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J. Biol. Chem., Vol. 280, Issue 1, 494-505, January 7, 2005

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The C-terminal Residues in the Alpha-interacting Domain (AID) Helix Anchor Ca_V Subunit Interaction and Modulation of Ca_V2.3 Channels^*

Laurent Berrou, Yolaine Dodier, Alexandra Raybaud¶, Audrey Tousignant||, Omar Dafi, Joelle N. Pelletier||, and Lucie Parent**

From the Département de Physiologie, Groupe d'étude des Protéines Membranaires, the ^¶Département de Physique, and the ^||Département de Chimie, Université de Montréal, P. O. Box 6128, Downtown Station, Montréal, Québec H3C 3J7, Canada

Received for publication, September 21, 2004 , and in revised form, October 25, 2004.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
The alpha-interacting domain (AID) in the I-II linker of high voltage-activated (HVA) Ca²⁺ channel 1 subunits binds with high affinity to Ca_V auxiliary subunits. The recently solved crystal structures of the AID-Ca_V complex in Ca_V1.1/1.2 have revealed that this interaction occurs through a set of six mostly invariant residues Glu/Asp⁶, Leu⁷, Gly⁹, Tyr¹⁰, Trp¹³, and Ile¹⁴ (where the superscript refers to the position of the residue starting with the QQ signature doublet) distributed among three -helical turns in the proximal section of the I-II linker. We show herein that alanine mutations of N-terminal AID residues Gln¹, Gln², Ile³, Glu⁴, Glu⁶, Leu⁷, and Gly⁹ in Ca_V2.3 did not abolish [³⁵S]Ca_V1b or [³⁵S]Ca_V3 subunit overlay binding to fusion proteins nor did they prevent the typical modulation of whole cell currents by Ca_V3. Mutations of the invariant Tyr¹⁰ with either hydrophobic (Ala), aromatic (Phe), or positively charged (Arg, Lys) residues yielded Ca_V3-responsive whole cell currents, whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca_V3 binding and modulation. In contrast, modulation and binding by Ca_V3 was significantly weakened in I14A (neutral and hydrophobic) and I14S (neutral and polar) mutants and eradicated in negatively charged I14D and I14E or positively charged I14R and I14K mutants. Ca_V3-induced modulation was only preserved with the conserved I14L mutation. Molecular replacement analyses carried out using a three-dimensional homology model of the AID helix from Ca_V2.3 suggests that a high degree of hydrophobicity and a restrained binding pocket could account for the strict structural specificity of the interaction site found at position Ile¹⁴. Altogether these results indicate that the C-terminal residues Trp¹³ (1) and Ile¹⁴ anchor Ca_V subunit functional modulation of HVA Ca²⁺ channels.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
The passage of Ca²⁺ in a selective manner across the lipid bilayer of the cellular plasma membrane occurs by way of several transport proteins. The voltage-dependent calcium channel (VDCC)¹ represents one key pathway that regulates the entry of extracellular calcium into electrically excitable cells. VDCC are multimeric proteins composed of the transmembrane pore-forming Ca_V1, the disulfide-linked dimer Ca_V2, the intracellular Ca_V subunits (1-4), and in some cases the Ca_V subunit (2). To this date, the primary structures for 10 distinct VDCC 1 subunits have been identified by molecular cloning and shown to belong to three main families: the classical L-type high voltage-activated (HVA) VDCC form the Ca_V1 class; Ca_V2 are non-L-type HVA VDCC; and Ca_V3 are the T-type VDCC that are activated at low voltage (LVA). Although a minimum VDCC can be formed by a single Ca_V1 subunit, co-expression with auxiliary Ca_V subunits is required for HVA Ca_V channels such as the cardiac L-type Ca_V1.2 (3, 4), brain N-type Ca_V2.1 (5), brain L-type Ca_V1.3 (6-8), and R-type Ca_V2.3 (4) to generate Ca²⁺ currents with time course and voltage dependence similar to native currents (9). Ca_V subunit modulation of LVA T-type (Ca_V3.1-3.3) channels has yet to be reported (10).

Ca_V subunits increase peak current density, in part by recruiting the Ca_V1 subunit to the plasma membrane (11-14), by hyperpolarizing the voltage dependence of activation and inactivation, and by increasing the channel opening probability at the single channel level (4, 15). Ca_V subunits increase inactivation kinetics in an isoform-specific manner with Ca_V3 > Ca_V1b > Ca_V1a > Ca_V4 >> Ca_V2a (4). The four known auxiliary Ca_V subunits can potentially associate with any of the six Ca_V1 pore-forming subunits of HVA VDCC (Ca_V1.1-1.4, Ca_V2.1-2.3) via the alpha interaction domain (AID) located on the I-II linker of the Ca_V1 subunit. The AID motif is absent from LVA T-type (Ca_V3.1-3.3) VDCC for which Ca_V subunit modulation has never been reported. About half of the residues of the AID helix (Gln¹-Gln²-X³-Glu⁴-X⁵-X⁶-Leu⁷-X⁸-Gly⁹-Tyr¹⁰-X¹¹-X¹²-Trp¹³-Ile¹⁴-X¹⁵-X¹⁶-X¹⁷-Glu¹⁸) are strictly conserved (in bold) among the six Ca_V1 subunits but homology was found to be higher within members of the Ca_V1 and Ca_V2 families. Nonetheless, positions 8, 11, and 15 are occupied by residues that could vary even within the same Ca_V family (Table I). The AID motif Gln¹-Gln²-X³-Glu⁴-X⁵-X⁶-Leu⁷-X₈-Gly⁹-Tyr¹⁰-X¹¹-X¹²-Trp¹³-Ile¹⁴-X¹⁵-X¹⁶-X¹⁷-Glu¹⁸ (where X can be any residue) might not be the unique determinant of the Ca_V1- interaction. In vitro binding experiments have revealed the presence of additional interaction sites of lower affinity on the cytoplasmic loops of Ca_V2.1 (16, 17) and on the C-terminal of Ca_V2.3 (18) but the AID appears to be the primary high affinity site of interaction (14, 15).

View this table: [in this window] [in a new window]   TABLE I AID domain in HVA CaV1 subunits Primary sequences of the AID domain are shown at each of the 18 positions of the AID region for six HVA CaV1-subunits corresponding to the CaV1.1 to the CaV2.3 families. Sequences were obtained from the rabbit CaV1.1 (GenBankTM M23919 [GenBank] ), rabbit CaV1.2 (GenBankTM X15539 [GenBank] ), rat CaV1.3 (GenBankTM D38101 [GenBank] ), rabbit CaV2.1 (GenBankTM X57477 [GenBank] ), rabbit CaV2.2 (GenBankTM D14157 [GenBank] ), and human CaV2.3 (GenBankTM L27745 [GenBank] ).

The AID peptide from the Ca_V1 subunit adopts an -helical structure upon binding to the Ca_V subunit, and the six AID residues from the three main -helical turns nestle into the hydrophobic groove found in the GK domain opposite to the SH3 domain in the Ca_V subunit. The hydrophobic face of the AID helix aptly named the "alpha-binding pocket" (ABP) contains 7 residues X³, X⁶, Leu⁷, Gly⁹, Tyr¹⁰, Trp¹³, and Ile¹⁴ (or Ile³⁷⁶, Glu³⁷⁹, Leu³⁸⁰, Gly³⁸², Tyr³⁸³, Trp³⁸⁶, and Ile³⁸⁷ in Ca_V2.3) that interact with the Ca_V subunit. The interaction between the AID helix and the Ca_V protein appears to be primarily secured by the projection of the AID C-terminal pairs Gly⁹/Tyr¹⁰ and Trp¹³/Ile¹⁴ onto the Ca_V fold (21). Both Tyr¹⁰ and Trp¹³ are buried completely within the Ca_V protein. Tyr¹⁰ makes multiple van der Waals interactions with Ca_V through its main chains while its side-chain hydroxyl group forms hydrogen bonds with two water molecules, the side-chain of Asp⁶, and the main chain of Ile¹⁴ (19). Trp¹³ plays a central role by forming six van der Waals interactions with residues of Ca_V subunits and one hydrogen bond with the main-chain carbonyl of a methionine residue in Ca_V subunit. Ile¹⁴ forms three van der Waals interactions with residues of Ca_V subunits (19, 20). Gly⁹ appears to be entrenched within the Ca_V fold like Tyr¹⁰ and almost to be as inaccessible as Trp¹³ and Ile¹⁴ (19), but the extent of its interaction with Ca_V remains debatable (20, 21). The extensive network of interactions between the two subunits, mostly hydrophobic in nature, probably account for the nanomolar affinity between the Ca_V1 and the Ca_V subunits (22, 23).

The three-dimensional structures vividly account for a wealth of structure-function data gathered over the last decade. Functional studies have long shown the YWI residues to be critical for Ca_V1b and/or Ca_V3 binding in Ca_V2.1 (24). We have recently shown that point mutations of the conserved Trp¹³ (Trp³⁸⁶) eliminated the functional modulation of Ca_V2.3 currents by Ca_V3 as well as disrupted the Ca_V3 subunit binding to the AID peptide (1). The stereochemical requirements at this position were strict since substitutions with either hydrophobic (Ala, Gly), hydrophilic (Gln, Arg, Glu), and aromatic (Phe, Tyr) residues led to the same results suggesting that Trp¹³ (Trp³⁸⁶) played a unique role in Ca_V subunit binding and modulation of Ca_V2.3 channels. Y10F preserved in part Ca_V3 and Ca_V2 binding but preserving the hydroxyl side-chain in Y10S and conserving the aromatic group in the Y10W mutant led nonetheless to a significant decrease in Ca_V2, Ca_V3, and Ca_V1b binding to AID_A (Ca_V2.1) (24, 25). This observation was also confirmed in Ca_V1.2 and Ca_V1.1 channels where the Y10S mutant disrupted the plasma membrane localization of the Ca_V1 subunit while preserving in part the Ca_V subunit-induced modulation of whole cell currents (26-28). In contrast, point mutations of Gln¹, Gln², Glu⁴, Leu⁷, Gly⁹, and Glu¹⁶ failed to abolish the binding of Ca_V3 to the denatured AID peptide from Ca_V2.1 (13) suggesting that the stereochemical requirements might not be as strict in these positions to achieve a strong interaction with Ca_V subunits. In contrast, there is very little information regarding the role of the N-terminal pair of residues Glu⁶/Leu⁷ in establishing the functional interaction with Ca_V subunits although the crystal structures have demonstrated a strong interaction between the two subunits at this site. It thus remains to be seen whether all of these Ca_VAID-Ca_V interaction sites are required to confer the typical Ca_V subunit-induced functional modulation of Ca_V1 and Ca_V2 currents.

Herein we show that the alanine mutation of each of the AID residues Gln¹, Gln², Glu³, Glu⁴, Glu⁶, Leu⁷, Gly⁹, and Glu¹⁸ did not eliminate [³⁵S]Ca_V1b or [³⁵S]Ca_V3 subunit overlay binding to fusion proteins nor did it prevent the typical modulation of Ca_V2.3 whole cell currents by Ca_V3. Mutations of Tyr¹⁰ with either aromatic (Phe) or positively charged (Arg, Lys) residues yielded whole cell currents that responded to Ca_V3 essentially like the wild-type channel whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca_V3 binding and modulation. The structural requirements were more stringent for Ile¹⁴ since the modulation and binding by Ca_V3 were only preserved with the conserved Iso to Leu mutation. Altogether these results suggest that the C-terminal residues Trp¹³ (1) and Ile¹⁴ play a more critical role than Gly⁹ and Tyr¹⁰ in Ca_V subunit functional modulation of HVA Ca²⁺ channels.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Recombinant DNA Materials—Standard methods of plasmid DNA preparation were used (29). cDNAs coding for the auxiliary rat Ca_V3 (GenBank^TM M88751 [GenBank] ) and rat brain Ca_V1b (GenBank^TM X61394 [GenBank] ) were kindly donated by Dr. E. Perez-Reyes (30-32). The wild-type human 1E or Ca_V2.3 (GenBank^TM L27745 [GenBank] ) was a gift from Dr Toni Schneider (33). The rat brain Ca_V2b subunit was provided by Dr. T. P. Snutch.

Point Mutations and RNA Transcription—Point mutations were performed with 40-mer synthetic oligonucleotides using the QuikChange^TM XL-mutagenesis kit (Stratagene, La Jolla, CA). Briefly, mutations were achieved by cassette cloning using the NotI/XhoI Ca_V2.3 fragment (positions 111 and 1275 nt) subcloned into pBluescript (Stratagene). Constructs were verified by restriction mapping after religation of the mutated fragment into the naturally occurring NotI/XhoI sites of the wild-type Ca_V2.3. The nucleotide sequence of the mutant channels was confirmed through automated fluorescent DNA sequencing (BioST, Lachine, Qué). cDNA constructs for wild-type and mutated Ca_V1 subunits were linearized at the 3'-end by HindIII digestion whereas the Ca_V3 subunit was digested by NotI. Run-off transcripts were prepared using the methylated cap analog m⁷G(5')ppp(5')G and T7 RNA polymerase with the mMessage mMachine® transcription kit (Ambion, Austin, TX). The final cRNA products were resuspended in DEPC-treated H₂O and stored at -80 °C. The integrity of the final product and the absence of degraded RNA were determined by denaturing agarose gel electrophoresis stained with ethidium bromide.

Ca_V3 and Ca_V1b Overlay Assays onto pGFP-uv Fusion Proteins—A fragment of 105 bp including the whole AID region of Ca_V2.3 (25-residue AID_E peptide) was generated by polymerase chain reaction and cloned in-frame into the HindIII-KpnI sites of pGFPuv vector (GenBank^TM U62636 [GenBank] ) (Clontech, BD Biosciences, Mississauga, Ontario) to give a fusion protein with an estimated molecular mass of 32 kDa. The AID-fusion protein was expressed in the chemically competent Escherichia coli strain DH5. The synthesis of the fusion proteins was induced at 37 °C using 0.5 mM isopropyl -D-thiogalactoside in an overnight liquid culture, and bacteria were collected by centrifugation. As the GFP protein cloned in-frame with the AID_E peptide has been modified to fluoresce under UV light, the fusion protein confers a green color to the DH5 bacterial extracts when monitored under UV light. For overlay assays, crude DH5 bacterial extracts (200 µl) were boiled for 2 min in Laemmli's loading buffer and separated on a denaturing 10% SDS-polyacrylamide gel. Total proteins were either visualized with Red Ponceau or were loaded in duplicate to be visualized by Coomassie staining in addition to the autoradiogram. This half of the gel was transferred onto a polyvinylidene difluoride membrane (Millipore, Fisher, Whitby, Ontario) using Towbin buffer (in mM: 25 Tris, 192 glycine, 20% methanol, and 0.05% SDS). The membrane was blocked with 1% bovine serum albumin in HBS-Tween (in mM: 137 NaCl, 3 KCl, 10 HEPES, and 0.05% Tween-20 pH 7.4), washed once with HBS-Tween and incubated for 1 h at room temperature in 5 ml of HBS-Tween with 20 µl of [³⁵S]methionine labeled Ca_V3- or Ca_V1b subunit prepared as described below. The blots were washed twice for 10 min with 1 mM CaCl₂ in HBS-Tween, air-dried, and radioactive signals were detected by autoradiography.

[³⁵S]Methionine-labeled Ca_V3 or Ca_V1b in pBluescript (0.5 µg) was synthesized by coupled in vitro transcription and translation (TNT; Promega, Madison, WI) in a 50-µl reaction volume for 1 h, and the reaction mixture was applied without further treatment to the overlay membrane.

Functional Expression of Wild-type and Mutants Channels—Oocytes were obtained from female Xenopus laevis clawed frog (Nasco, Fort Atkinson, WI) as described previously (1, 34, 35). Briefly, stage VI oocytes free of follicular cells were injected with 46 nl of a solution containing 28 ng of cRNA coding for the wild-type or mutated Ca_V1 subunit. The Ca_V1 subunit was always co-injected with cRNA coding for the rat brain Ca_V2b (6) and with or without the rat brain Ca_V3 (36) in a 6:2:3 or 6:2 weight ratio. The final amount of exogenous Ca_V3 injected corresponds to 14 ng, which represents a 23-fold enrichment over the estimated concentration of 0.56 ± 0.02 ng of endogenous Ca_V3xo per oocyte (22). Oocytes were incubated at 19 °C for 1-5 days after injection in Barth's solution (in mM): 100 NaCl; 2 KCl; 1.8 CaCl₂; 1 MgCl₂; 5 HEPES; 2.5 pyruvic acid; 100 units/ml of penicillin; 50 µg/ml gentamicin; pH 7.6.

Electrophysiological Recordings in Oocytes—Wild-type and mutant channels were screened at room temperature for macroscopic Ba²⁺ currents using a two-electrode voltage clamp amplifier (OC-725C, Warner Instruments, Hamden, CT) as described earlier (1, 4, 34). In order to minimize series resistance problems associated with voltage clamping large currents in Xenopus oocytes, experiments were performed on cells with peak currents smaller than -5 µA in most cases. Hence, the wild-type channel was recorded 3-4 days after cRNA injection whereas whole cell Ba²⁺ currents were generally measured 1-2 days after injection for AID mutant channels in the absence of Ca_V3 and 2-3 days in the presence of Ca_V3. Oocytes were first impaled in a modified Ringer solution (in mM): 96 NaOH; 2 KOH; 1.8 CaCl₂; 1 MgCl₂; 10 HEPES titrated to pH 7.4 with methanesulfonic acid CH₃SO₃H (MeS), then the bath was perfused with the 10 Ba²⁺ solution (in mM; 10 Ba(OH)₂; 110 NaOH; 1 KOH; 20 Hepes titrated to pH 7.3 with MES). To minimize kinetic contamination by the endogenous Ca²⁺-activated Cl^- current, oocytes were injected with 18.4 nl of a 50 mM EGTA (Sigma) 0.5-1 h before the experiments. Oocytes were superfused by gravity flow at a rate of 2 ml/min that was fast enough to allow complete chamber fluid exchange within 30 s. Experiments were performed at room temperature (20-22 °C).

Electrophysiological Data Acquisition and Analysis—PClamp software, Clampex 6.02 and Clampfit 6.02 (Axon instruments, Foster City, CA) was used for on-line data acquisition and analysis as previously described (1, 34, 35). Unless stated otherwise, data were sampled at 10 kHz and low pass-filtered at 5 kHz using the amplifier built-in filter. For all recordings, a series of voltage pulses were applied from a holding potential of -80 mV at a frequency of 0.2 Hz from -40 to +60 mV. Isochronal inactivation data (h or hinf) were obtained from tail currents generated at the end of a 5-s prepulse (4, 37). Tail current amplitudes were estimated using the function Analyze in Clampfit 6.0 from the peak current arising during the first 10 ms after the capacitive transient (20 data points). Each of these currents was then normalized to the maximum current obtained before the prepulse voltage (I/I_max) and was plotted against the prepulse voltage. For the isochronal inactivation figures,

(Eq. 1)

pooled data points (mean ± S.E.) were fitted to Equation 1 using user-defined functions and the fitting algorithms provided by Origin 6.1 and 7.0 (Microcal Software, Northampton, MA) analysis software. Equation 1 accounts for the fraction of non-inactivating current with E_0.5,inact mid-point potential; z, slope parameter; Y₀, fraction of non-inactivating current; V_m, the prepulse potential, and RT/F with their usual meanings. The fitting process generated values estimating errors on the given fit values.

Activation potentials were estimated from the normalized I-V curves obtained for each channel combination (22). Although this calculation was not exempt from gating contamination, it provided a qualitative approximation of the Ca_V3 modulation on I-V parameters. The I-V relationships were normalized to the maximum amplitude and were fitted to Equation 2, a Boltzmann equation coupled to a linear function.

(Eq. 2)

E_0.5,act is the potential for 50% activation; G_rel is the normalized conductance; z, slope parameter; V_m, the test potential, V_rev, the apparent reversal potential and RT/F with their usual meanings.

Inactivation kinetics were quantified using R300 values, that is the ratio of the whole cell current remaining at the end of a 300-ms pulse (34, 35, 38). As inactivation kinetics can vary with current density, comparisons between constructs and mutants were generally restricted to whole cell currents lower than -5 µA as much as possible. Furthermore, this range of current densities made it easier to voltage clamp the oocyte uniformly thus decreasing the possibility of series resistance artifacts contaminating the current kinetics data. Capacitive transients were erased for clarity in the final figures. Statistical analyses and Student's t test were performed using the fitting routines provided by Origin 6.1 and 7.0 (Microcal Software, Northampton, MA).

Homology Modeling of Wild-type and Mutant Ca_V2.3—Computations were performed using the InsightII package (version 2000, Accelrys) as described elsewhere (39, 40). Briefly, the BIOPOLYMER and BUILDER modules were used in particular to build or modify molecular structures, and all energy minimizations were performed with the DISCOVER module using the consistent valence force field (CVFF). For all minimizations, one thousand steps of Steepest Descent minimization were performed, followed by a Conjugate gradient minimization until a convergence of 0.001 kcal/mol/Å was reached.

The starting coordinates were taken from the PDB file 1T0J [PDB] .pdb (Ca_V2a + AID from Ca_V1.2) (19). Molecular replacement of the following eight residues (L430I, E432R, D433E, K435N, L438R, D439A, T442D, and Q443D) to their reciprocal residues in the human Ca_V2.3 was undertaken using BIOPOLYMER to obtain the complex Ca_V2a + AID wt from Ca_V2.3. The crystallographic water molecules were not retained. Hydrogen atoms were added using the BIOPOLYMER module at the normal ionization state of the amino acids at pH 7.0. Potentials as well as partial and formal charges were fixed with CVFF. A first series of energy minimizations was then performed with the DISCOVER module using the CVFF with a distance-dependent dielectric constant of 80 (implicit H₂O). Minimizations were carried out while keeping the heavy atoms of the protein fixed. The structure obtained was then submitted to a second series of energy minimizations performed as explained above, after removing all constraints on the atoms and with a 20 Å radius sphere of explicit water molecules centered around AID residue Asn⁴³⁵. These two series of minimizations yielded the energy-minimized configuration of Ca_V2a + AID wt from Ca_V2.3. This wild-type configuration was then used to produce six individual mutants: Y437F, Y437R, Y437D, I441L, I441R, and I441D that were also minimized as explained above with a 20 Å radius sphere of explicit water molecules.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
A Three-dimensional Model of the AID Peptide from Ca_V2.3—By homology with the three crystal structures obtained by x-ray diffraction of the AID region in Ca_V1.1 and Ca_V1.2 channels, the AID region of the Ca_V2.3 channel (AID_E) should form an -helix upon binding to Ca_V subunits (19-21). The primary sequence of AID_E, Gln¹-Gln²-Ile³-Glu⁴-Arg⁵-Glu⁶-Leu⁷-Asn⁸-Gly⁹-Tyr¹⁰-Arg¹¹-Ala¹²-Trp¹³-Ile¹⁴-Asp¹⁵-Lys¹⁶-Ala¹⁷-Glu¹⁸ conserves 9 out of 18 residues when compared with the AID helix from Ca_V1.1 and Ca_V1.2 1 subunits (Table I). The three-dimensional representations of AID_E obtained by homology modeling with either Ca_V1.1/Ca_V2a (21) (not shown), Ca_V1.2/Ca_V3 (20) (not shown), or Ca_V1.2/Ca_V2a (19) (Fig. 1) all show that AID_E assumes an -helical structure in the presence of Ca_V. The orientation of the residues facing the hydrophilic side of the AID helix (Gln², Glu⁴, Arg⁵, Asn⁸, Arg¹¹, Ala¹², Asp¹⁵, and Lys¹⁶) was seen to vary slightly but the residues mostly located on the hydrophobic face of the AID_E helix that includes the invariant LGWYI residues (Glu⁶, Leu⁷, Gly⁹, Tyr¹⁰, Trp¹³, and Ile¹⁴) are well buried in the Ca_V subunit fold. As seen in Fig. 1, the front (A), the back (B), and the bottom (C) perspectives of the AID_E model obtained using the crystal coordinates of Ca_V1.2/Ca_V2a (19) clearly show that the side-chains of Tyr¹⁰, Trp¹³, and Ile¹⁴ are entrenched within the Ca_V subunit. Hence, the C-terminal residues appear to interact extensively with the Ca_V subunit. The Gly⁹ residue is significantly enclosed by the Ca_V subunit and could be needed to provide some degree of flexibility to the AID helix. The crystal structures further show that the aromatic side-chain of Tyr¹⁰ is significantly surrounded by residues of the Ca_V subunit suggesting that the interaction might require an aromatic residue at this position. Located on the adjacent turn, the extreme concealment of Trp¹³ is compatible with our previous observations that mutations considered to be even conservative (W13F, W13Y), eliminated Ca_V3 binding and modulation of Ca_V2.3 (1). In contrast, Ile¹⁴ situated next to Tyr¹³ and the N-terminal residues Glu⁶ and Leu⁷ appear to form fewer interactions with Ca_V suggesting these three positions might be more compliant to structural substitutions than Tyr¹⁰ and Trp¹³. We thus undertook a systematic analysis of the physicochemical requirements of key AID positions to achieve functional modulation of Ca_V2.3 by Ca_V subunits.

View larger version (36K): [in this window] [in a new window]   FIG. 1. Three-dimensional model of the AID helix of the human CaV2.3 (AIDE). Model was obtained with INSIGHT II using the atomic coordinates for the human CaV1.2 AIDC crystal structure co-crystallized with CaV2a at a 2.0 Å resolution (1T0J. pdb) as a template (19). The AIDC peptide (Gln1-Gln2-Leu3-Glu4-Glu5-Asp6-Leu7-Lys8-Gly9-Tyr10-Leu11-Asp12-Trp13-Ile14-Thr15-Lys16-Ala17-Glu18) underwent 8 substitutions to obtain the primary sequence of AIDE (Gln1-Gln2-Ile3-Glu4-Arg5-Glu6-Leu7-Asn8-Gly9-Tyr10-Arg11-Ala12-Trp13-Ile14-Asp15-Lys16-Ala17-Glu18) as described under "Experimental Procedures" (see also Table I). The core of the AIDE helix is colored in white. The following side-chains on the hydrophobic face of the helix are color-coded: E379 (yellow), L380 (orange), G382 (turquoise), Y383 (green), W386 (red), I387 (magenta). The CaV2a subunit is shown in blue. A, front view with the N-terminal end projecting toward the top of the figure. B, back view with the N-terminal end projecting toward the top of the figure. C, bottom view. The BIOPOLYMER module was used to build the molecular structure, and all energy minimizations were performed with the DISCOVER module as described under "Experimental Procedures."

View larger version (54K): [in this window] [in a new window]   FIG. 2. Alanine mutations in the N-terminal end of the AID helix do not impede CaV binding and modulation. A, primary sequence of the AID region (Q1 Q2 I3 E4 R5 E6 L7 N8 G9 Y10 R11 A12 W13 I14 D15 K16 A17 E18) is shown for CaV2.3 (AIDE). The N-terminal residues mutated in this figure are underlined. B, autoradiogram of in vitro translated [35S]CaV3 overlays on GFPuv-AIDE mutants immobilized on nitrocellulose. Strong signals were detected (from left to right) for the wild-type AID peptide, Q374A (Q1A), Q375A (Q2A), I376A (I3A), E377A (E4A), E379A (E6A), and L380A (L7A). [35S]CaV3 binding could not be detected for the empty vector pGFPuv and the AID mutant W386A (WA). C, autoradiogram of in vitro translated [35S]CaV1b overlays on GFPuv-AIDE mutants immobilized on nitrocellulose. Strong signals were detected (from left to right) for the wild-type AIDE peptide, Q374A (Q1A), Q375A (Q2A), I376A (I3A), E377A (E4A), E379A (E6A), and L380A (L7A). [35S]CaV1b binding could not be detected for the empty vector pGFPuv and the AIDE mutant W386A. D, typical whole cell current traces obtained after the expression of the E379A (E6A) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). Time and current scales are identical for D and E. E, typical whole cell current traces obtained after the expression of the L380A (L7A) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). F, mean R300 ratios (the fraction of the whole cell current remaining at the end of a 300-ms pulse) are shown ± S.E. from 0 to +20 mV for the wild-type and the mutant CaV2.3 channels (Q374A, Q375A, I376A, E377A, E379A, L380A, and W386A) in the absence of CaV3. G, R300 graph for the wild-type and the mutant CaV2.3 channels (Q374A, Q375A, I376A, E377A, E379A, L380A, and W386A) after co-injection with CaV3. As seen, the VDI kinetics of the N-terminal mutants are significantly accelerated in a manner comparable to the wild-type channel. In contrast, the kinetics of W386A were not increased by CaV3 suggesting that the first turn of the AID helix is more tolerant to alanine mutations. Whole cell currents were recorded using the two-electrode voltage clamp technique in the presence of 10 mM Ba2+ after injection of EGTA. Holding potential was -80 mV. Xenopus oocytes were pulsed from -40 mV to +60 mV using 10 mV steps for 450 ms. All mutants expressed significant inward currents with typical current-voltage properties. Capacitive transients were erased for the first ms after the voltage step. The mutant properties (mid-potentials of activation and inactivation as well as peak currents) are provided in Table II.

Alanine Scan of the GYI Residues in the -Helix C-terminal Turns in Ca_V2.3—The role of the two remaining pairs of critical residues Gly⁹/Tyr¹⁰ and Trp¹³/Ile¹⁴ located on the C-terminal -helical turns was also investigated after mutation with alanine. As explained earlier, Ca_V subunit binding was investigated in overlay assays whereas modulation by Ca_V subunits was studied after functional expression in Xenopus oocytes. Fig. 3A shows the overlay binding assays for Y383A, G382A, and I387A performed in the 25-residue AID_E peptide inserted in the pGFPuv vector. Ca_V subunit binding is shown for [³⁵S]methionine-labeled Ca_V1b (Fig. 3A). The empty pGPFuv vector and wild-type AID_E peptide were tested along as additional controls. Strong bands were obtained at the expected molecular weight of 32 kDa for the wild-type AID_E peptide as well as for G382A indicating that the alanine mutation at position Gly⁹ did not impede the interaction with [³⁵S]Ca_V1b under denaturing conditions. In contrast, [³⁵S]Ca_V1b binding onto Y383A and I387A peptides was similar to the background level as compared with the lane loaded with the empty vector pGPFuv suggesting that substitution with alanine disrupted the interaction between Tyr³⁸³ or Ile³⁸⁷ and Ca_V1b. Similar results were obtained with [³⁵S]Ca_V3 (not shown). The binding data were compared with the electrophysiological data obtained for the same mutants in the presence and in the absence of Ca_V3. As expected from the binding studies, Ca_V3 modulated the whole cell currents of G382A in a fashion reminiscent of the wild-type channel (Fig. 3B) with a 5-fold decrease in the R300 values in the presence of Ca_V3 (p < 10^-5) (Fig. 3D). Table II reports the estimated mid-potentials of activation and inactivation under the same conditions.

View larger version (45K): [in this window] [in a new window]   FIG. 3. CaV subunit binding and modulation of G382A, Y383A, and I387A channels. A, Coomassie Blue-stained SDS-PAGE gel showing the wild-type AIDE and the alanine mutants Y383A, G382A, and I387A (in that order) from CaV2.3 is juxtaposed with the autoradiogram of the same mutants with the in vitro translated [35S]methionine CaV1b. As seen, gel loading was equivalent in each lane. The fusion proteins have a molecular mass of 29 kDa (no insert) or 32 kDa (the 25-residue AID peptide). The lanes were loaded equally except for I387A that shows a larger concentration of proteins. Autoradiogram of in vitro translated [35S]methionine CaV1b overlays on GFPuv-AIDE mutants immobilized on nitrocellulose. The empty vector pGFPuv (no insert) is shown as a control (lane 1). Whereas a strong signal was obtained for the wild-type AIDE peptide (lane 2) and the G382A mutant (lane 4), [35S]CaV1b binding could not be detected for Y383A (lane 3) and for I387A (lane 5). Similar results were obtained with [35S]CaV3 (not shown). B, typical whole cell current traces obtained after the expression of the G382A mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). Time and current scales are identical for panels B and C. C, typical whole cell current traces obtained after the expression of the I387A (I14A) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). D, mean R300 ± S.E. values of G382A were significantly decreased by the co-injection of CaV3 (p < 10-8 at 0 < Vm < 20 mV). R300 values for the wild-type channel have been added for comparison. E, mean R300 ± S.E. values of Y383A were decreased by the co-injection of CaV3 (p < 10-3 at 0 < Vm < 20 mV). R300 values for the wild-type channel have been added for comparison. F, mean R300 ± S.E. values of I387A were decreased by the co-injection of CaV3 (p < 0.05 at 0 < Vm < 20 mV). R300 values for the wild-type channel have been added for comparison. The numbers to the right of the mutants refer to the numbers of experiments used for statistical analysis. The voltage dependence of activation and inactivation are detailed in Table II.

Aromatic and Positively Charged Residues at Tyr¹⁰ Preserve Ca_V Binding and Modulation—Because the substitution of the aromatic tyrosine residue Tyr¹⁰ at position 383 by the small and hydrophobic alanine (Ala) residue was shown to disrupt Ca_V3 subunit binding as well as decreasing the Ca_V-induced modulation of Ca_V2.3, the structural requirements for Ca_V subunit binding and modulation were further investigated after substitutions with aromatic (Phe, Trp) as well as positive (Arg, Lys) and negative (Asp, Glu) residues. Ca_V subunit-induced modulation of inactivation was also preserved with the Y10S mutant in L-type Ca_V1.1 and Ca_V1.2 channels (26) whereas in vitro binding of Ca_V4 to AID_A was demonstrated in the presence of the Y10S and Y10F mutants (25). We had previously reported that the substitution with the neutral but polar serine (Ser) residue at the same position yielded a channel with VDI gating slower than the wild-type channel in the presence of Ca_V3 (1). Nonetheless, the voltage dependence of inactivation of Y383S was clearly shifted in the hyperpolarized direction by -20 mV in the presence of Ca_V3 (1). These data were replicated over a 2-year period, and Table II provides the complete set of numerical values for this and the other Tyr³⁸³ mutants.

[³⁵S]Ca_V1b binding to pGFPuv-AID_E fusion proteins mutated to Y383F, Y383E, Y383K, and Y383R is shown in Fig. 4A whereas [³⁵S]Ca_V3 binding to pGFPuv-AID_E mutants Y383F, Y383S, Y383E, Y383R, and Y383W in Fig. 4B. For the Ca_V1b data, the wild-type AID_E fusion protein was used as a positive control whereas W386A was loaded to assess the background signal. E391A (E18A) was used as a positive control in the presence of Ca_V3 since it remained functionally modulated by Ca_V3 in a wild-type fashion (Table II). Altogether, the signal intensity for the AID_E fusion peptides ranked from AID_E wt E391A >> Y383F Y383W Y383K Y383R >Y383S >> W386A. The binding signal for Y383E was somewhat stronger in the presence of Ca_V3. As seen, the signal was similar for the two aromatic mutations Y383F and Y383W confirming that the aromatic side-chain is a key structural requirement. Furthermore, positively charged residues were more tolerated than the negatively charged ones suggesting that electrostatic-based interactions are important for [³⁵S]Ca_V1b and [³⁵S]Ca_V3 binding at least under our experimental conditions where the AID helix has been denatured.

View larger version (56K): [in this window] [in a new window]   FIG. 4. CaV subunit binding and modulation of Tyr383 mutants. A, autoradiogram of in vitro translated [35S]methionine-labeled CaV1b overlays on GFPuv-Y383 mutants immobilized on nitrocellulose. The W386A (WA) mutant is shown to assess the background signal (lane 2). Whereas a strong signal was obtained for the wild-type AIDE peptide (lane 3), [35S]CaV1b binding ranked from AID >> Y383F (lane 1) > Y383K (lane 5) Y383R (lane 6) > Y383E (lane 4). B, autoradiogram of in vitro translated [35S]methionine CaV3 overlays on GFPuv-Tyr383 mutants immobilized on nitrocellulose. The W386A mutant is shown as a negative control to assess the background signal (lane 2). Whereas a strong signal was obtained for the E391A (E18A) (lane 1), the intensity of the signal went from Y383F (lane 3) > Y383W (lane 7) Y383R (lane 6) > Y383E (lane 5) > Y383S (lane 4) >> W386A (lane 2). C, typical whole cell current traces obtained after the expression of the Y383R (Y10R) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). Time and current scales are identical for C and D. D, typical whole-cell current traces obtained after the expression of the Y383D (Y10D) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). E, bar graph showing the mean R300 ± S.E. values for the wild-type, Y383F, Y383A, Y383K, Y383R channels (from left to right) with the R300 value obtained with CaV3 plotted before the value obtained in the absence of the subunit. VDI gating were significantly decreased by the co-injection of CaV3 for these mutants at p < 10-4 for 0 < Vm < 20 mV. The Y383W did not yield detectable inward Ba2+ currents in response to membrane depolarization (see Table II). F, bar graph showing the mean R300 ± S.E. values for the wild-type, Y383S, Y383D, Y383E channels (from left to right) with the R300 value obtained with CaV3 plotted before the value obtained in the absence of the subunit. The VDI gating of Y383D and Y383E were not significantly altered by the co-injection of CaV3 (p > 0.05) for 0 < Vm < 20 mV. For Y383S, the R300 values were decreased in the presence of CaV3 (p < 0.01) for 0 < Vm < 20 mV but remained significantly higher than for the wild-type channel (p < 0.001) for 0 < Vm < 20 mV. The numbers to the right of the mutants refer to the numbers of experiments used for statistical analysis. The voltage dependence of inactivation for these mutants were computed using the Boltzmann Equation 1 after 5-s prepulses. The estimated values for the mid-potentials of activation and inactivation are provided in Table II.

The Iso to Leu Substitution Is Tolerated at Position Ile¹⁴—The stereochemical requirements for Ca_V binding and modulation at position Ile¹⁴ were investigated after substitutions with the conserved leucine residue (Leu), the neutral but polar serine (Ser) as well as positive (Lys, Arg) and negative (Asp, Glu) residues. As seen earlier, the substitution of the hydrophobic isoleucine residue at this position by the likewise hydrophobic but smaller alanine residue was shown to disrupt Ca_V3 subunit binding as well as decreasing the Ca_V-induced modulation of Ca_V2.3 suggesting that the volume of the residue plays a critical role in establishing essential interactions with Ca_V subunits.

[³⁵S]Ca_V1b binding to pGFPuv-AID_E fusion proteins mutated to I387D, I387R, and I387K is shown in Fig. 5A. As shown before, the wild-type AID_E fusion protein and W386A were used as positive and negative controls, respectively. The signal was unambiguous for AID_E wt but was indistinguishable from the background noise for I387D I387R I387K W386A. Similar data were obtained with [³⁵S]Ca_V3 with I387S I387D I387E I387R I387K (not shown). As seen, none of the polar mutations at this position, including the mutation by the neutral serine, preserved binding to [³⁵S]Ca_V under denaturing conditions.

View larger version (51K): [in this window] [in a new window]   FIG. 5. CaV subunit binding and modulation of Ile387 mutants. A, autoradiogram of in vitro translated [35S]methionine CaV1b overlays on GFPuv Ile- mutants immobilized on nitrocellulose. The W386A mutant is shown to assess the background signal (lane 2). Whereas a strong signal was obtained for the wild-type AID peptide (lane 1), the [35S]CaV1b binding to I387R (lane 4) was modest whereas binding to I387D (lane 3), and I387K (lane 5) was comparable to the W386A (lane 2) peptide. Similar data were obtained with [35S]CaV3 (not shown). B, typical whole cell current traces obtained after the expression of the I387L (I14L) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). Time and current scales are identical for panels B and C. C, typical whole cell current traces obtained after the expression of the I387K (I14K) mutant in Xenopus oocytes with CaV2b without exogenous CaV3 (-3) and after co-injection with CaV3 (+3). D, mean R300 ± S.E. values of I387L were significantly decreased by the co-injection of CaV3 (p < 10-10 at 0 < Vm < 20 mV) but the modulation was milder for I387A and I387S mutants (p < 0.05 at 0 < Vm < 20 mV). E, mean R300 ± S.E. values of negatively and positively charged I387 mutants were unaffected by the co-injection of CaV3 at most voltages. However, the R300 for I387E and I387K were significantly decreased by CaV3 at 0 mV (p < 10-3). The numbers to the right of the mutants refer to the numbers of experiments used for statistical analysis. The voltage dependence of inactivation for these mutants estimated from 5-s prepulses are provided in Table II.

Substitutions with positively charged (Arg, Lys) or negatively charged (Asp, Glu) residues yielded functional channels with typical I-V properties that were ineffectually modulated by Ca_V3 (Fig. 5E). Although Ca_V3 appeared to significantly speed up the VDI kinetics of I387E at 0 mV (p < 10^-5), this effect vanished at +20 mV. Furthermore the R300 values for I387E + Ca_V3 were systematically larger than the wild-type channel under the same conditions. Finally, the voltage dependence of inactivation of the charged mutants was not significantly hyperpolarized by the co-injection with Ca_V3 (Table II). Altogether these data suggest that the position Ile¹⁴ specifically requires a bulky, neutral, and hydrophobic residue to achieve Ca_V-induced modulation of Ca_V channels.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
The C-terminal Turn of the AID Helix Anchors the Interaction with Ca_V—The four known auxiliary Ca_V (Ca_V1-4) subunits can potentially associate with any of the six Ca_V1 pore-forming subunits of HVA VDCC (Ca_V1.1-1.3; Ca_V2.1-2.3) via AID located on the I-II linker of the Ca_V1 subunit. About half of the residues of the AID helix Gln¹-Gln²-X³-Glu⁴-X⁵-X⁶-Leu⁷-X⁸-Gly⁹-Tyr¹⁰-X¹¹-X¹²-Trp¹³-Ile¹⁴-X¹⁵-X¹⁶-X¹⁷-Glu¹⁸) are strictly conserved among the six Ca_V1 subunits but homology was found to be higher within members of the Ca_V1 and Ca_V2 families (Table I). The crystal structures of the Ca_V/AID peptide complex from L-type Ca_V1.1 and Ca_V1.2 channels have recently revealed that the AID region forms an -helix upon binding Ca_V subunits (19-21). The interaction between the AID helix and the Ca_V protein appears to be primarily secured by the projection of three pairs of hydrophobic residues onto the Ca_V fold with Tyr¹⁰ and Trp¹³ being almost completely buried within the Ca_V protein (19-21). The three-dimensional model of AID_E obtained by homology modeling with Ca_V1.2/Ca_V2a (19) suggests similarly that the -helical structure of the AID peptide is preserved in Ca_V2.3 with the three pairs of residues Glu⁶/Leu⁷, Gly⁹/Tyr¹⁰, and Tyr¹³/Ile¹⁴ projecting into Ca_V residues with Leu⁷, Gly⁹, Tyr¹⁰, Trp¹³, and Ile¹⁴ being invariant in all HVA Ca_V1 channels. The sixth position is always occupied by a negatively charged residue with the aspartate residue in Ca_V1 being replaced by a glutamate residue in Ca_V2 channels. The three-dimensional model of AID_E shows that Tyr¹⁰ and Trp¹³ are deeply enmeshed within residues of the Ca_V subunit suggesting that these residues could play a more critical role in Ca_V modulation of Ca_V2.3 currents than Gln¹, Gln², Ile³, Glu⁴, Glu⁶, Leu⁷, Gly⁹, and Ile¹⁴.

Our mutational analysis herein demonstrated that alanine mutants of Gln¹, Gln², Ile³, Glu⁴, and Glu¹⁸ did not abolish [³⁵S]Ca_V1b or [³⁵S] Ca_V3 subunit overlay binding to fusion proteins nor did they prevent the typical modulation of Ca_V2.3 whole cell currents by Ca_V3. These results suggest that the N-terminal residues of the AID helix located on the hydrophilic face of the AID helix do not play a determinant role in the interaction between Ca_V1 and Ca_V subunits in agreement with the predictions of the crystal structure. In general, the signal intensity to the AID_E peptide was found to be more robust for [³⁵S]Ca_V1b than for [³⁵S]Ca_V3 binding in agreement with previous reports showing that Ca_V1b displayed a significantly higher affinity for the AID_A peptide than Ca_V3 in Ca_V2.1 channels (17, 41).

Furthermore, alanine mutations of Glu⁶ and Leu⁷ located in the first hydrophobic -helical turn of the AID_E helix did not affect significantly Ca_V binding and modulation of Ca_V2.3 currents. These results suggest that the binding pocket at these positions can accommodate alanine residues. The central -helical turn of the AID helix was slightly less tolerant to mutations than the N-terminal residues. In fact, mutation of Gly⁹ by alanine enhanced Ca_V-induced modulation indicating that the helical propensity conferred by the alanine residue fastened the interaction with Ca_V whereas increased flexibility at this position could loosen the interaction. Mutations of Tyr¹⁰ with either aromatic (Phe) or positively charged (Arg, Lys) residues yielded whole cell currents that responded to Ca_V3 essentially like the wild-type channel whereas mutations with negatively charged residues (Asp, Glu) disrupted Ca_V3 binding and modulation.

In contrast, mutations of the C-terminal residues Trp¹³ and Ile¹⁴ disrupted significantly Ca_V binding and modulation. We had previously provided evidence that position Trp¹³ could not be successfully substituted with hydrophobic (Ala, Gly), hydrophilic (Gln, Arg, Glu), or aromatic (Phe, Tyr) residues (1). Herein, we further demonstrate that modulation and binding by Ca_V3 was significantly weakened in I14A (neutral and hydrophobic) and I14S (neutral and polar) mutants and completely eradicated in negatively charged I14D and I14E or positively charged I14R and I14K mutants. The conservative mutation I14L was found to be the only substitution that preserved the Ca_V3-induced modulation of Ca_V2.3 currents. Hence, in contrast to the N-terminal residues, we have compellingly established that Ca_V binding as well as Ca_V-induced modulation of HVA Ca_V currents are determined by the C-terminal pair of residues of the AID helix.

Computer Modeling and Molecular Replacements of Tyr¹⁰ (Tyr³⁸³) and Ile¹⁴ (Ile³⁸⁷) Positions—The analysis of the accessible surface area in the Ca_V1.2 AID-Ca_V2a complex showed Tyr¹⁰ to be slightly more buried within the Ca_V fold than Ile¹⁴ suggesting that the binding pocket formed at the Tyr¹⁰ position could impose harsher structural requirements than the binding pocket at Ile¹⁴ (19). On the contrary, our data revealed that mutations at position Ile¹⁴ impacted more significantly on Ca_V3 binding and functional modulation of Ca_V2.3 channel function than mutations at Tyr¹⁰. To visualize the changes in the environment caused by the AID_E mutations, molecular replacements were produced in the three-dimensional model of the wild-type AID_E helix obtained for Ca_V2.3 (see "Experimental Procedures"). The resulting energy-minimized configuration was then compared with the three-dimensional model for the wild-type AID_E helix.

The three-dimensional model of the AID_E helix implies that Ile¹⁴ could make van der Waals interactions with Ca_V2a hydrophobic residues Leu³⁵², Val²⁴¹, Met²⁴⁴, and Met²⁴⁵. From the wild-type model, Ile¹⁴ appears to be confined to a smaller hydrophobic pocket than Tyr¹⁰ suggesting that steric hindrance could become a key parameter at this position. As a result, mutations with hydrophobic residues of comparable size are predicted to be better tolerated than mutations with bulkier residues. Indeed, there is an excellent superposition of the C, C, and C between the isoleucine and the similar leucine residues at position 14 of the AID_E helix. The van der Waals interactions are also highly conserved and the I14L mutation does not appear to alter the helix conformation of the AID peptide (not shown).

The I14R mutation that introduces a bulkier and hydrophilic residue in the highly hydrophobic binding pocket produced stronger effects with a distinct deviation of the side-chain at the C that pushes residues from Ca_V2a away from the AID_E helix and results into a significantly increased r.m.s.d. value (1.53Å as compared with 0.55Å for I14L) especially for the four hydrophobic Ca_V2a residues (Fig. 6). Hence, the introduction of an arginine residue could result in a significantly loosening of the interaction with Ca_V2a residues. The I14L and I14R models thus appear to correlate well with our experimental data both in terms of in vitro binding and Ca_V-induced functional modulation.

View larger version (37K): [in this window] [in a new window]   FIG. 6. Three-dimensional models of the AID helix of the human CaV2.3 obtained with INSIGHT II using the atomic coordinates for the human CaV1.2 AID crystal structure co-crystallized with CaV2a at a 2.0 Å resolution (1T0J.pdb) as a template (19). The backbone of the AID helix is colored in white. Key residues are shown in CPK representation. A, front view of the AID helix with the native isoleucine residue at position 14. B, front view of the AID helix with the bulkier arginine residue at position 14. The atoms of the residue at position 14 are color-coded with carbon atoms (green), oxygen (red), and nitrogen (blue). The hydrogen atoms are not shown. The residue at position 14 makes van der Waals interactions with four hydrophobic residues from CaV2a shown in CPK representation with a single color with the residue: Leu352 (cyan), Val241 (magenta), Met244 (gray), and Met245 (violet). The I14R mutation pushes CaV2a residues away from the AIDE helix and results in a significantly increased r.m.s.d. value (1.53 Å as compared with 0.55 Å for I14L) for the four hydrophobic CaV2a residues. The BUILDER module was used to construct the molecular replacements onto the CaV2.3 model and all energy minimizations were carried out with the DISCOVER module as described under "Experimental Procedures."

Since the binding pocket formed by Tyr¹⁰ appears to be deeper than the Ile¹⁴ site, bulky residues are expected to be better tolerated at Tyr¹⁰ than at Ile¹⁴. The substitution of the tyrosine by a phenylalanine at position 10 results in the loss of two hydrogen bonds with the Ca_V2a residues Ser³⁴⁴ and Glu³⁸⁹. Nonetheless, the aromatic rings of the tyrosine and the phenylalanine residues remain in the same orientation and the carbon atoms of the side-chains for both residues are in the same plane. The r.m.s.d. value for the AID_E helix (0.18 Å) remained small suggesting that the Y10F mutation should not affect the Ca_V-Ca_VAID interaction as we have observed experimentally. Given that the binding pocket at this position is relatively deeper than the one at position 14, the arginine residue could eventually adopt an energetically favorable elongated configuration where its carbon atoms (, , and ) could espouse the same conformation as the equivalent carbon atoms from the native tyrosine residue. Indeed, the arginine residue appears to be as comfortably buried within Ca_V2a as the phenylalanine or the native tyrosine residues, which is substantiated by the small r.m.s.d. value observed with this mutant (0.23 Å). Finally, the arginine residue forms also a hydrogen bond with Ca_V2a like the native wild-type tyrosine residue does, albeit with a different residue (Asp³⁸⁸). All these factors could account for the observation that the Y383R mutation did not affect significantly Ca_V binding or Ca_V-induced modulation. In contrast, the aspartate residue at this position (Y10D) was too short to fully interact with Ca_V2a or to form any hydrogen bond with Ca_V2a residues. The shorter side-chain of the aspartate residue does not however appear to significantly affect the conformation of the AID_E peptide since the r.m.s.d. value (0.24 Å) remained comparable to the values obtained for the arginine and the phenylalanine at this position. Hence the overall lower hydrophobicity as well as the absence of the hydrogen bond could explain in part the lack of Ca_V-induced modulation of the Y383D mutant in Ca_V2.3.

	CONCLUSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 
Our data proved that mutations of residues located on the N-terminal -helical turn of the AID helix (Glu⁶, Leu⁷) impact less significantly on Ca_V channel modulation by Ca_V than mutations on the C-terminal end of the AID helix (Trp¹³, Ile¹⁴). These data agree with the three-dimensional model of Ca_V2.3/Ca_V2a built using the crystal coordinates from Ca_V1.2/Ca_V2a. However, Ca_V binding to the denatured AID peptide did not appear to completely account for the interaction between the Ca_V1 and the Ca_V subunits. In particular, Ca_V binding to the AID peptide was ablated after conserved and nonconserved mutations of the Tyr¹⁰ whereas Ca_V-induced modulation of whole cell currents was mostly preserved with Y10F, Y10A, Y10K, and Y10R mutations. It can be speculated that the interaction is conserved when both the AID helix and Ca_V are in their native conformation. It remains also possible that functional modulation of HVA Ca_V currents by Ca_V involves more partners than just the AID helix. Nonetheless, binding and functional modulation data were quite well correlated for the C-terminal Ile¹⁴ residue. We have thus proposed that fewer structural constraints such as the deeper interaction site and the lower degree of hydrophobicity at Tyr¹⁰ could account in part for these differences. Altogether, the Ca_V-induced modulation data are compatible with a region-specific specialization of the AID helix: the N-terminal residues playing a critical role in a hinged-lid type inactivation mechanism (44) while the C-terminal residues anchoring Ca_V subunit interaction.

	FOOTNOTES

 
^* This work was supported by the Canadian Heart and Stroke Foundation and by Canadian Institutes of Health Research Grant MOP 13390 (to L. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^** To whom correspondence should be addressed. Tel.: 514-343-6673; Fax: 514-343-7146; E-mail: lucie.parent{at}umontreal.ca.

¹ The abbreviations used are: VDCC, voltage-dependent calcium channel; MES, 4-morpholineethanesulfonic acid; r.m.s.d., root mean-squared deviation; GFP, green fluorescent protein; AID, alpha-interacting domain; HVA, high voltage-activated; LVA, low voltage-activated; VDI, voltage-dependent inactivation; SH3, Src homology 3 domain; GK, guanylate kinase.

	ACKNOWLEDGMENTS

 
We thank Dr. Toni Schneider for the human Ca_V2.3 channel, Dr. Ed Perez-Reyes for Ca_V3 and Ca_V1b subunits, Dr. Rémy Sauvé for critical reading, Drs. Pierre Bissonnette, and Hélène Klein for discussions, Claude Gauthier for artwork, and Julie Verner for dedicated oocyte culture.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION CONCLUSION REFERENCES

 

1. Berrou, L., Klein, H., Bernatchez, G., and Parent, L. (2002) Biophys. J. 83, 1429-1442[Medline] [Order article via Infotrieve]
2. Catterall, W. A. (2000) Annu. Rev. Cell Dev. Biol. 16, 521-555[CrossRef][Medline] [Order article via Infotrieve]
3. Singer, D., Biel, M., Lotan, I., Flockerzi, V., Hofmann, F., and Dascal, N. (1991) Science. 253, 1553-1557[Abstract/Free Full Text]
4. Parent, L., Schneider, T., Moore, C. P., and Talwar, D. (1997) J. Membr. Biol. 160, 127-140[CrossRef][Medline] [Order article via Infotrieve]
5. Williams, M. E., Brust, P. F., Feldman, D. H., Patthi, S., Simerson, S., Maroufi, A., McCue, A. F., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Science. 257, 389-395[Abstract/Free Full Text]
6. Williams, M. E., Feldman, D. H., McCue, A. F., Brenner, R., Velicelebi, G., Ellis, S. B., and Harpold, M. M. (1992) Neuron 8, 71-84[CrossRef][Medline] [Order article via Infotrieve]
7. Bell, D. C., Butcher, A. J., Berrow, N. S., Page, K. M., Brust, P. F., Nesterova, A., Stauderman, K. A., Seabrook, G. R., Nurnberg, B., and Dolphin, A. C. (2001) J. Neurophysiol. 85, 816-827[Abstract/Free Full Text]
8. Beguin, P., Nagashima, K., Gonoi, T., Shibasaki, T., Takahashi, K., Kashima, Y., Ozaki, N., Geering, K., Iwanaga, T., and Seino, S. (2001) Nature. 411, 701-706[CrossRef][Medline] [Order article via Infotrieve]
9. Catterall, W. A. (1991) Science. 250, 1499-1500
10. Perez-Reyes, E. (2003) Physiol. Rev. 83, 117-161[Abstract/Free Full Text]
11. Brice, N. L., Berrow, N. S., Campbell, V., Page, K. M., Brickley, K., Tedder, I., and Dolphin, A. C. (1997) Eur. J. Neurosci. 9, 749-759[CrossRef][Medline] [Order article via Infotrieve]
12. Tareilus, E., Roux, M., Qin, N., Olcese, R., Zhou, J., Stefani, E., and Birnbaumer, L. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 1703-1708[Abstract/Free Full Text]
13. Bichet, D., Cornet, V., Geib, S., Carlier, E., Volsen, S., Hoshi, T., Mori, Y., and DeWaard M. (2000) Neuron 25, 177-190[CrossRef][Medline] [Order article via Infotrieve]
14. Dolphin, A. C. (2003) J. Bioenerg. Biomembr. 35, 599-620[CrossRef][Medline] [Order article via Infotrieve]
15. Birnbaumer, L., Qin, N., Olcese, R., Tareilus, E., Platano, D., Costantin, J., and Stefani, E. (1998) J. Bioenerg. Biomembr. 30, 357-375[CrossRef][Medline] [Order article via Infotrieve]
16. Cens, T., Restituito, S., Vallentin, A., and Charnet, P. (1998) J. Biol. Chem. 273, 18308-18315[Abstract/Free Full Text]
17. Walker, D., Bichet, D., Campbell, K. P., and De Waard M. (1998) J. Biol. Chem. 273, 2361-2367[Abstract/Free Full Text]
18. Qin, N., Platano, D., Olcese, R., Costantin, J. L., Stefani, E., and Birnbaumer, L. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 4690-4695[Abstract/Free Full Text]
19. Van Petegem, F., Clark, K. A., Chatelain, F. C., and Minor, D. L., Jr. (2004) Nature. 429, 671-675[CrossRef][Medline] [Order article via Infotrieve]
20. Chen, Y. H., Li, M. H., Zhang, Y., He, L. L., Yamada, Y., Fitzmaurice, A., Shen, Y., Zhang, H., Tong, L., and Yang, J. (2004) Nature. 429, 675-680[CrossRef][Medline] [Order article via Infotrieve]
21. Opatowsky, Y., Chen, C. C., Campbell, K. P., and Hirsch, J. A. (2004) Neuron 42, 387-399[CrossRef][Medline] [Order article via Infotrieve]
22. Canti, C., Davies, A., Berrow, N. S., Butcher, A. J., Page, K. M., and Dolphin, A. C. (2001) Biophys. J. 81, 1439-1451[Medline] [Order article via Infotrieve]
23. Opatowsky, Y., Chomsky-Hecht, O., Kang, M. G., Campbell, K. P., and Hirsch, J. A. (2003) J. Biol. Chem. 278, 52323-52332[Abstract/Free Full Text]
24. DeWaard M., Scott, V. E., Pragnell, M., and Campbell, K. P. (1996) FEBS Let. 380, 272-276[CrossRef][Medline] [Order article via Infotrieve]
25. Witcher, D. R., De, W. M., Liu, H., Pragnell, M., and Campbell, K. P. (1995) J. Biol. Chem. 270, 18088-18093[Abstract/Free Full Text]
26. Neuhuber, B., Gerster, U., Doring, F., Glossmann, H., Tanabe, T., and Flucher, B. E. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 5015-5020[Abstract/Free Full Text]
27. Neuhuber, B., Gerster, U., Mitterdorfer, J., Glossmann, H., and Flucher, B. E. (1998) J. Biol. Chem. 273, 9110-9118[Abstract/Free Full Text]
28. Gerster, U., Neuhuber, B., Groschner, K., Striessnig, J., and Flucher, B. E. (1999) J. Physiol. (Lond) 517, 353-368[Abstract/Free Full Text]
29. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (2nd Ed.) Cold Spring Harbor Laboratory, Cold Spring Harbor, NY
30. Castellano, A., Wei, X., Birnbaumer, L., and Perez-Reyes, E. (1993) J. Biol. Chem. 268, 3450-3455[Abstract/Free Full Text]
31. Pragnell, M., Sakamoto, J., Jay, S. D., and Campbell, K. P. (1991) FEBS Lett. 291, 253-258[CrossRef][Medline] [Order article via Infotrieve]
32. Perez-Reyes, E., Castellano, A., Kim, H. S., Bertrand, P., Baggstrom, E., Lacerda, A. E., Wei, X. Y., and Birnbaumer, L. (1992) J. Biol. Chem. 267, 1792-1797[Abstract/Free Full Text]
33. Schneider, T., Wei, X., Olcese, R., Costantin, J. L., Neely, A., Palade, P., Perez-Reyes, E., Qin, N., Zhou, J., Crawford, G. D., Smith, R. G., Appel, S. H., Stefani, E., and Birnbaumer, L. (1994) Recept. Channels 2, 255-270[Medline] [Order article via Infotrieve]
34. Berrou, L., Bernatchez, G., and Parent, L. (2001) Biophys. J. 80, 215-228[Medline] [Order article via Infotrieve]
35. Bernatchez, G., Berrou, L., Benakezouh, Z., Ducay, J., and Parent, L. (2001) Bioch. Biophys. Acta 1514, 217-229[Medline] [Order article via Infotrieve]
36. Castellano, A., Wei, X., Birnbaumer, L., and Perez-Reyes, E. (1993) J. Biol. Chem. 268, 12359-12366[Abstract/Free Full Text]
37. Parent, L., Gopalakrishnan, M., Lacerda, A. E., Wei, X., and Perez-Reyes, E. (1995) FEBS Lett. 360, 144-150[CrossRef][Medline] [Order article via Infotrieve]
38. Bernatchez, G., Sauvé, R., and Parent, L. (2001) J. Memb. Biol. 184, 143-159[CrossRef][Medline] [Order article via Infotrieve]
39. Chica, R. A., Gagnon, P., Keillor, J. W., and Pelletier, J. N. (2004) Protein Sci. 13, 979-991[CrossRef][Medline] [Order article via Infotrieve]
40. Doucet, N., De Wals, P. Y., and Pelletier, J. N. (2004) J. Biol. Chem. 279, 46295-46303[Abstract/Free Full Text]
41. Walker, D., Bichet, D., Geib, S., Mori, E., Cornet, V., Snutch, T. P., Mori, Y., and De Waard M. (1999) J. Biol. Chem. 274, 12383-12390[Abstract/Free Full Text]
42. Kangas, E., and Tidor, B. (1998) J. Chem. Phys. 109, 7522-7545[CrossRef]
43. Sheinerman, F. B., and Honig, B. (2002) J. Mol. Biol. 318, 161-177[CrossRef][Medline] [Order article via Infotrieve]
44. Dafi, O., Dodier, Y., Berrou, L., Raybaud, A., Sauve, R., and Parent, L. (2004) Biophys. J. 87, 3181-3192[CrossRef][Medline] [Order article via Infotrieve]

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