©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Calcium Binding in the Pore of L-type Calcium Channels Modulates High Affinity Dihydropyridine Binding (*)

(Received for publication, May 9, 1995; and in revised form, June 14, 1995)

Blaise Z. Peterson William A. Catterall (§)

From the Department of Pharmacology, SJ-30, School of Medicine, University of Washington, Seattle, Washington 98195

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The pore-forming alpha(1) subunit of L-type voltage-gated Ca channels contains a Ca-binding site that is allosterically coupled to the receptor site for dihydropyridine (DHP) Ca antagonists. Site-directed mutations of conserved Phe and Glu residues in the pore-lining SS1/SS2 segments greatly reduced Ca enhancement of DHP binding. Substitution of Phe-1013 in the alpha(1) subunit from rabbit skeletal muscle (alpha) with Gly (F1013G) as in DHP-insensitive Ca channels caused a 4-fold decrease in sensitivity to Ca. Mutation of the Ca-binding residues Glu-1014 in domain III and Glu-1323 in domain IV to Gln (E1014Q and E1323Q) caused 11- and 35-fold decreases in sensitivity to Ca, respectively, as well as decreases in the maximal DHP binding affinities attained at optimal concentrations of Ca. DHP binding to the charge-reversal mutation, E1014K, had no sensitivity to Ca. Our results demonstrate that high affinity Ca binding to the Glu residues in the SS1/SS2 segments of domains III and IV of alpha stabilizes the DHP receptor site in its high affinity state. We propose a three-state model in which the affinity for DHPs is dependent on the presence of 0, 1, or 2 bound Ca ions at sites in the pore.


INTRODUCTION

Calcium entry through voltage-gated Ca channels is essential for regulation of excitation-contraction coupling, excitation-secretion coupling, electrical excitability, and other cellular functions(1) . Voltage-gated Ca channels purified from skeletal muscle are heteropentamers consisting of alpha(1), alpha(2), beta, and subunits (reviewed in (1) and (2) ). The pore-forming alpha(1) subunit is composed of four homologous domains consisting of six transmembrane segments (S1-S6)(3) . The loops that connect transmembrane segments S5 and S6 in each domain contain the short SS1/SS2 segments that are thought to form the ion selectivity filter(4, 5, 6, 7, 8) . Conserved glutamate residues in the SS2 segments (one in each domain) form two Ca binding sites that are essential for ion permeation and selectivity(4, 5, 6, 7, 8) .

The alpha(1) subunit also contains the high affinity receptor site for the dihydropyridine (DHP) (^1)Ca antagonists(9, 10, 11) . DHPs act by stabilizing the Ca channel in different gating states; agonists stabilize a state with high open probability, while antagonists stabilize a state with low or null open probability(12) . The DHP receptor site has been localized by photoaffinity labeling and antibody mapping to peptides that correspond to extracellular portions of the S6 segments of domains III and IV(9, 10, 11) . In addition, mutations in the extracellular portion of segment IVS6 affect the action of DHP Ca channel agonists(13) . The DHP receptor site is thought to exist in at least two affinity states, and the high affinity state for antagonists is stabilized by Ca binding with a dissociation constant (K) less than 1 µM(14) . Here, we demonstrate that high affinity Ca binding to the glutamate residues in the SS2 segments of domains III and IV stabilizes the DHP receptor site in its high affinity state, and we propose a three-state model in which the affinity for DHPs is dependent on the binding of 0, 1, or 2 Ca ions to sites in the pore.


EXPERIMENTAL PROCEDURES

Construction and Expression of Mutant Ca Channels

DNAs encoding the alpha(1) subunit of the Ca channel from rabbit skeletal muscle (15) in the expression plasmid ZemRVSP6(16) , the beta subunit (17) in pRc/CMV, and the alpha(2) subunits (15) in ZemRVSP6 (16) were constructed. (^2)Site-directed mutagenesis was performed using established procedures(18) . tsA-201 cells were cotransfected (19) with wild-type or mutant ZemCaCh, and cDNA encoding the beta and alpha(2) subunits at a molar ratio of 1:1:1. 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/ml leupeptin, and 2.0 µg/ml aprotinin, pH 8.0) containing 1 mM EDTA. Cells were scraped and homogenized in the same buffer using a glass-Teflon homogenizer. The homogenate was centrifuged at 700 g for 5 min. The resulting pellet was discarded, and the supernatant was centrifuged 30 min at 100,000 g. The supernatant was discarded, and the membrane pellet was washed and homogenized in Buffer A (with no added EDTA).

Radioligand Binding

Equilibrium binding assays were performed in Buffer A on 20-100 µg of membrane protein, 0.1-10 nM (+)-[^3H]PN200-110 (NEN Dupont), and the indicated concentrations of free Ca for 90 min at 22 °C. Nonspecific binding was determined in the presence of 1 µM (±)-PN200-110, and bound and free ligands were separated by vacuum filtration over GF/C glass fiber filters. All data are means ± S.E., and Student's t test with p < 0.05 was defined as the criterion of significance.

Experiments that determine the sensitivity of DHP binding to cations were performed on wild-type and mutant membranes using concentrations of (+)-[^3H]PN200-110 corresponding the K of each channel for (+)-[^3H]PN200-110 determined by Scatchard analysis in 1 mM free Ca. Experiments measuring Ca sensitivity were performed in Buffer A containing 5 mM EDTA, 5 mM HEDTA, 5 mM nitrilotriacetic acid, and various concentrations of CaCl(2). Experiments measuring Ba sensitivity were performed in Buffer A containing 1 mM EGTA and various concentrations of BaCl(2). The levels of free Ca and free Ba in these experiments were determined using published cation binding constants for the various chelating agents(20) . Experiments measuring Cd sensitivity were performed in Buffer A, 100 µM CaCl(2), and the indicated concentrations of CdCl(2).

Data Analysis

DHP binding as a function of cation concentration was analyzed according to a three-state model as illustrated in Fig. S1(see ``Results and Discussion''). The total amount of DHP binding at a given DHP concentration (B) is the sum of the binding to all three states: B = [DHP bullet alpha] + [DHP bullet alpha bullet Ca(1)] + [DHP bullet alpha bullet Ca(2)]. The fractional occu-pancy at each affinity state is described by a rectangular hyperbola; therefore Y, the fraction of all channels with bound DHP is given by:














Figure S1:


where K(0), K(1), and K(2) are the dissociation constants for DHP for each affinity state when 0, 1, and 2 Ca ions are bound to the channel, and P[alpha], P[alphabulletCa(1)], and P[alphabulletCa(2)] represent the fraction of channels in each affinity state.

The probability of each DHP affinity state depends on whether 0, 1, or 2 Ca ions are bound. The fractional occupancy of Ca binding sites 1 and 2 by Ca ions is given by p = [Ca]/([Ca] + K

The expressions for p and q above can be substituted into to yield an expression defining DHP binding as a function of free Ca. K(1) was determined empirically as the dissociation constant for DHP binding in the presence of 1 mM Ca. K(2) was assumed to be 430 nM which is the dissociation constant for low affinity block of skeletal muscle Ca channels by PN200-110 determined electrophysiologically (21) because two Ca ions must bind for permeation.


RESULTS AND DISCUSSION

A comparison of the amino acid sequences of the SS1/SS2 segments of voltage-gated Ca channels reveals that all DHP-sensitive channels have Phe in the SS1/SS2 segment of domain III at the position corresponding to 1013 (Phe-1013) in alpha, while all DHP-insensitive channels have a Gly at this position (Fig. 1). The adjacent Glu at position 1014 is a component of the selectivity filter(4, 5, 6, 7) , and this amino acid is conserved in all voltage-gated Ca channels. We hypothesized that Phe-1013 might be involved in the allosteric coupling of Ca binding and DHP binding due to its close proximity to the Ca-binding Glu residue in the pore. A mutant that replaces Phe-1013 with Gly was constructed, and both wild-type and mutant (F1013G) constructs were transiently expressed in tsA-201 cells. Membranes derived from these cells were tested for the binding of the DHP antagonist (+)-[^3H]PN200-110 in the presence of various concentrations of free Ca. Increasing the free Ca causes a substantial increase in the level of (+)-[^3H]PN200-110 binding to both wild-type and F1013G membranes. However, for the mutant channel, the EC for this stimulation is shifted to a 4.1-fold higher free Ca concentration, and the amount of (+)-[^3H]PN200-110 binding at 1 nM free Ca is decreased below the level of detection (Fig. 2A).


Figure 1: Sequence alignment of the P-region from DHP-sensitive (alpha, alpha, alpha) and DHP-insensitive (alpha, alpha, alpha) Ca channels. Amino acids corresponding to positions 1012-1016 (Domain III) and 1321-1325 (Domain IV) of alpha are shown. Glu residues involved in selectivity and permeation are indicated in bold. Phe (DHP-sensitive) and Gly (DHP-insensitive) residues corresponding to position 1013 of domain III are indicated with a box.




Figure 2: Ca dependence of dihydropyridine binding in wild-type and F1013G membranes. A, stimulation of (+)-[^3H]PN200-110 binding by increasing free Ca in wild-type and F1013G membranes. Wild-type (): EC = 0.560 ± 0.144 µM. F1013G (): EC = 2.30 ± 0.77 µM, p < 0.05. B and C, Scatchard transformation of equilibrium binding data at 1 mM (), 10 µM (), 1 µM (), 100 nM (), and 1 nM () free Ca in wild-type (B) and F1013G (C) membranes. D, DHP affinity, 1/K(nM), as a function of free Ca from experiments shown in B and C, above.



In order to analyze the changes in Ca sensitivity and DHP affinity quantitatively, Scatchard analyses were performed at various Ca concentrations (Fig. 2, B-D). These experiments demonstrate that 1) increasing the concentration of free Ca causes the DHP binding site to shift from a low affinity state to a high affinity state in both wild-type and F1013G; 2) F1013G requires higher levels of free Ca to stabilize this high affinity state; 3) the K for DHP binding to the high affinity state of mutant F1013G is not significantly different from that of wild-type (p > 0.05); and 4) DHP binding to the low affinity state of mutant F1013G is not detectable by radioligand binding, while wild-type has a measurable K of 2.1 ± 0.1 nM. From these experiments, we conclude that the high affinity state of the DHP receptor site is not altered in F1013G, but this mutant has reduced affinity for DHPs in the low affinity state and has a decreased affinity for Ca.

In general, the effects of divalent cations on DHP binding are biphasic with enhanced binding at intermediate concentrations followed by reduced binding as the concentration of cation is increased(14, 22) . This biphasic response is illustrated for Ba in Fig. 3A. The EC for the Ba-dependent stimulation of DHP binding is 18-fold higher in F1013G (EC =109 µM) than in wild-type (EC = 6.0 µM), while the IC for Ba-dependent inhibition of DHP binding is not significantly different (IC = 1-3 mM; p > 0.05). Similar results were obtained with Co, La, Mg, Mn, and Ni. These results can most easily be explained by a three-state model describing a single DHP receptor site that exists in three interconvertible affinity states as proposed by Glossmann et al.(14) . Our results are most consistent with the assumption that equilibrium among these three states is dependent on the number of cations bound to the channel (Fig. S1).


Figure 3: The effects of divalent cations on DHP binding are biphasic. A, stimulation and inhibition of (+)-[^3H]PN200-110 binding by increasing free Ba in wild-type () and F1013G () membranes. Wild-type (): EC = 5.96 ± 4.86 µM, IC = 2.67 ± 1.13 mM. F1013G (): EC = 108.9 ± 2.0 µM, p < 0.05; IC = 1.1 ± 0.85 mM, p > 0.05. B, inhibition of (+)-[^3H]PN200-110 binding by increasing free Cd in wild-type () and F1013G () membranes. Wild-type (): IC = 6.58 ± 1.86 µM, n(H) = -2.59 ± 0.71. F1013G (): IC = 4.18 ± 0.79 µM, n(H) = -2.23, p > 0.05.



K

Of the ions tested, Ca and Cd represented two extreme cases. The range of Ca concentrations that stabilized the high affinity DHP binding site was very broad suggesting K

The results with the mutant F1013G suggest that Ca binding to the Glu residues within the pore of the Ca channel may be necessary to stabilize the high affinity state of the DHP receptor site. To test this hypothesis directly, we altered the Ca-binding Glu residues in domains III and IV because photoaffinity labeling experiments suggest that portions of these two domains are important determinants of DHP binding(9, 10, 11) . Glu-1014 in domain III was replaced with an uncharged Gln (E1014Q) or a positively charged Lys (E1014K). A Lys is located at this position in voltage-gated sodium channels(4) . Glu-1323 in domain IV was also replaced with a Gln (E1323Q) or a Lys (E1323K), but E1323K exhibited no detectable (+)-[^3H]PN200-110 binding and was not studied further.

Fig. 4A shows the Ca dependence of (+)-[^3H]PN200-110 binding in membranes derived from cells expressing wild-type, E1014Q, E1014K, and E1323Q. Membranes were incubated in the presence of a constant level of (+)-[^3H]PN200-110 and the indicated concentrations of Ca. The concentration of (+)-[^3H]PN200-110 used was roughly equal to the dissociation constant for high affinity binding of (+)-[^3H]PN200-110 determined by Scatchard analysis of DHP binding at 1 mM free Ca for each mutant. For example, in the experiment of Fig. 4A, the concentrations of (+)-[^3H]PN200-110 used for wild-type, E1014Q, E1014K, and E1323Q were 0.28, 1.4, 1.9, and 1.4 nM, respectively, and each gave a DHP site occupancy of approximately 0.5 at 1 mM Ca. The data were fit using a mathematical model based on Fig. S1(see ``Experimental Procedures''), and the parameters determined by this fit are summarized in Table 1. K


Figure 4: Ca dependence of (+)-[^3H]PN200-110 binding in wild-type, E1014Q, E1014K, and E1323Q membranes. A, membranes were incubated with the indicated concentrations of free Ca and (+)-[^3H]PN200-110 was measured. Data are normalized to B(max) and fit using a mathematical model based on Fig. S1as described under ``Experimental Procedures.'' The results of this fit are summarized in Table 1. B, normalized stimulation of DHP binding from A illustrates relative shifts in EC values for wild-type and mutants. C, inhibition of (+)-[^3H]PN200-110 binding by increasing free Cd. D, DHP affinity (1/K in nM) determined by Scatchard analysis as a function of free [Ca]. For all panels: wild-type, ; E1014Q, ; E1014K, ; and E1323Q, .





Table 1shows that mutation E1014Q results in a significant increase in K(1), while mutation E1323Q results in significant increases in both K(0) and K(1). This implies that, unlike mutation F1013G, mutations E1014Q and E1323Q affect both Ca affinity and DHP affinity. In order to study these effects in more detail, DHP binding affinities (1/K) were determined from saturation binding experiments and Scatchard analyses on wild-type, E1014Q, E1014K, and E1323Q membranes in the presence of a range of concentrations of free Ca (Fig. 4D). With the exception of E1014K, all exhibited increased affinity for (+)-[^3H]PN200-110 with increasing Ca, although the highest affinity reached as well as the concentration of Ca required to reach maximum affinity varied substantially. The maximum DHP affinities attained in 1 mM free Ca (K(1)) for E1014Q, E1014K, and E1323Q are significantly less than for wild-type (Fig. 4D and Table 1). The K of the low affinity state (K(0)) measured in the presence of 1 nM free Ca was not significantly different among wild-type, E1014Q, and E1014K (p > 0.05). In contrast, E1323Q, like F1013G (Fig. 2D), has an affinity too low to measure by radioligand binding methods at 1 nM Ca. These results are consistent with K(0) values estimated from the model of Fig. S1and the results of Fig. 4A (see Table 1) where wild-type, E1014Q, and E1014K varied only slightly, while K(0) for E1323Q was 3-fold higher than for wild-type. The simplest explanation to account for these observations is that wild-type, E1014Q, and E1014K can bind (+)-[^3H]PN200-110 with a low but measurable affinity without Ca bound, while F1013G and E1323Q have lost this ability as a result of an alteration in the structure of the DHP receptor site or its allosteric coupling to the Ca binding site in the pore.

Permeation of Ca through the pore of the Ca channel is thought to require the binding of two Ca ions (5, 6, 23, 24) . The first Ca ion binds with a K of 0.7 µM and blocks the pore while the second binds with a K of about 100 mM and allows permeation(23) . Similarly, two Ba ions are thought to bind with Kvalues of 16 µM(25) and 6 mM(26) , respectively. Our results indicate that Glu residues in the pore of the Ca channel are required to bind one Ca or Ba ion to generate the high affinity state for DHP antagonists and that the K for Ca binding (K


FOOTNOTES

*
This work was supported by National Institutes of Health Research Grant P01 HL 44948 (to W. A. C.), a research grant-in-aid from the American Heart Association (to W. A. C.), and a predoctoral training fellowship from National Institutes of Health Training Grant T32 HL07312 (to B. Z. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed.

^1
The abbreviation used is: DHP, dihydropyridine.

^2
Y. Lai, P. Gallombardo, K. S. De Jongh, B. Z. Peterson, and W. A. Catterall, unpublished experiments.

^3
B. Z. Peterson and W. A. Catterall, unpublished results.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.