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, 29 (23), 3924-38

Multiple C-terminal Tail Ca(2+)/CaMs Regulate Ca(V)1.2 Function but Do Not Mediate Channel Dimerization

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Multiple C-terminal Tail Ca(2+)/CaMs Regulate Ca(V)1.2 Function but Do Not Mediate Channel Dimerization

Eun Young Kim et al. EMBO J.

Erratum in

  • EMBO J. 2010 Dec 1;29(23):4062

Abstract

Interactions between voltage-gated calcium channels (Ca(V)s) and calmodulin (CaM) modulate Ca(V) function. In this study, we report the structure of a Ca(2+)/CaM Ca(V)1.2 C-terminal tail complex that contains two PreIQ helices bridged by two Ca(2+)/CaMs and two Ca(2+)/CaM-IQ domain complexes. Sedimentation equilibrium experiments establish that the complex has a 2:1 Ca(2+)/CaM:C-terminal tail stoichiometry and does not form higher order assemblies. Moreover, subunit-counting experiments demonstrate that in live cell membranes Ca(V)1.2s are monomers. Thus, contrary to previous proposals, the crystallographic dimer lacks physiological relevance. Isothermal titration calorimetry and biochemical experiments show that the two Ca(2+)/CaMs in the complex have different properties. Ca(2+)/CaM bound to the PreIQ C-region is labile, whereas Ca(2+)/CaM bound to the IQ domain is not. Furthermore, neither of lobes of apo-CaM interacts strongly with the PreIQ domain. Electrophysiological studies indicate that the PreIQ C-region has a role in calcium-dependent facilitation. Together, the data show that two Ca(2+)/CaMs can bind the Ca(V)1.2 tail simultaneously and indicate a functional role for Ca(2+)/CaM at the C-region site.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1
Figure 1
Structure of the Ca2+/CaM–CaV1.2 PreIQ–IQ domain complex. (A) Two CaV1.2 PreIQ helices (red and salmon) form a crystallographic dimer cross-bridged by Ca2+/CaMs. Ca2+/CaM N-lobe and C-lobe are coloured green and blue, respectively. N- and C-termini of PreIQ–IQ domain are indicated. Cartoon representation shows a schematic asymmetric unit. (B) Ca2+/N-lobeAC PreIQ A-region-binding site. (C) Ca2+/C-lobeAC PreIQ C-region-binding site. In (B, C), PreIQ and Ca2+/CaM lobes are shown in stick and surface representation, respectively. Important contact residues are highlighted in white and labelled. Hydrophobic Ca2+/CaM lobe-binding interface residues are indicated and displayed in yellow. Due to poor Ca2+/C-lobeAC electron density, Ca2+/C-lobe from copy A of 2BE6 (Van Petegem et al, 2005) superimposed on the Ca2+/C-lobeAC model was used for the figure. (D) Schematic diagram of PreIQ anti-parallel coiled-coil interactions. a–g positions of heptad repeats and residue numbers are indicated. Right hand side shows the PreIQ coiled-coil interaction. Sidechains are shown as sticks. Interface residues targeted for mutation experiments are shown in space filling representation.
Figure 2
Figure 2
Sedimentation equilibrium analysis of Ca2+/CaM–CaV1.2 tails. (A) Equilibrium distribution of 100 μM Ca2+/CaM–CaV1.2 PreIQ–IQ domain complex at 11 000 r.p.m. and 4°C and measured at 293 nm. Raw data (black open circles) and single species fit (black line) are compared with predicted curves for complexes having Ca2+/CaM:CaV1.2 tail ratios of 1:1 (red), 2:1 (yellow), 2:2 (green), and 4:2 (blue). Inset is random distribution of residuals as a function of radial distance. (B) Comparison of the measured molecular weight of Ca2+/CaM–CaV1.2 PreIQ–IQ domain based on sedimentation equilibrium experiments performed at different protein concentrations and centrifugation speeds. Molecular weights for 2:1 (42.8 kDa) and 4:2 (85.6 kDa) complexes are indicated by the dotted lines. (C) Equilibrium distribution of 15 μM Ca2+/CaM–CaV1.2 C–IQ complex. A representative curve is shown; 15 μM of Ca2+/CaM–CaV1.2 C–IQ complex at 11 000 r.p.m. and 4°C and measured at 281 nm. Solid lines are calculated as in (A).
Figure 3
Figure 3
Subunit counting of CaV1.2 in cell membrane. (A) Inactivation properties of GFP-tagged CaV1.2 co-expressed with CaVβ2a in Xenopus oocytes. Normalized Ca2+ and Ba2+ currents recorded from Cav1.2–GFP (dark blue and light blue, respectively) are compared to those of wild type (black and grey, respectively). The pulse protocol is indicated above the graph and scale bars represent 200 nA Ca2+ current. (B) A representative TIRF image showing the CaV1.2–GFP fluorescent spots in the cell surface of X. laevis oocytes. Bright spots are CaV1.2–GFP single molecules when the shutter is first opened at the beginning of the bleaching experiment. Blue circles mark the selected molecules for subunit counting. (C) Time courses of photobleaching from single fluorescent spots. Two examples are shown. (D) Distribution of fluorescent spots that bleach in one or more steps. Percentile of each bleaching step is indicated.
Figure 4
Figure 4
The Ca2+/CaM–CaV1.2 PreIQ–IQ domain complex contains two types of Ca2+/CaM. (A) Phenylsepharose chromatograph of the Ca2+/CaM–CaV1.2 PreIQ–IQ complex. Peak 1 is the flow through fraction and Peak 2 is eluate obtained with 10 mM EGTA. Absorbance and conductivity are shown as blue and green, respectively. (B) SDS–PAGE analysis of Peak 1 and Peak 2 from (A). Peak 1 contains both CaM and CaV1.2 PreIQ–IQ peptide. Peak 2 contains only CaM. (C) Sedimentation equilibrium analysis of the Ca2+/CaM–CaV1.2 PreIQ–IQ complex post phenylsepharose treatment. Representative data obtained at 4°C and measured at 14 000 r.p.m. and 236 nm using 10 μM complex is shown. (D) Sedimentation equilibrium analysis of Ca2+/CaM–CaV1.2 C–IQ complex after phenylsepharose chromatography. Representative data measured at 11 000 r.p.m. and 297 nm using 90 μM protein is shown. Fits in (C) and (D) are presented as in Figure 2A and C.
Figure 5
Figure 5
Characterization of Ca2+/CaMAC–CaV1.2 PreIQ interactions. (A) 100 μM Ca2+/N-lobe titrated into 10 μM HMT-tagged A-region. Panels show injections of 10 μl titrant to the target (top) and binding isotherms (bottom). (B) Pulldown assay using 20 μM HMT alone and 20 μM HMT-tagged A-region variants with 100 μM Ca2+/N-lobe. M indicates molecular weight standards. (C) 100 μM Ca2+/C-lobe titrated into 10 μM HMT-tagged A-region. Panels show injections of 10 μl titrant to the target (top) and binding isotherms (bottom). (DF) Binding isotherms for titration of 100 μM Ca2+/C-lobe into (D) 10 μM wild type, (E) Trp1593Ala, and (F) Trp1593Glu 1:1 Ca2+/CaM–CaV1.2 C–IQ complexes. (G) Overlaid binding isotherms of (DF). Cartoon representations depict components of each experiment. A-region, C-region, and IQ domain are denoted as PreIQ A, PreIQ C, and IQ, respectively. Stars indicate mutation sites.
Figure 6
Figure 6
Inactivation of CaV1.2 and CaV1.2 bearing mutations in (A) the crystallographic anti-parallel coiled-coil interface, (B) Ca2+/N-lobeAC–A-region binding sites, and (C) Ca2+/C-lobeAC–C-region binding sites. CaV1.2s were co-expressed with CaVβ2a and CaVα2δ in Xenopus oocytes. Data shows currents for 450 ms with a test pulse from −90 to +20 mV. Normalized Ca2+ and Ba2+ currents for wild type (black and grey, respectively) and indicated mutants (dark blue and light blue, respectively) are shown. Scale bars indicate 200 nA Ca2+ current.
Figure 7
Figure 7
C-region mutations affect CDF. Blocking both dimer interface and Ca2+/C-lobeAC–C-region interaction almost completely abolished Ile1624Ala CDF. Ca2+ currents were recorded in a 3-Hz 40-pulse train (50 ms pulse from −90 to +20 mV) and normalized to Imax of the first trace (black) for comparison. For clearer view, every fourth trace is shown in grey with an exception of the 40th trace (red).
Figure 8
Figure 8
(A) Sequence alignments of the PreIQ and IQ domains of human CaV1.2 1561–1647 (Cavα1c77) CAA84346; CaV1.1 1466–1552, AAI33672; CaV1.3 1534–1620, NP_001077085; CaV1.4, 1526–1612 NP_005174, Ciona CaV1 1466–1552, XP_002123864; CaV2.1 1905–1994 NP_001095163; CaV2.2 1794–1883, NP_000709.1; and CaV2.3 1760–1849,Q07652. PreIQ, IQ Domain, A-region, and C-regions are indicated. Conservation of the C-region tryptophan is highlighted blue. Yellow circles indicate residues L1585 and I1588. (B) Cartoon model depicting the possible relative positions of CaV intracellular elements. ‘?' signifies the potential for a Ca2+/CaM anchored at the C-region site to make bridging interactions with other components of the CaV complex. Yellow indicates the positions of residues L1585 and I1588 that augment CDF effects. Relative orientation of the C-terminal tail is chosen to display the key elements. Its orientation relative to the other intracellular components is not known.

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