3). Data are means ± S.E.M., and statistical significance was set at P < 0.05 (^*).

View larger version (15K): [in this window] [in a new window]   Fig. 3. Ca2+ binding to the selectivity filter is allosterically coupled to DHP binding. A and B, individual dissociation constants, KD1 (A) and KC1 (B), were determined for [3H]PN200-110 and Ca2+ binding to wild-type and the indicated mutant channels as described under Materials and Methods. KD1 and KC1 values for all glutamine and lysine substitutions are significantly different from wild type. KC1 for the glutamine substitutions in domains II, III, and IV are significantly different from E292Q. KD1 values for E292Q and E1014Q are significantly different from E1323Q but not E614Q. C, values for the coupling factor were determined as described under Materials and Methods using Fig. 2B, Scheme 1 (also see Fig. 2A).

	Results

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Figure 2B, Scheme 1, is thermodynamically constrained such that the ratio of dissociation constants for DHP binding in the absence and presence of Ca²⁺ must equal to the ratio of the dissociation constants for Ca²⁺ binding in the absence and presence of DHP. Therefore, the individual dissociation constants for DHP and Ca²⁺ binding are completely determined by K_D1, the dissociation constant for DHP binding to a channel whose selectivity filter is occupied by a single Ca²⁺ ion, and K_C1, the dissociation constant for Ca²⁺ binding to DHP-occupied Ca²⁺ channels, respectively, and the allosteric factors and .

Ca²⁺ Binding to the Outer Pore Is Allosterically Coupled to DHP Binding. The pore segments from each domain of DHP-sensitive (Ca_V1.1-4) and -insensitive (Ca_V2.1-3) Ca²⁺ channels are aligned in Fig. 1B. To determine whether Ca²⁺ binding to the pore is allosterically coupled to DHP binding, each of the negatively charged glutamate residues that collectively form the selectivity filter (Fig. 1B, boxes) was replaced by a neutral glutamine or a positively charged lysine residue, generating the mutants E292Q, E614Q, E1014Q, E1323Q, E292K, E614K, E1014K, and E1323K. E614K and E1323K exhibited no DHP binding and were not studied further. K_D1 for each mutant was determined by saturation binding in the presence of 1 mM free Ca²⁺ and increasing concentrations of (+)-PN200-110. K_D1 values for wild-type and each of the mutant channels are summarized in Fig. 3A and Table 1. The sensitivity of the wild-type and mutant channels to Ca²⁺ was determined by incubating membranes in a concentration of (+)-PN200-110 equal to the measured K_D1 value for that particular channel (resulting in an occupancy of 0.5) plus increasing concentrations of free Ca²⁺, as described under Materials and Methods. K_C1 and were determined by fitting these data using Fig. 2B, Scheme 1, with K_D1 and free (+)-PN200-110 serving as fixed parameters. K_D1, K_C1, and for wild-type and each mutant channel are summarized in Fig. 3 and Table 1.

DHP binding, the sensitivity of DHP binding to Ca²⁺, and the coupling factor are altered for the mutant channels. The values determined for K_D1 are increased 5.5- to 10-fold by these mutations (Fig. 3A; Table 1). Although the four glutamate residues are not contiguously localized on the primary sequence, each functions as an important DHP binding determinant in the presence of Ca²⁺, suggesting that the selectivity filter acts as a unified locus that modulates DHP binding. Alteration of the four glutamate residues affects K_D1, K_C1, and to different extents. These differences indicate that the binding site for Ca²⁺ ions is asymmetrical, as is the coupling of each glutamate residue to DHP binding (Fig. 3).

As expected for mutant channels with amino acid substitutions in their selectivity filters, dramatic changes in K_C1 were observed (Fig. 3B). The smallest change in K_C1 was observed with E292Q, whose binding of Ca²⁺ is reduced by less than 12-fold. K_C1 for the other glutamate mutants was increased by 90- to 150-fold.

The allosteric coupling factor , which reflects the effect of binding of one Ca²⁺ ion on DHP affinity, was reduced from 15.5 for wild type to values ranging from 1.3 to 9.8 for the glutamate substitution mutants (Fig. 3C). E292Q, whose value for was increased to 54.5, was the only exception to this trend. The role plays in determining DHP binding properties is discussed in greater detail below. Together, the changes in K_D1, K_C1, and in these mutants indicate that the DHP receptor site is allosterically coupled to the selectivity filter of L-type Ca²⁺ channels. Therefore, it is plausible that the molecular details that underlie DHP activity may involve structural rearrangements in the outer pore and selectivity filter of the channel.

Uncharged Residues in the Outer Pore Are Critical for DHP and Ca²⁺ Binding. A comparison of the amino acid sequences in the outer pore segments of each repeat of the Ca²⁺ channel isoforms reveals several residues that are unique to DHP-sensitive channels and are adjacent to the Ca²⁺ binding glutamate residues in the selectivity filter (Fig. 1B). To determine whether these residues are important DHP- and/or Ca²⁺ binding determinants, the mutants C288A, F1013G, Q1018E, Q1018M, Y1021K, C1319A, Q1326H, and E1327Q were constructed and analyzed as described above and under Materials and Methods (Fig. 4; Table 2). C288A, Q1018E, Q1018M, C1319A, Q1326H, and E1327Q have DHP and Ca²⁺ binding profiles similar to those of wild type and are not discussed further.

View larger version (14K): [in this window] [in a new window]   Fig. 4. Nonglutamate residues in the pore segment of domain III are critical for DHP and Ca2+ binding. A to C, nonglutamate residues in the pores of repeats I, III, and IV were altered and analyzed as described in Fig. 3.

In contrast to the glutamate mutants, high-affinity binding of DHP and Ca²⁺ is enhanced for mutant F1013G, in which a glycine conserved in all Ca_V2 channels is substituted for a phenylalanine residue that is present in all Ca_V1.1 channels. K_D1 for F1013G is slightly lower than that of wild type (0.22 verses 0.31 nM, respectively), and K_C1 for F1013G decreased 2.3-fold from 40 to 17.4 nM (Fig. 4, A and B). However, the most interesting change in the binding profile for F1014G is the 12.5-fold increase in magnitude of the coupling factor which results in an 11-fold increase in K_D1 compared with wild type (Fig. 4C).

High-affinity binding of DHP and Ca²⁺ is also modified for mutant Y1021K, in which a lysine residue found in Ca_V2.1 and Ca_V2.2 channels is substituted for a tyrosine residue found in all Ca_V1.1 channels (Fig. 4). First, in contrast to F1013G, K_D1 for Y1021K is 3-fold larger than K_D1 for wild type (Fig. 4A; Table 2). Second, K_C1 for Y1021K is more than 30-fold larger than K_C1 for wild type (Fig. 4B). Finally, although the coupling factor for F1013G is 12.5-fold larger than that of wild type, for Y1021K is only one-half that of wild type (Fig. 4C).

The results with F1013G and Y1021K indicate that non-glutamate residues in the outer pore loop function as important binding determinants for DHPs and Ca²⁺ ions. The qualitative differences in the effects of these mutations on ligand binding suggest that the roles Phe-1013 and Tyr-1021 play in DHP and Ca²⁺ binding are distinct.

Ca²⁺ Promotes Energetic Coupling between Phe-1013 and Tyr-1021. Qualitatively divergent effects of Ca²⁺ on DHP binding affinity were observed with the mutants F1013G and Y1021K (Fig. 4; Table 2), even though these two substitutions typically coexist in Ca_V2 channels within the same nine-amino acid segment of the outer pore loop of domain III (Fig. 1B). This prompted us to construct a mutant channel in which Phe-1013 and Tyr-1021 have been replaced with glycine and lysine, respectively, resulting in the double mutant FY/GK. The double mutation causes a much larger increase in K_D1 than either single mutation (Fig. 4A; Table 2), whereas the values of K_C1 and are intermediate between the two single mutants (Fig. 4, B and C; Table 2). Thus, the parameters for DHP and Ca²⁺ binding to FY/GK differ substantially from the sum of those for the single mutants, indicating that the two residues interact energetically in their function as DHP and Ca²⁺ binding determinants.

Thermodynamic mutant cycle analysis (Carter et al., 1984; Hidalgo and MacKinnon, 1995) was used to quantitate the energetic interaction between Phe-1013 and Tyr-1021 in DHP binding (Fig. 5). The coupling energy (RTln) between Phe-1013 and Tyr-1021 was calculated in nominally zero Ca²⁺ using K_D1 values (Fig. 5A) and in 1 mM Ca²⁺ using K_D1 values (Fig. 5B) by subtracting the G that results from replacing Phe-1013 with glycine in a wild-type background from the G that results from the same substitution made in a Y1021K background. These analyses demonstrate that the degree of coupling between Phe-1013 and Tyr-1021 in DHP binding is highly dependent on the occupancy of the selectivity filter by Ca²⁺. The coupling energy in nominally zero Ca²⁺ of 0.08 kcal/mol is negligible (Fig. 5A). This near-zero coupling energy indicates that, in the absence of Ca²⁺ (i.e., state [₁] of Fig. 2B, Scheme 1), Phe-1013 and Tyr-1021 are not energetically coupled. In contrast, the coupling energy between Phe-1013 and Tyr-1021 determined in 1 mM Ca²⁺ is substantial. Replacing Phe-1013 with glycine is highly dependent on whether the substitution is placed in a wild-type backbone (+0.22 kcal/mol) versus the Y1021K backbone (-1.23 kcal/mol). Therefore, when the selectivity filter is occupied by a single Ca²⁺ ion (i.e., state [Ca/₁] of Fig. 2B, Scheme 1), Phe-1013 and Tyr-1021 are strongly coupled with an energy of 1.45 kcal/mol [(+0.22 kcal/mol) - (-1.23 kcal/mol) = 1.45 kcal/mol]. This "coupled state", in which Phe-1013 and Tyr-1021 are energetically coupled and a single Ca²⁺ ion is bound, may represent a stably blocked, nonconducting state of the channel, as is addressed in the discussion below.

View larger version (15K): [in this window] [in a new window]   Fig. 5. Ca2+ binding to the selectivity filter promotes energetic coupling between Phe-1013 and Tyr-1021. Double mutant cycle analysis was used to measure the coupling energy between Phe-1013 and Tyr-1021 in the absence (A) and presence (B) of a single Ca2+ ion in the selectivity filter. PN200-110 binding to its receptor site is driven by a negative change in Gibbs free energy, G. G changes for mutant channels whose affinity for DHP binding is altered and this change in G, designated G, is calculated using the dissociation constants for drug binding to the wild-type and mutant channels, such that G =-RTln(KDmut/KDwt), where R is the gas constant and T is the absolute temperature in Kelvin. G values (kilocalories per mole) for each mutant channel are noted by numbers adjacent to arrows. Thermodynamic mutant cycle analysis is based on the principle that the G value resulting from the simultaneous alteration of two amino acids is equal to the sum of the G values for the individual substitutions that lead to the double mutant. For example, note in A that the G for the double mutant FY/GK (-1.76 kcal/mol) is equal to the sum of the G values for the individual steps that lead to FY/GK [i.e., {(-1.42) + (-0.34)} and {(-0.26) + (-1.50)}]. The coupling energy (RTln) between two amino acids can be determined by calculating the difference between the G values associated with the horizontal (or vertical) arrows as described in the text. Notice that Phe-1013 and Tyr-1021 are strongly coupled only when a single Ca2+ ion is bound to the selectivity filter (also see Fig. 2B, vertical box).

The Coupling Factor Plays an Important Role in Determining the Ca²⁺ Dependence of DHP Binding. The effects of the coupling factor on the Ca²⁺ dependence of DHP binding to the mutant Ca²⁺ channels are illustrated in Fig. 6. These relationships between DHP binding affinity (K_D1^-1 and K_D1^-1 in nanomolar^-1) and Ca²⁺ concentration were simulated from the binding model in Fig. 2B, Scheme 1 and the binding parameters from Table 1. The DHP affinity of E292Q is 11-fold less than that of wild type with no Ca²⁺ ions bound to selectivity filter, but because of a 3.5-fold increase in compared with wild type, the affinity is decreased by only 3-fold when one Ca²⁺ ion is bound (Fig. 6A). In contrast to E292Q, values for the other glutamate mutants are smaller than those of wild type (Fig. 3). Consequently, the increases in DHP affinity upon binding of a single Ca²⁺ ion to these mutants are much less than for E292Q or wild type, as illustrated for E292K in Fig. 6A. In the absence of Ca²⁺, the DHP binding affinity for mutants E1014K and E1014Q are nearly identical to that of wild type (Fig. 6B). Increasing Ca²⁺ concentration has even less effect on DHP binding for E1014Q and E1014K (Fig. 6B) than for E292Q (Fig. 6A).

View larger version (15K): [in this window] [in a new window]   Fig. 6. Substitution of positively charged lysine residues in the selectivity filter partially mimics a bound Ca2+ ion. Simulations of [3H]PN200-110 binding affinity (nanomolar-1) versus free Ca2+ for wild type (dashed lines) and the mutants E292Q and E292K (A), E1014Q and E1014K (B), and F1013G and Y1021K (C) are plotted using the binding parameters from Tables 1 and 2. The expression levels of E614K and E1323K were too low to use in these analyses. The -fold differences between the DHP binding affinities of wild-type and/or mutant channels are indicated by numbers adjacent to arrows.

	Discussion

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Ca²⁺ binding to the pore of the L-type Ca²⁺ channels is allosterically coupled to DHP binding, which suggests that the binding of DHPs and Ca²⁺ promotes reciprocal structural rearrangements in the outer pore that alter the functional behavior of the channel. To better understand the roles amino acid residues residing in the outer pore have on DHP binding, permeation and gating, the binding properties of 16 mutant Ca²⁺ channels were assessed using Fig. 2B, Scheme 1. In addition to dramatic changes in the dissociation constants for DHP and Ca²⁺ binding, we found that the coupling factor plays a major role in determining the pharmacological properties of the mutant channels. For example, for the nonglutamate mutant F1013G is increased nearly 200-fold, whereas for Y1021K is only one-half that of wild type. Thermodynamic mutant cycle analysis of these mutants indicates that Phe-1013 and Tyr-1021 are energetically coupled only when the outer pore is occupied by a single Ca²⁺ ion. Our findings are discussed in the context of current theoretical models for permeation (Nonner et al., 1998; Boda et al., 2001; Lipkind and Fozzard, 2001; Wang et al., 2005). We propose that DHPs block monovalent and divalent currents by stabilizing a nonconducting blocked state that is structurally and functionally analogous to a channel with a single Ca²⁺ ion in its selectivity filter.

Binding of DHP Antagonists and Ca²⁺ Stabilize a Blocked Conformation of the Outer Pore. In the absence of Ca²⁺, the outer pore is held in an open conformation ([₁]) by electrostatic repulsion between the four glutamate residues of the EEEE locus. When a single Ca²⁺ ion enters the selectivity filter, it acts as a countercharge and draws the four glutamate residues together to form a blockedconformation ([Ca/₁]) (Lipkind and Fozzard, 2001). According to Fig. 2B, Scheme 1, DHP binding interacts allosterically with Ca²⁺ to stabilize the blocked conformation, thereby preventing Ca²⁺ conductance. Neutralizing any one of the four glutamate residues would destabilize this blocked conformation by reducing the magnitude of the attractive forces between the bound Ca²⁺ ion and the partially neutralized selectivity filter. In Fig. 3, the K_D1 values for the glutamate-to-glutamine mutants are all larger than that of wild type. We postulate that neutralization of the residues in the selectivity filter would destabilize the open conformation of the outer pore as well, because the magnitude of the repulsive forces between the four glutamate residues would be decreased. The simulations in Fig. 6 show reduced DHP binding in the absence of Ca²⁺, indicating that this postulate holds true.

Introduction of Positively Charged Lysine Residues Mimics Binding of Ca²⁺. Replacing the substituted glutamine residues with positively charged lysine residues would be expected to partially mimic a Ca²⁺ ion by acting as a countercharge in the selectivity filter. If the charge-reversal substitutions were to perfectly mimic a single bound Ca²⁺ ion in the pore, K_D1 would equal K_D1, and the coupling factor would equal 1. We were pleased to find that the charge-reversal substitutions did follow this predicted trend, but as might be expected, inserting a lysine with a valence of only +1 into a constrained position in the selectivity filter does not perfectly mimic a free divalent Ca²⁺ ion in the pore. The affinity for DHP binding in nominal Ca²⁺ (i.e., 1/K_D1) to E292K and E1014K membranes is 3- and 2-fold higher than their charge-neutralized counterparts (Fig. 6). These findings indicate that the introduction of a positive charge in the selectivity filter makes the [₁] state of the outer pore behave more like the Ca²⁺-bound [Ca/₁] state.

The charge-reversal mutants are still sensitive to Ca²⁺, but the -fold change in binding affinity that occurs as the channel transitions from [₁] to [Ca/₁] (i.e., the coupling factor ) is reduced. This reduction occurs because replacing the neutral glutamine residues with positively charged lysine residues introduces repulsive forces between the positively charged amine of the lysine residue and the incoming Ca²⁺ ion. Consequently, values for E292K and E1014K are decreased 8.5- and 3.3-fold compared with their glutamine-substituted counterparts. The combined effects on K_D1 and indicate that the introduction of a positive charge in the pore partially mimics a single Ca²⁺ ion coordinated in the selectivity filter.

Mechanism for DHP Action. The cooperativity between DHP and Ca²⁺ binding suggests that DHPs modulate channel gating by promoting conformational changes in the outer pore. We used a "volume exclusion/charge neutralization" model to explain the reduced conductance of Ba²⁺ but not Ca²⁺ through the pores of Ca_V1.2 correlates of F1013G, Y1021K, and FY/GK (Wang et al., 2005). The crystal diameters of Ca²⁺ and Na⁺ ions are nearly identical (2.00 versus 2.04 Å, respectively), yet each Ca²⁺ ion carries twice as much countercharge as a Na⁺ ion. Therefore, Ca²⁺ binds tightly to the selectivity filter because it is able to neutralize the highly charged EEEE locus without overcrowding it with counterions. Ba²⁺ and Ca²⁺ ions carry the same charge, but the ionic diameter of Ba²⁺ is approximately 36% larger than that of Ca²⁺. Thus, Ba²⁺ ions exhibit a higher degree of crowding, lower binding affinity and consequential faster exit rate (i.e., larger conductance) than Ca²⁺ ions. These results suggest that Ba²⁺ conductance is reduced because Ba²⁺ ions in mutant pores are less prone to overcrowding (Wang et al., 2000).

According to Fig. 2B, Scheme 1, Ca²⁺ and DHP binding shifts the configuration of the outer pore from an open state to a blocked state. [₁] or [2Ca/₁] are designated as conducting states in Fig. 2B, Scheme 1, because they represent channels conducting monovalent and divalent currents, respectively. The blocked state, [Ca/₁], is designated as a nonconducting state because single Ca²⁺ ions are known to block both monovalent and divalent (i.e., Ba²⁺) currents (Almers et al., 1984; Hess and Tsien, 1984; Hille, 2001). Here, we combine the volume exclusion/charge neutralization model with Fig. 2B, Scheme 1, and propose that DHPs block mono- and divalent currents through the open [₁] and [2Ca/₁] states of the outer pore by stabilizing a nonconducting blocked state that is structurally and functionally analogous to [Ca/₁].

This postulate is consistent with the structural model of Lipkind and Fozzard (2001). In this model, the eight carboxyl groups from the EEEE locus are thought to form three binding sites: a central high-affinity divalent cation binding site formed by four carboxyl groups flanked by two low-affinity sites, each composed of two carboxyl groups (Lipkind and Fozzard, 2001). Divalent cation permeation through such a pore would depend on the occupancy of the two low-affinity sites, thus producing a conducting state ([2Ca/₁]). Ion permeation would be prevented if the central, high-affinity site were occupied by a single Ca²⁺ ion, thus producing a non-conducting state ([Ca/₁]). In the absence of divalent cations, repulsive forces in the EEEE locus would open the pore to greater than 4 Å and allow the passage of monovalent cations through the pore, thus producing a conducting state for monovalent cations ([₁]).

Given this scenario, the outer pore of an activated channel would switch between conducting ([2Ca/1]) and nonconducting, blocked ([Ca/₁]) states. The overall open probability of a channel would be determined by the probability that the inner gate is open and the probability that the channel is not in state [Ca/₁], which is dependent on the dwell time of the single blocking Ca²⁺ ion residing in the selectivity filter. We propose that DHP antagonists increase this dwell time and that the open probability of the channel decreases as a consequence. It will require single-channel measurements to determine whether the mean open times decrease in the presence of DHP antagonists. Such experiments are very challenging using Ca_V1.1 channels, so this hypothesis will be tested using the cardiac Ca_V1.2 channel.

Energetic Coupling Mediated by Bound Ca²⁺. The conformational changes that underlie the transitions between the open and blocked states of the outer pore and modulate DHP binding affinity are dependent on the magnitude of the coupling factor . We used thermodynamic mutant cycle analysis to assess the transition of the outer pore between the open and blocked states, [₁] and [Ca/₁], respectively. Our results demonstrate that Phe-1013 and Tyr-1021 are strongly coupled only when the selectivity filter is occupied by a single Ca²⁺ ion and not when the selectivity filter is unoccupied by Ca²⁺ (Fig. 2B, Scheme 1, box). It seems that the bound Ca²⁺ ion serves to mediate energetic coupling between these two aromatic amino acid residues, which are located on either side of Glu-1014 (Fig. 1). This coupling may result from electronic interactions between the two aromatic residues through the bound Ca²⁺ ion, structural rearrangements in the selectivity filter caused by Ca²⁺ binding, or both. We postulate that Phe-1013 and Tyr-1021 are energetically coupled when the pore is in a blocked, nonconducting state, and that DHP antagonists block the channel by stabilizing this same nonconducting conformational state. The positive energetic coupling between Phe-1013 and Tyr-1021 revealed here is likely to make a major contribution to the stability of this Ca²⁺-bound, blocked state.

	Acknowledgements

 
We thank Gregory M. Lipkind for insightful comments on the manuscript.

	Footnotes

 
This work was supported by American Heart Association grant 0230298N (to B.Z.P.) and National Institutes of Health grants R01-HL074143 (to B.Z.P.) and P01-HL44948 (to W.A.C.).

ABBREVIATIONS: DHP, dihydropyridine; PN200-110, 4-(4-benzofurazanyl)-1,4-dihydro-2,6-dimethyl-3.5-pyridinedicarboxylic acid methyl 1-methyl ester.

Address correspondence to: Dr. Blaise Z. Peterson, Cellular and Molecular Physiology, H166, Penn State Milton S. Hershey Medical Center, College of Medicine, 500 University Dr., Room C6603, P.O. Box 850, Hershey, PA 17033-0850. E-mail: bpeterson{at}psu.edu

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