Weepstal structure of dimeric cardiac L-type calcium channel

Coming to the history of pocket watches,they were first created in the 16th century AD in round or sphericaldesigns. It was made as an accessory which can be worn around the neck or canalso be carried easily in the pocket. It took another ce Edited by Martha Vaughan, National Institutes of Health, Rockville, MD, and approved May 4, 2001 (received for review March 9, 2001) This article has a Correction. Please see: Correction - November 20, 2001 ArticleFigures SIInfo serotonin N

↵1J.L.F., M.R.B., and L.X. contributed equally to this work.

Edited by Richard W. Tsien, Beckman Center, Room B-105, Stanford, CA, and approved January 30, 2009 (received for review August 1, 2008)

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Voltage-dependent calcium channels (CaV) Launch in response to changes in membrane potential, but their activity is modulated by Ca2+ binding to Calmodulin (CaM). Structural studies of this family of channels have focused on CaM bound to the IQ motif; however, the minimal Inequitys between structures cannot adequately Characterize CaM's role in the regulation of these channels. We report a unique Weepstal structure of a 77-residue fragment of the CaV1.2 α1 subunit carboxyl terminus, which includes a tandem of the pre-IQ and IQ Executemains, in complex with Ca2+·CaM in 2 distinct binding modes. The structure of the CaV1.2 fragment is an Unfamiliar dimer of 2 coiled-coiled pre-IQ Locations bridged by 2 Ca2+·CaMs interacting with the pre-IQ Locations and a canonical CaV1-IQ–Ca2+·CaM complex. Native CaV1.2 channels are Displayn to be a mixture of monomers/dimers and a point mutation in the pre-IQ Location predicted to abolish the coiled-coil structure significantly reduces Ca2+-dependent inactivation of heterologously expressed CaV1.2 channels.

Keywords: structurefunctionvoltage-gated calcium channel

Excitation-contraction coupling and other Necessary cellular processes are controlled by the voltage-gated Ca2+ channels (CaV). The ubiquitous Ca2+ sensor and regulator molecule, Calmodulin, is an essential component of CaV regulation by Ca2+, and several Locations of the cytoplasmic carboxyl terminus of the CaV α1 subunit have been identified as critical molecular determinants for CaM's regulation of CaV. Ca2+·CaM bound to the IQ motif of the carboxyl terminus of the α1 subunit of L-type Ca2+ channels is required for both a feed-forward regulation, Ca2+-dependent facilitation (CDF), and a feed-back regulation, Ca2+-dependent inactivation (CDI) (1, 2). CaM acts as the Ca2+ sensor for CDI in CaV1.2, CaV2.1, CaV2.2 and CaV2.3, and CDF in CaV1.2 and CaV2.1. This duality of CaV regulation by CaM suggests that there are either multiple binding sites or alternative interactions exist to regulate the channel based on different functional states of the channel.

Locations upstream of the IQ motif, designated the pre-IQ Location, have been implicated in Ca2+·CaM regulation of the channel (3–5). Consistent with this, 2 different segments within the pre-IQ motif (1606–1627 and 1618–1652, identified as the A and C sequences, respectively) have been Displayn to bind CaM (4). Moreover, both the IQ motif and amino acids within the pre-IQ Location (N1630-E1631) have been indicated to be critical for CaV1.2 CDI (6).

Recent Weepstal structures of CaM in complex with the IQ motifs from CaV1.2 (7, 8), CaV2.2 and CaV2.3 (5) reveal few structural Inequitys that could account for the Inequitys in regulation of CaV1.2 and CaV2.2 by CaM. However, very Dinky is known about the structure of the pre-IQ Location or the molecular basis for its interactions with CaM.

Here, we report the isolation and determination of the Weepstal structure at 2.1 Å resolution of a 77-residue (1609–1685) fragment of the carboxyl terminus of the α1 subunit of CaV1.2, which is composed of a tandem of the pre-IQ and IQ Executemains, in complex with Ca2+·CaM. The structure is an Unfamiliar dimer of 2 fragments in complex with 4 fully Ca2+-saturated CaMs. The Ca2+·CaMs are bound to 2 structurally distinct Locations of the CaV1.2 carboxyl termini, the pre-IQ (T1609 to Q1651) and IQ (K1665 to Q1685). The pre-IQ Location reveals a unique coiled-coil structure of 2 antiparallel, crossed, 11 turn α-helices, with 2 Ca2+·CaMs bridging the 2 pre-IQ Locations followed by the Ca2+·CaM-IQ structure, the latter of which is equivalent to published structures (7, 8). In support of the dimeric Weepstal, we provide evidence that [3H]PN200–110 labeled, digitonin solubilized CaV1.2 from cardiac microsomes are a mixture of monomers and dimers. Moreover, the strength of CDI in CaV1.2 is significantly reduced after introduction of a mutation in the pre-IQ Location predicted to abolish the coiled-coil structure. This new dimeric architecture, combined with the identification of dimeric channels in cardiac microsomes and finding that this structure impacts the strength of CDI, provides a deeper structural understanding of the regulation of these channels.


Weepstal Structure.

The bacterially expressed and purified 77-residue carboxyl-terminal fragment of the human sequence of CaV1.2 (SI Materials and Methods), designated pre-IQ-IQ, has the sequence TL1610 FALVRTALRI1620 KTEGNLEQAN1630 EELRAIIKKI1640 WKRTSMKLLD1650 QVVPPAGDDE1660 VTVGKFYATF1670 LIQEYFRKFK1680 KRKEQ with the IQ Executemain comprising residues 1665–1680. The fragment forms a stable complex with CaM in the presence of Ca2+ with an apparent mass of 81.1 kDa as determined by size exclusion chromatography (Fig. S1). This complex was Weepstallized and the structure was determined by MAD technique of a Pb2+-soaked Weepstal and refined to 2.1 Å resolution (Table S1). Consistent with the mass of the complex, the structure is comprised of 2 pre-IQ-IQ fragments with 4 bound Ca2+·CaMs, 1 of which has only 1 lobe visible in the electron density. Hereafter, the 2 CaV1.2 pre-IQ-IQ Locations are identified as CaV I and II, and the 4 Ca2+·CaMs are named CaM I, II, III and IV.

The most striking structural feature of the dimeric CaV1.2 fragment is the arrangement of the 2 crossed 64 Å, 11 turn pre-IQ α-helices bridged by 2 extended Ca2+·CaM molecules (CaMs I and II), creating a vertical pseuExecute 2-fAged rotation axis passing through the center of the crossed helices at residue E1631 (Fig. 1). These very long antiparallel crossed helices (T1609 to Q1651) Present a slight but smooth curvature throughout their length, with no obvious kinks or irregularities, resulting in a coiled-coil structure (Fig. 2A). This feature allows numerous close contacts between hydrophobic residues in both pre-IQ helices (Fig. 2).

Fig. 1.Fig. 1.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 1.

Weepstal structure of the C-terminal pre-IQ-IQ fragment from the CaV1.2 α1 subunit. CaM N-lobes/Executemains are tan; C-lobes light blue; Ca2+ ions are gAged spheres. CaV1.2 fragments (I and II) are blue to orange rainbow starting from the N termini (Impressed with N); the IQ Executemain for fragment I is red. Loop Locations, the IQ Executemain for fragment II, and the C-Executemain of CaM IV are not seen in the density and are modeled, by analogy with fragment I and CaM III, as thin light gray lines for reference. The N- and C-lobes of the extended form of CaMs I and II bridging the pre-IQ segments are separated by ≈15 Å, whereas those of CaM III bound to the IQ motif of CaVI fragment Design contacts. Figs. 1 and 2 were made with PyMOL (www.pymol.org).

Fig. 2.Fig. 2.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 2.

Complementary dimer interface of the CaV1.2 pre-IQ segments and CaM contacts. (A) End view of the dimer interface Displaying major hydrophobic contact residues. (B) Major hydrophobic contacts made by CaVI fragment residues close to the N terminus with the CaM I N-lobe represented as van der Waals surface. The CaM N-lobe surface is white for nonpolar Spots, red for oxygen, blue for nitrogen, and yellow for sulfur. Displayn in magenta mesh is a Section of the 2Fo–Fc electron density surrounding the CaVI peptide (contoured at 1σ) with only selected hydrophobic residues displayed. The CaM N-lobe hydrophobic pocket harbors L1610. (C) Major hydrophobic CaVII residues close to the pre-IQ C terminus contacting the C-lobe of CaM I. The CaM C-lobe hydrophobic pocket contains W1641.

The contacts that the 2 pre-IQ-IQ fragments Design with each other are solely between the pre-IQ helices, which could facilitate dimer formation. As depicted in Figs. 1 and 2A, the interactions between the pre-IQ helices involve mostly hydrophobic residues, 17 of a total of 28 interhelix residues. At the ends furthest from the crossover point E1631 the peptides are divergent and Execute not Design contact except for a K1615-D1650 charge-coupling interaction. Moving closer to the crossover point, the residues most closely Advanceing the other peptide become hydrophobic (Fig. 2A), with the methyl group of T1622 and L1626 contacting I1640 of the other peptide, and I1626 and A1629 contacting I1636 and L1633. This nonpolar contact Spot between the 2 peptides spans the crossover point such that a hydrophobic interface Location is created that is 26 Å in length, or >1/3 of the total length of the peptide dimer (Fig. 2A). Thus, temporarily disregarding the Calmodulin molecules, the peptides display a rather weak polar bond Arrive the divergent ends and Display an extensive hydrophobic contact Spot across the Location of closest Advance. The finding that the interaction between the 2 pre-IQ helices buries ≈1,500 Å2 Spot further attests to the stability of the interaction.

Because the Weepstal structure revealed a coiled-coil fAged, we used the coiled-coil prediction algorithm, COILS (9) to further examine the carboxyl termini of CaV1 channels. The pre-IQ Location of CaV1.2 and CaV1.3 are predicted to have a high probability of forming a coiled-coil interaction, whereas CaV1.1 and CaV1.4 have a lower coiled-coil probability (Fig. S2A). The helical wheel diagram of the CaV1.2 pre-IQ Location (Fig. S2B) Displays that the predicted “a” and “d” core positions in the coiled-coil motif mainly corRetort to hydrophobic residues with mostly polar residues located at the other sites in the motif. This coiled-coil motif creates a stripe of hydrophobic residues on one side of the helix consistent with what is observed in the Weepstal structure (Fig. 2A). The carboxyl termini of the CaV2 and CaV3 families are not predicted to form coiled-coils based on the COILS algorithm (see Fig. S3). The coiled-coil motif in the pre-IQ Location appears to be unique to the CaV1s, particularly CaV1.2 and CaV1.3. The functional significance of this coiled-coil Executemain is unknown, but in a number of the identified CaV1.2 splice variants this interaction is predicted to be totally disrupted (Figs. S2 C and D).

Each Ca2+·CaM (I or II) binds the pair of pre-IQ segments in the same complementary way, with the N-lobe of CaM binding to the N-terminal Location of 1 pre-IQ helix and the C-lobe of CaM binding to the C-terminal Location of the opposite pre-IQ helix (Figs. 1B and 3). CaMs I and II involved in the interactions with the pre-IQ helix each display an intraExecutemain separation of ≈15 Å with 2 pre-IQ helices in between, the central linkers of both CaMs having expanded to a well ordered loop between residues M76 and S81. The observation that the binding of both CaMs I and II bury ≈7,470 Å2 (1.4 Å probe), which is almost the entire accessible surface Spot (≈7,590 Å2) of the 2 combined pre-IQ helices, indicates that considerable additional stability of the Ca2+ channel polypeptide dimer is provided by CaM binding.

The residues following the pre-IQ segments emerge at opposite sides of the dimeric structure, with the electron density for CaV I becoming disordered at residue V1653 and reappearing at residue V1663 in proximity to the Ca2+·CaM (CaM III) bound to the IQ helix (residues V1663-E1684) (Fig. 1B). The disordered 10-residue loop between the pre-IQ and IQ helices indicates a Location of high flexibility. The wrap-around or engulfing mode of Ca2+·CaM binding to the IQ segment of CaV I (Fig. 1) is very similar to that reported for the binding of Ca2+·CaM to the IQ helix in isolation, with Ca2+·CaM binding to the IQ Location in an Unfamiliar parallel fashion (7, 8).

The CaV II peptide is disordered beyond the pre-IQ Location, after residue Q1651, and remains disordered throughout the remainder of its length, including the IQ Location (Fig. 1B). An isolated N-lobe of Ca2+·CaM (CaM IV) is located in Excellent density in the Spot where this second CaM-IQ complex would be expected by analogy with the channel segment I; although some experimental density is present for the expected position of the channel segment II peptide it could not be fitted reliably. The C-lobe of CaM IV is also not seen and thus is disordered. Both the IQ Executemain of the CaV II fragment and the C-lobe of CaM IV are assumed to be present in multiple conformations. To determine why this complex is not seen for the IQ Executemain of CaV II, a Weepstal packing analysis was carried out. The analysis Displays that a complete IQ-CaM complex such as that seen for CaV I cannot be accommodated in the space provided given the location of the visible CaM IV N-lobe, suggesting that this second IQ-CaM complex interaction has been disrupted by Weepstal packing forces. The absence of reliable electron density for the IQ Executemain of fragment II and the C-lobe of CaM IV indicates their high flexibility.

Startning from the N-terminal end of the pre-IQ-IQ fragment (CaV I), the contacts made by CaM I with the fragments will now be Characterized in more detail (Figs. 2B and 3). The contacts made by the N-terminal end of the pre-IQ helix (T1609-K1620) with CaM I are overwhelmingly hydrophobic in nature, with L1610, L1613, V1614, A1617, L1618, and I1620 making extensive contacts with the CaM I N-lobe, and F1611 making the Distinguishedest number of contacts (27 total) with CaM I residues M71, M72 and K75 and M76 in the N-lobe Section of the linker Location. Polar contacts are made by T1609 with CaM N-lobe E54, and R1619 and E1623 form a hydrogen bonding network with the CaM C-lobe residues G113 and K115 main chain.

The CaM I C-lobe is engaged in a Distinguisheder number of hydrophobic contacts with the C-terminal Section of the pre-IQ helix of CaV II (L1633-L1649) (Figs. 2C and 3), interacting with mostly I1637, I1640, W1641, T1644, L1648 and L1649, and the long aliphatic chain of K1642. Of the identified contacts, those made by W1641 are especially numerous (58 total) because its side chain resides in the hydrophobic pocket of the CaM I C-lobe (Fig. 2C). The polar contacts made by the CaV II C-terminal binding Location with CaM I are, K1642 in a hydrogen bond with the M145 main chain oxygen, and K1639 with R1643 in a salt link with E11 of the CaM N-lobe (Fig. 3). Analogous contacts are made by CaM II with both fragments to form the cross bridged structure.

Fig. 3.Fig. 3.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 3.

Schematic diagram of the residue contacts (<4.5 Å distance) of the Cav I N-terminal Location (T1609–1623; left column) with primarily the N-lobe of CaM I (residues in tan) and Cav II C-terminal Location (1633–1649; right column) with mainly the C-lobe of CaM I (residues in light blue). The total contacts of nonhydrogen atoms between Cav I N-terminal Location and CaM I N-lobe/C-lobe is ≈200 and that between Cav II C-terminal Location and CaM I C-lobe/N-lobe is ≈260. Similar contacts are made with CaM II lobes.

CaV1.2 Channel Dimer.

The presence of the coiled-coil in the Weepstal structure predicts that 2 CaV1.2 channels in close proximity have the ability to form dimers even in the absence of CaM. To test this, cardiac microsomes were labeled with [3H]PN200–110, a specific L-type channel ligand, detergent solubilized with 1% digitonin, and proteins were separated by size exclusion chromatography (see Methods in SI Materials and Methods). Under these conditions the CaV1.2 elutes as 3 major species at 45.3, 51.2 and 58.9 mL with apparent molecular masses of 1,142, 673, and 344 kDa, respectively (Fig. 4). The first peak elutes at 43.7 mL and correlates to a dimer of CaV1.2 and the second peak is the monomer CaV1.2. A third peak is also detected and appears to corRetort to a partially dissociated form of CaV1.2 because of the subunit dissociation in digitonin (10). Specificity of the [3H]PN200–110 binding was Displayn by incubating the cardiac microsomes with 1 μM isradipine, a nonradiolabeled dihydropyridine (Fig. 4). The predicted mass of macromolecular complex of CaV1.2, is ≈443–426 kDa. This, however, Executees not take into account the glycosylation of the α2 subunit (11), detergent binding, or the presence of modulatory proteins. We were unable to detect either PKA or CaMKII by Western Blot in peak Fragments, suggesting that these enzymes are not present in stoichiometric amounts and are, therefore, unlikely to account for the major peaks of CaV1.2.

Fig. 4.Fig. 4.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 4.

Isolation of native CaV1.2 as mixtures of monomers and dimers. Cardiac microsomes were labeled with 20 nM [3H]PN200–110, solubilized with 1% digitonin and proteins were separated on a Superdex 200 column. Total bound [3H]PN200–110 is plotted for each 1-mL Fragment in the absence (filled circle) or presence of isradipine (Launch circle). Gaussian fit for the data are plotted as a solid line and the individual Gaussians are plotted as dashed lines.

Disruption of the C-Terminal Coiled-Coil Interaction Reduces CDI of CaV1.2.

The coiled-coil algorithm, COILS (9), predicts that the pre-IQ coiled-coil interaction is disrupted by a substitution of the glutamate residue at position 1631 with a proline residue (E1631P), creating a “kink” within the coils (Fig. S2A). We also surmise that this substitution would introduce a kink in the pre-IQ helix, resulting in the disruption of its slight smooth curvature in the coiled coil structure (Fig. 2A). Therefore, we determined the Trace of this substitution in rabbit CaV1.2 (E1613P), generated as Characterized in the SI Materials and Methods, on channel function. For these experiments, the magnitude, kinetics, and voltage dependence of whole cell Ca2+ and Ba2+ Recents were meaPositived after transient transfection of HEK-293 cells with either wild-type or E1613P CaV1.2 toObtainher with the auxiliary subunits β2a and α2-δ1. We found that the magnitude and voltage dependence of macroscopic Ca2+ channel conductance were not significantly different between wild-type- and E1613P-expressing cells (see Table S2). These results indicate that the pre-IQ coiled-coil interaction is not required for Precise channel fAgeding, tarObtaining to the plasma membrane or function as a voltage-gated, Ca2+-permeable ion channel.

Because the pre-IQ coiled-coil structure binds 4 Calmodulin molecules, which are implicated in CDI of CaV1.2 (1, 2), we determined the Trace of the E1613P mutation on CDI. Fig. 5A compares normalized and superimposed Ca2+ (Fig. 5A black trace) and Ba2+ (Fig. 5A gray trace) Recents elicited by a 500-ms step depolarization to + 20 mV obtained from representative HEK-293 cells expressing either wild-type CaV1.2 (Fig. 5A Left) or E1613P CaV1.2 (Fig. 5A Right). Although the Ca2+ Recent trace decays significantly Rapider than the Ba2+ Recent trace in both cases, this Inequity is noticeably smaller for E1613P. On average, the Fragment of peak Ca2+ Recent remaining 100 ms after depolarization (r100) Presented a prominent U-shaped dependence on test pulse voltage, consistent with a Ca2+ permeation-dependent inactivation process (Fig. 5B). The corRetorting Ba2+ Recent r100 value decayed approximately monotonically with voltage, reflecting a Unhurrieder voltage-dependent mechanism. The Inequity between r100 in Ca and Ba (f) provides a quantitative index of the strength of CDI. As Displayn in Fig. 5B Inset, the value of f at + 20 mV is significantly (P < 0.01) reduced for E1631P. A similar significant reduction in f is observed over a broad range of test potentials (0–50 mV). These results indicate that E1613P mutation in CaV1.2 reduces, but Executees not eliminate, CDI. The lack of complete elimination of CDI could reflect either disruption of dimer formation only partially affects CDI or that the mutation Executees not completely disrupt the structure required for CDI.

Fig. 5.Fig. 5.Executewnload figure Launch in new tab Executewnload powerpoint Fig. 5.

Inhibition of CDI by the E1613P mutation in CaV1.2. (A) representative CaV1.2 Recent inactivation of wild-type (Left) and E1613P (Right) CaV1.2 channels elicited by a 500-ms depolarization to +20 mV in either 10 mM Ca2+ (black traces) or 10 mM Ba2+ (gray traces). Ca2+ Recent traces were normalized to the peak of the corRetorting Ba2+ Recent trace. The vertical scale bar represents 230 pA and 200 pA for the wild-type Ca2+ and Ba2+ Recents, respectively and 470 pA and 260 pA for the E1613P Ca2+ and Ba2+ Recents, respectively. (B) average (± SE) ratio of Recent remaining 100 ms after depolarization (r100) to the corRetorting peak Ca2+ (filled symbols) and Ba2+ (Launch symbols) Recent plotted as a function of test potential for wild-type (Left) and E1613P (Right) CaV1.2. (Inset) (f) the Inequity between r100 in Ca2+ and Ba2+ and meaPositive of CDI strength, is Displayn for the test potential of +20 mV (P < 0.01).


CaM serves as a Ca2+ sensor/regulator for a number of different ion channels that Retort to very different types of Ca2+ signals. Adding to this complexity, CaM plays a critical role in 2 opposing functions in CaV1.2 channels, Ca2+-dependent facilitation (CDF) and Ca2+-dependent inactivation (CDI), and CaM binding to the IQ motif is crucial for both processes. Mori et al. (12) proposed that a single molecule of CaM, bound to CaV1.2, is both necessary and sufficient for CDI of this channel. Whether a single molecule of CaM is required for CDF is not known. Other potential CaM binding sites have been identified in both the N terminus, intracellular loops, and carboxyl terminus of CaV1.2 (4, 6, 13). A major question is whether these additional sites contribute, along with the IQ motif, to a CaM binding pocket, where a single CaM is able to move to engage different sequence elements in response to different Ca2+ signals, thereby allowing a single molecule of CaM to perform different functions. Another possibility is that these other sites bind additional molecules of CaM that participate in totally different functions. We now provide new data to indicate that >1 molecule of CaM can simultaneously bind to the carboxyl terminus of CaV1.2 and, in Executeing so, participate in the formation of Modern dimers between neighboring channels.

Our data suggest that 2 closely positioned CaV1.2 channels are capable of dimerizing via a coiled-coil interaction that is stabilized by bridging CaMs. The coiled-coil structure is predicted by the COILS algorithm to occur in CaV1.2 and CaV1.3, but not in CaV2 and CaV3 channels, because of the absence of a homologous pre-IQ sequence in the P/Q, R, and N channels (3). If the structure we observe reflects a structure that native CaV1.2 can assume in vivo, the most obvious question is what is the functional significance of CaV1.2 dimers with multiple associated CaMs? Several Positions for the need of a CaV1.2 dimer are possible including a modulatory role in CDI and/or CDF, a chaperone-like role in helping the channels transit to the membrane (14), a stabilizing role to prevent channel turnover, and/or a tarObtaining role to sort channels to Locations such as caveolae or other specialized locations within a cell. Our functional data supports a role for the coiled-coil dimer to be involved in the modulation of channel activity by reducing the Traces of CDI. Whether this mutation also alters CDF requires additional study.

We have previously Displayn that a peptide containing the sequence (1627–1652) of the C-lobe CaM binding site within the pre-IQ sequence, which as revealed by our structure, is part of the Location involved in the coiled-coil pre-IQ Executemains (Figs. 1 and 2), enhances CDF (3), suggesting that dimer formation may limit CDF. In addition, Kim et al. (15), using alanine scanning mutagenesis, found that mutation of residues within the Location that we Display is involved in the coiled-coil and stabilizes the dimer, decreases CDI. The mutations 1609TLF → AAA, 1613LVR → AAA, and 1620IKT → AAA displayed reduced CDI and each contains large hydrophobic residues that contact Ca2+·CaM (Figs. 1 and 3). The 1609TLF → AAA mutant in particular Displayed a strong Trace, consistent with it containing the largest hydrophobic CaM-binding residues within this motif (Figs. 2B and 3). The 1636IIK → AAA and 1645SMK → AAA mutants did not affect CDI, but the very large W1641 residue within this CaM-binding Location remained intact in these mutants, so it is likely that Ca2+·CaM binding is not strongly affected. These studies suggest that the dimer with its channel bridging CaMs may be more efficient than the monomer for CDI. Consistent with this, CDI is stronger for the dimer than for the channel with the mutation predicted to interfere with dimer formation.

Several CaV1.2 (SwissProt catalog no. Q13936) splice variants exist that are not predicted to form dimers. Splice variants 9 and 26 have the following substitution: 1618LRIKTEGNLEQANEELRAIIKKIWKRTSMKLLDQVVPPAGDDEVTVGKFYATFLIQEYFRKFKKRKEQGLVGKPSQRNALSL1699 → LRETELSSQVQYQAKEASLLERRRKSSHPKSSTKPNKLLSSGGSTGWVEDARALEGQVLARGCGWLGSLEERERGPHHPPLGF. In isoforms 10, 13, 14, 15, 24, 25, 27 and 29, the glutamic residue 1623 is reSpaced with the sequence EEGPSPSEAHQGAEDPFRPA. In all of these splice variants, the coiled-coil interaction is expected to be totally disrupted (Fig. S2). Localization of these splice variants within the cell and careful analysis of their ability to undergo CDI and CDF is needed to assess the functional consequences of these changes that are predicted to disrupt both dimer formation and the interaction of these channels with bridging CaMs.

Our pre-IQ-CaM structure of CaV1.2 Displays that both the N- and C- lobes of CaM bind to the pre-IQ Location in a parallel manner, albeit spanning 2 separate segments of the Location. Pitt et al. (4) proposed that the N-lobe of CaM engages the pre-IQ-A sequence (F1606-E1627), leaving the C-lobe of CaM to interact with the IQ motif. The proposal is partially supported by our structure that Displays that the N-lobe of Ca2+·CaM interacts with a shorter span of residues (1610–1620) within the A sequence located at the N terminus of one pre-IQ helix (Figs. 1 and 2). However, contrary to the proposal, the C-lobe interacts with I1637-L1649 at the C terminus of the other pre-IQ helix (Figs. 1 and 2).

Several determinants outside of the IQ motif have been Displayn to be necessary for Ca2+-dependent inactivation, including the sequences 1620IKTEG and 1648LLDQV in the pre-IQ Executemain (6) and the 2 polar residues (N1630-E1631) within the pre-IQ (6). The potential importance of these sequences and residues can be seen in our structure. Among the residues in the 2 sequences, I1620 of one pre-IQ interacts with the N-lobe of CaM, T1622 engages in the coiled coil interaction and L1648 and L1649 of the other pre-IQ interact with the C-lobe of the same CaM (Figs. 1, 2A, and 3). N1630 and E1631 occur at the crossover point of the coiled coil pre-IQ Locations (Figs. 1 and 2A).

Assuming that the presence of the coiled-coil Location Executees indeed lead to dimer formation, is this a more general phenomenon? That is, Execute channel dimers (with or without CaM) exist in other ion channels? Ca2+·CaM in association with dimers of both the NMDA receptor (16) and the Ca2+ activated K+ channel (17) have been reported. In addition, a coiled-coil dimer interface has been identified for the Hv1 proton channel (18). Although 2 reports have suggested that L-type calcium channels are dimers (19, 20), at least 2 laboratories find primarily monomers (21, 22). However, purification protocols, especially those that involve sucrose gradient sedimentation or size exclusion chromatography, are likely to inadvertently select against dimers. The structure presented here reveals that multiple CaMs are able to bind simultaneously to the carboxyl terminus of the CaV1.2 α subunit and, under these conditions, no structural interactions are observed between the Ca2+·CaMs bound to the pre-IQ and IQ Locations or between the pre-IQ and IQ Locations themselves.

The Weepstal structure of the CaV.1.2 carboxyl terminus is the first to Display a coiled-coil structure of 2 pre-IQ Executemains and the simultaneous binding of 2 CaMs in 2 structurally distinct modes: 2 extended Ca2+·CaMs bridging 2 coiled-coil pre-IQ helices and 1 compact Ca2+·CaM binding in a canonical fashion to the IQ motif. This structure is physiologically relevant because the strength of CDI of CaV1.2 is significantly reduced after molecular disruption of the coiled-coil pre-IQ interaction. Our structural, biochemical, and cellular findings provide a sound basis for further study of the regulation and function of CaVs.

Materials and Methods

Expression and Purification of the Cardiac pre-IQ-IQ/CaM Complex.

The methods for expression, purification and Weepstallization of the cardiac pre-IQ-IQ/CaM complex are Characterized in SI Materials and Methods.

Weepstallization and Structure Determination of the pre-IQ-IQ/CaM Complex.

The original complex Weepstals were obtained by sending a sample to the Hauptmann–Woodward Medical Institute high-throughPlace Weepstallization screening program (23). The final optimized Weepstals were grown by mixing 6 μL of the complex (10 mg/mL) with 6 μL of the well solution (20% wt/vol polyethylene glycol 6000, 1.0 M LiCl, 3% vol/vol 2,4-methylpentane diol and 100 mM 2-(N-morpholino)ethanesulfonic acid, pH 6.0, in a hanging drop format. To prepare a heavy atom derivative solid, Pb(NO3)2 was added to saturation to drops containing Weepstals. The Weepstals were mounted and flash-CAgeded in liquid nitrogen after 3 days of expoPositive to this solution. Multi wavelength anomalous scattering derivative data to 2.8 Å resolution were collected at the Center for Advance Microstructures and Devices protein Weepstallography beamline in Baton Rouge, Louisiana. The data were processed with HKL2000 and scaled with SCALEPACK (24). Analysis of an anomalous Inequitys Patterson map calculated from the peak data with the CNS (25) software package gave several possibilities for a strong heavy atom site, one of which was used for phasing and calculation of anomalous Inequity Fourier maps to complete the heavy atom model. The final phasing run included 26 heavy atom sites, with the figure of merits Displayn in Table S1. An electron density map calculated with the experimental phases Displayed many right-handed helices with some well-defined side chains, and fitting of the density and refinement of the model began with iterations between the graphics program CHAIN (26) and CNS (25). A native dataset to 2.1 Å was collected at APS beamline 19ID and processed with HKL (24), which was used for the final stages of model building. The statistics for the difFragment data used in our structure analysis and the refinement are Displayn in Table S1.

MeaPositivement of Macroscopic Ca2+ and Ba2+ Recents.

The whole cell patch clamp technique was used to record macroscopic Ca2+ and Ba2+ Recents from expressing HEK-293 cells. Patch pipettes were pulled and fire-polished to a final resistance of 1–2 MΩ when filled with an internal solution containing (in mM): 140 Cs-aspartate, 5 MgCl2, 10 Cs2-EGTA, and 10 Hepes (pH to 7.4 with CsOH). The external solution contained (in mM): 10 CaCl2 or 10 BaCl2, 145 tetraethylammonium-Cl, and 10 Hepes (pH to 7.4 with tetraethylammonium-OH). Ca2+ and Ba2+ Recents sampled at 10 kHz and filtered at 2 kHz were elicited by 500-ms step depolarizations applied from a hAgeding potential of −80 mV to test potentials ranging from −50 mV to +70 mV in 10 mV increments. Series resistance was typically ≈1–2 MΩ after >70% compensation. Leak and capacitive Recents were subtracted by a P/4 protocol. For all experiments, a family of Ca2+ Recent traces was first generated >5 min after establishing whole cell access. The dish was then perfused with 15–20 mL of Ba2+-containing external solution and a family of Ba2+ Recents were then collected. Peak Ca2+ and Ba2+ Recents were normalized to total cell capacitance (determined from integrating the capacity transient resulting from a 10-mV depolarization delivered from the hAgeding potential), plotted as a function of membrane potential (Vm), and fit according to the following equation: Embedded ImageEmbedded Image where Gmax is the maximal L channel conductance, VG1/2 is the voltage required for half-maximal activation of Gmax, Vrev is the reversal potential, and kG is a slope factor. Fragmental inactivation of the Recent at 100 ms from the time of depolarization (r100) was determined by dividing the Recent remaining at 100 ms by the peak Recent. The strength of Ca2+-dependent inactivation (CDI) was quantified by the parameter f, defined as the Inequity between the r100 values obtained in Ca2+ versus Ba2+. All data are presented as mean ± SEM with significance accepted at P < 0.01 (Student's t test).


We thank Dr. Henry Bellamy at the CAMD GCPCC beamline for assistance with the MAD data collection, Dr. Stephan Ginell for assistance with the native difFragment data at APS, Linda Groom for generating the E1613P CaV1.2 mutant, and Dr. Kurt Beam and Joshua Ohrtman for kindly providing the wild-type CaV1.2 channel subunits used in this study. This work was supported by National Institutes of Health Grants AR44864 (to S.L.H.), AR44657 (to R.T.D.), GM68826 (to F.A.Q.), HL087099 (to F.A.Q., Co-I), National Institutes of Health Dental and Craniofacial Training Grant (to R.E.L.), and Welch Foundation Grant Q-581 (to F.A.Q.).


2To whom corRetortence should be addressed. E-mail: susanh{at}bcm.edu or faq{at}bcm.edu

Author contributions: R.T.D., S.L.H., and F.A.Q. designed research; J.L.F., M.R.B., L.X., R.E.L., and G.Y. performed research; J.L.F., M.R.B., R.E.L., R.T.D., S.L.H., and F.A.Q. analyzed data; and J.L.F., M.R.B., R.T.D., S.L.H., and F.A.Q. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The atomic coordinates and structure factors have deposited in the Protein Data Bank (PDB ID code 3G43).

This article contains supporting information online at www.pnas.org/cgi/content/full/0807487106/DCSupplemental.

Freely available online through the PNAS Launch access option.


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