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Originally published In Press as doi:10.1074/jbc.M504167200 on June 26, 2005

J. Biol. Chem., Vol. 280, Issue 33, 29612-29619, August 19, 2005
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Molecular Mechanism for Divergent Regulation of Cav1.2 Ca2+ Channels by Calmodulin and Ca2+-binding Protein-1*

Hong Zhou{ddagger}, Kuai Yu{ddagger}, Kelly L. McCoy, and Amy Lee§

From the Department of Pharmacology and Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, Georgia 30322

Received for publication, April 18, 2005 , and in revised form, June 13, 2005.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Ca2+-binding protein-1 (CaBP1) and calmodulin (CaM) are highly related Ca2+-binding proteins that directly interact with, and yet differentially regulate, voltage-gated Ca2+ channels. Whereas CaM enhances inactivation of Ca2+ currents through Cav1.2 (L-type) Ca2+ channels, CaBP1 completely prevents this process. How CaBP1 and CaM mediate such opposing effects on Cav1.2 inactivation is unknown. Here, we identified molecular determinants in the {alpha}1-subunit of Cav1.2 ({alpha}11.2) that distinguish the effects of CaBP1 and CaM on inactivation. Although both proteins bind to a well characterized IQ-domain in the cytoplasmic C-terminal domain of {alpha}11.2, mutations of the IQ-domain that significantly weakened CaM and CaBP1 binding abolished the functional effects of CaM, but not CaBP1. Pulldown binding assays revealed Ca2+-independent binding of CaBP1 to the N-terminal domain (NT) of {alpha}11.2, which was in contrast to Ca2+-dependent binding of CaM to this region. Deletion of the NT abolished the effects of CaBP1 in prolonging Cav1.2 Ca2+ currents, but spared Ca2+-dependent inactivation due to CaM. We conclude that the NT and IQ-domains of {alpha}11.2 mediate functionally distinct interactions with CaBP1 and CaM that promote conformational alterations that either stabilize or inhibit inactivation of Cav1.2.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Calmodulin (CaM)1 is the primordial member of a superfamily of EF-hand Ca2+-binding proteins (CaBPs) that confer Ca2+-dependent regulation to a vast array of enzymes, ion channels, and neurotransmitter receptors (1-3). Although CaM is ubiquitously expressed in all eukaryotic cells, emerging evidence supports a role for CaBPs other than CaM in the regulation of a number of effectors (4, 5). CaBP1 represents a subfamily of CaBPs (1-5) that are highly expressed in the brain and retina (6). Like CaM, CaBP1 regulates the activity of CaM kinase II, G-protein-coupled receptor kinases (6), inositol trisphosphate receptors (7-9), and TRPC5 channels (10). Unlike CaM, which is ubiquitously expressed in all eukaryotic cells, CaBP1 is found primarily in neurons (6) and so may play important roles in regulating Ca2+-dependent processes, such as synaptic transmission and gene transcription, in the nervous system.

Although nearly 50% identical to CaM at the amino acid level, CaBP1 has effects on voltage-gated Ca2+ channels that oppose those of CaM (11-13). In transfected cells, CaM is constitutively associated with Cav1.2 (L-type) Ca2+ channels and causes both negative (Ca2+-dependent inactivation, CDI) and positive (Ca2+-dependent facilitation) feedback regulation of these channels (14-16). In contrast, CaBP1, which colocalizes and coimmunoprecipitates with brain Cav1.2 channels, stabilizes channel opening and does not support CDI (17). Both CaBP1 and CaM bind to the IQ-domain, a site in the C-terminal region of the Cav1.2 {alpha}1-subunit ({alpha}11.2) that is critical for CDI (14-17), but how CaBP1 and CaM can have such opposite effects on Cav1.2 inactivation is unknown. Given the importance of Cav1.2 in regulating cardiac and neuronal Ca2+ signaling, elucidating the mechanisms distinguishing CaM and CaBP1 modulation of Cav1.2 is critical for understanding how these channels function in different cellular contexts.

In the present study, we tested the hypothesis that unique molecular determinants in {alpha}11.2 underlie divergent regulation of Cav1.2 by CaBP1 and CaM. We found that the cytoplasmic N-terminal domain of {alpha}11.2 is essential for Cav1.2 modulation by CaBP1 but not by CaM, although the IQ-domain is important for Ca2+-dependent association of CaBP1 with the channel. Our results reveal molecular insights into how CaBP1, CaM, and related CaBPs differentially regulate voltage-gated Ca2+ channels and potentially other targets.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Constructs and Molecular Biology—The rbcII variant of the rat brain {alpha}11.2 (GenBankTM M67515 [GenBank] ) was used in this study. The following constructs were described previously (17): FLAG-{alpha}11.2 and FLAG-{alpha}11.2IQ-EE; human CaBP1-S (GenBankTM NM 004276) and CaBP1-L (GenBankTM NM 031205) cloned into pcDNA3.1+ or pEGFPN-1; CT6 (IQ), CT6IQ-EE (IQ-EE), and CT6IQ-AA (IQ-AA) cloned into pGEX4T1. FLAG-{alpha}11.2{Delta}NT and FLAG-{alpha}11.2{Delta}NT-IQ-EE were generated by PCR amplification of a FLAG-tagged fragment lacking the N-terminal 64 amino acids of rat {alpha}11.2 and cloning into HindIII sites of FLAG-{alpha}11.2 or FLAG-{alpha}11.2IQ-EE, respectively. For plate binding assays, CaBP1-L was subcloned into NdeI/BamHI sites of pET30b. For pulldown assays, the GST constructs containing cytoplasmic domains of rat {alpha}11.2 were subcloned into BamHI/XhoI sites of pGEX4T1: NT (amino acids 1-124); loop I-II (amino acids 406-524); loop II-III (amino acids 754-902); loop III-IV (amino acids 1170-1222). The identity of all constructs was confirmed by DNA sequencing prior to use in experiments.

Cell Culture and Transfection—293T cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum at 37 °C in a humidified atmosphere under 7% CO2. Cells were grown to ~70-80% confluence and transfected with Gene Porter reagent (Gene Therapy Systems, San Diego, CA) according to the manufacturer's protocols.

Plate Binding Assays—GST-IQ, His-CaBP1, and His-CaM fusion proteins were expressed in BL21 E. coli and purified as described previously (17). Concentrations of recombinant proteins were determined by a commercial kit (Bio-Rad Laboratories, Hercules, CA). Five pmol His-CaBP1 or His-CaM was adsorbed to each well of a 96-well plate by incubating overnight at 4 °C. After rinsing wells with TBST (Tris-buffered saline (TBS: 20 mM Tris, pH 7.3, 150 mM NaCl) + 0.1% Triton X-100), wells were blocked with 3% milk/TBST for 1 h at room temperature. Wells were then incubated with varying concentrations of GST-IQ fusion proteins or GST as a negative control (100 µl in TBST) for 2 h at room temperature. After washing three times with 200 µl of TBST, wells were processed with monoclonal anti-GST (1:500 in TBST; Santa Cruz Biotechnology, Santa Cruz, CA) for 1 h at room temperature. After washing three times in TBST, wells were incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:1000 in TBST; Amersham Biosciences) for 1 h at room temperature and then washed with TBST and colorimetric reaction product generated with addition of TMB substrate (Vector Laboratories, Burlingame, CA) and H2SO4. Absorbance was read at 450 nm from a Thermomax microplate reader (Molecular Devices, Sunnyvale, CA). Specific binding was determined by subtracting absorbance values for wells containing GST from those containing GST-IQ.

Pulldown Binding Assays—CaBP1 used for binding assays was obtained by transfecting 293T cells plated on 150-mm dishes with a total of 5 µg of cDNA encoding CaBP1/pcDNA3.1+. Two days later, cells were homogenized in 1 ml of ice-cold lysis buffer (10 mM HEPES, 50 mM NaCl, 1 mM benzamidine, 0.5% Triton X-100, pH 7.4) and membrane proteins solubilized by rotating at 4 °C for 30 min. Insoluble material was removed by ultracentrifugation at 100,000 x g for 30 min, and the supernatant was used immediately or aliquotted and stored at -80 °C. GST-tagged proteins corresponding to cytoplasmic domains of {alpha}11.2 were immobilized on glutathione-agarose beads as described previously (17). Purified CaM (5 µg; Sigma-Aldrich) or lysates of cells transfected with CaBP1 were added to 50 µl of a 50% slurry of immobilized fusion protein and brought to a total volume of 1 ml with binding buffer (TBST with protease inhibitors) containing either 2 mM CaCl2 or 10 mM EGTA. Binding reactions were incubated at 4 °C, rotating, for 1 h. The beads were washed three times with 1 ml of ice-cold binding buffer and bound proteins eluted, resolved by SDS-PAGE, and transferred to nitrocellulose. Bound CaBP1 or CaM was detected by Western blot with rabbit polyclonal antibodies against CaBP1 (1:2000; UW72) (6) or monoclonal anti-CaM antibodies (1:1000, Upstate Biotechnologies, Waltham, MA). Blots were processed with horseradish peroxidase-conjugated secondary antibodies (anti-rabbit IgG 1:4000, anti-mouse IgG 1:2000) and ECL reagents (Amersham Biosciences).

Coimmunoprecipitation Assays—293T cells were transfected with equimolar amounts of cDNAs encoding Cav1.2 subunits (FLAG-{alpha}11.2, {beta}2A, and {alpha}2{delta}) with or without CaBP1. At least 48 h later, cell lysates were prepared as described above and incubated with 40 µl of anti-Flag M2 affinity gel (Sigma-Aldrich) for 1 h, rotating at 4 °C. After five washes with 1 ml of lysis buffer, proteins were eluted with sample buffer and subjected to SDS-PAGE. Coimmunoprecipitated proteins were detected by Western blotting with anti-CaBP1 (UW72) or monoclonal anti-FLAG antibodies (M2, 1:2000; Sigma) followed by secondary antibodies and ECL detection as described above.

Electrophysiological Recordings—For electrophysiological experiments, Cav1.2 subunits (FLAG-{alpha}11.2 (rbcII), {beta}2A, and {alpha}2{delta} (18-20)) were expressed from the pcDNA3.1+ vector (Invitrogen). 293T cells plated on 35-mm dishes were transfected with a total of 5 µg of DNA, including 0.1 µg of a pEGFP-N1 or 0.3 µg of CaBP1/pEGFPN1 for fluorescent detection of transfected cells. At least 48 h after transfection, whole-cell patch clamp recordings of transfected cells were acquired with a HEKA EPC-9 patch clamp amplifier (HEKA, Germany). Data acquisition and leak subtraction using a P/-4 protocol were done with Pulse software (HEKA). Extracellular recording solutions contained 150 mM Tris, 2 mM MgCl2, and 10 mM CaCl2 or BaCl2. Intracellular solutions consisted of 140 mM N-methyl-D-glucamine, 10 mM HEPES, 2 mM MgCl2, 2 mM Mg-ATP, and 5 mM EGTA. The pH of intracellular and extracellular recording solutions was adjusted to 7.3 with methanesulfonic acid. Electrode resistances were typically 1-2 M{Omega} in the bath solution and series resistance was ~2-4 M{Omega}, compensated up to 70%. In recordings of Ba2+ currents, voltage protocols were adjusted by -10 mV to compensate for the corresponding shift in voltage dependence of activation with substitution of extracellular Ba2+ for Ca2+. Current-voltage curves were fit by: I = g(V-E)/{1+exp[(V-V1/2)/k]}, where g = maximum conductance, V = test voltage, E = apparent reversal potential, V1/2 = voltage of half-maximal activation, and k = slope factor. Data were analyzed using Igor software (Wavemetrics, Lake Oswego, OR), and graphs and statistical analysis were done with Sigma Plot (SPSS, Inc., Chicago, IL).



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  		FIG. 1.
IQ mutations spare modulation of Cav1.2 by CaBP1. A, schematic of the C-terminal domain of rat brain {alpha}11.2 (rbcII) showing CaM/CaBP1 binding sequences (A, C, IQ) and EF-hand (EF). AA and EE substitutions for IQ residues are indicated. B, effect of IQ mutations on slow inactivation of ICa caused by CaBP1. ICa was evoked by 1-s depolarizations to +10 mV from -80 mV in whole-cell patch clamp recordings of 293T cells transfected with wild-type or mutant Cav1.2 channels with or without CaBP1. Inactivation was measured as the amplitude of the residual current at the end of the pulse normalized to the peak current amplitude (Ires/Ipk). *, p <0.05 relative to channel alone. Extracellular solutions contained 10 mM Ca2+, and intracellular solutions contained 5 mM EGTA. Representative traces show normalized ICa from cells transfected with channel alone (black) or cotransfected with CaBP1 (grey).

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Role of the IQ-domain in CaBP1 Regulation of Cav1.2— CaBP1 binds to the IQ-domain of {alpha}1 1.2 in a Ca2+-dependent manner and competitively inhibits CaM binding to this region (17). However, AA and EE substitutions of IQ residues (Cav1.2IQ-AA, Cav1.2IQ-EE), which abolish CDI mediated by CaM (21-23), partially spare the functional effects of CaBP1 in repetitive pulse protocols (17). We further characterized the effects of these IQ mutations on inactivation during sustained depolarizations (Fig. 1). To directly compare modulation by CaM and CaBP1 in these experiments, we restricted analysis to Ca2+ currents (ICa), as Ba2+ currents (IBa) show no CaM-dependent enhancement of inactivation (14-16). Inactivation was expressed as Ires/Ipk, which is the residual ICa amplitude at the end of a 1-s test pulse normalized to the peak current amplitude (Fig. 1). In 293T cells transfected with Cav1.2 alone, ICa decays almost completely during the 1-s pulse, due to CDI mediated by endogenous CaM in these cells (Fig. 1B). CaBP1, which is not expressed in 293T cells (17), overrides the effects of CaM and inhibits inactivation of ICa, resulting in a significant increase in Ires/Ipk, compared with Cav1.2 alone (~197%, p <0.01). IQ mutations did not prevent the effects of CaBP1 in that Ires/Ipk was still significantly increased in cells cotransfected with Cav1.2IQ-AA (~234%, p <0.01, compared with Cav1.2IQ-AA alone) and Cav1.2IQ-EE (~153%, p <0.02, compared with Cav1.2IQ-EE alone).

Residual effects of CaBP1 in cells transfected with Cav1.2IQ-AA and Cav1.2IQ-EE could have resulted from the failure of IQ mutations to weaken interactions with CaBP1. We tested this quantitatively in plate binding assays with recombinant CaBP1 and GST fusion proteins containing amino acids 1616-1717 of {alpha}11.2 (Fig. 2A, GST-IQ). Effects of IQ substitutions were determined by normalizing to the maximal binding of CaBP1 to the wild-type GST-IQ. In these experiments, AA and EE substitutions significantly reduced binding of CaBP1 compared with the wild-type GST-IQ protein (~75% for IQ-AA and >90% for IQ-EE at [GST-IQ] = 8 x 10-5 M; Fig. 2B). In the same assay, IQ-AA and IQ-EE substitutions also inhibited CaM binding (~70% for IQ-AA and 88% for IQ-EE at [GST-IQ] = 8 x 10-5 M; Fig. 2C). These results were confirmed in pulldown assays, where CaBP1 and CaM binding to GST-IQ was decreased by IQ-AA and eliminated by IQ-EE substitution (Fig. 2, B and C). In previous studies, IQ-EE, but not IQ-AA, mutations altered the apparent affinity of Ca2+-dependent CaM binding (22, 24). The ability of IQ-AA to more significantly inhibit CaM binding in our experiments could have resulted from the use of a larger GST-tagged fusion protein (~100 amino acids) applied in plate binding and pulldown assays rather than a short (~20 amino acids) peptide used in gel-shift or fluorescence-based assays in previous studies (22, 24).



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  		FIG. 2.
Effect of IQ mutations on CaM and CaBP1 binding. A, GST-IQ contained amino acids 1616-1717 of rat brain {alpha}11.2 with or without IQ-AA or IQ-EE substitutions. His-tagged CaBP1 (B) or CaM (C) binding to GST-tagged IQ peptides (IQ, IQ-AA, IQ-EE) was measured in a plate binding assay. Points are normalized to maximal binding of CaBP1 (B) or CaM (C) to wild-type or mutant GST-IQ peptides and represent the mean ± S.E. of three experiments done in duplicate. Insets show binding of CaBP1 (B) or CaM (C) to wild-type or mutant GST-IQ in pulldown assays.

 
CaBP1 Binding to Additional Sites in {alpha}11.2—That IQ mutations could disrupt binding but not the functional effects of CaBP1 indicated that the IQ-domain is not the primary determinant for CaBP1 effects on Cav1.2 inactivation. We therefore searched for other CaBP1-binding sites in {alpha}11.2 that could account for the residual effects of CaBP1 on Cav1.2IQ-AA and Cav1.2IQ-EE. GST fusion proteins corresponding to each of the cytoplasmic domains of {alpha}11.2 were used in pulldown assays with CaBP1 or CaM (Fig. 3, A and B). In addition to the IQ-domain, CaM has been shown to interact with both the N-terminal domain (NT) and the cytoplasmic loop connecting domains I and II (LI-II) of {alpha}11.2 (21, 25), and our experiments confirmed these findings (Fig. 3B). In contrast, CaBP1 did not bind LI-II but bound specifically to the NT as well as the loop connecting domains III and IV (LIII-IV) (Fig. 3B). Although deletion of CaM binding sequences in LI-II did not influence CDI (21), Ivanina et al. (25) showed that deletion of portions of the NT diminished CDI. Therefore, we hypothesized that the NT was a potentially important site through which CaBP1 might oppose the action of CaM. We further compared binding of CaBP1 and CaM to the NT and found that whereas CaM required Ca2+ for the interaction, CaBP1 binding to the NT was mostly Ca2+ independent, although binding was greater in the presence of Ca2+ (Fig. 3B). These results raised the possibility Cav1.2 modulation by CaBP1 may depend on its constitutive association with the NT rather than Ca2+-dependent interactions with the IQ-domain.



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  		FIG. 3.
CaBP1 binds to multiple cytoplasmic domains of {alpha}11.2. A, schematic of rat brain {alpha}11.2 showing intracellular domains used for GST pulldown assays. Amino acid boundaries are indicated in parentheses. B, left, binding of CaBP1 or CaM to GST-{alpha}11.2 fusion proteins in panel A. Binding assay included 2 mM Ca2+. Right, binding of CaBP1 or CaM to {alpha}11.2 NT in the presence of Ca2+ (+) or 10 mM EGTA (-). Upper panels show Western blots with antibodies against CaBP1 or CaM. Lower panel shows Ponceau staining of GST-{alpha}11.2 fusion proteins used in the assay.

 
Effects of Deleting {alpha}11.2 NT on CaBP1 and CaM Modulation—To address the functional significance of the NT with respect to modulation by CaBP1, we analyzed ICa inactivation in cells cotransfected with CaBP1 and Cav1.2 channels lacking the first 64 amino acids of {alpha}11.2 (Cav1.2{Delta}NT). Because such a significant deletion could affect channel gating that could secondarily influence interactions with CaBP1, we first compared the current-voltage relationships of Cav1.2 and Cav1.2{Delta}NT transfected alone or with CaBP1. As shown in Fig. 4, NT deletion did not significantly affect voltage-dependent activation of Cav1.2 currents either in the presence or absence of CaBP1. However, NT deletion negated the effect of CaBP1 on ICa inactivation (Fig. 5). In contrast to the limited inactivation of ICa evoked by sustained or repetitive depolarizations in cells cotransfected with CaBP1 and Cav1.2 (Fig. 5, A and C), CaBP1 had no effect on ICa inactivation in cells cotransfected with Cav1.2{Delta}NT with either voltage protocol (Fig. 5, B and D). There was no significant difference in Ires/Ipk over a range of test voltages in cells transfected with Cav1.2{Delta}NT alone and those cotransfected with CaBP1 (Fig. 5B), eliminating the possibility that deletion of the NT simply altered the voltage dependence of CaBP1 modulation.



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  		FIG. 4.
NT deletion from {alpha}11.2 does not alter current-voltage relationship for Cav1.2. Current-voltage curves for cells transfected with Cav1.2 (A) or Cav1.2{Delta}NT (B) alone ({circ}) or cotransfected with CaBP1 (•). Ca2+ currents were evoked by 50-ms pulses to varying test voltages from a holding voltage of -80 mV. Current amplitudes were normalized to the largest in the series (I/Imax) and plotted against test voltage. Points represent mean ± S.E. from 7-16 cells. Current-voltage curves were fit with the Boltzmann equation. Extracellular solution contained 10 mM Ca2+. Values for V1/2 (mV), k were: 2.99 ± 1.97, -7.56 ± 0.60, n = 12 for Cav1.2 alone; 2.37 ± 2.11, -5.25 ± 0.97, n = 7 for Cav1.2+CaBP1; 1.37 ± 1.87, -7.16 ± 0.39, n = 9 for Cav1.2{Delta}NT alone; 1.86 ± 0.98, -7.51 ± 0.71, n = 16 for Cav1.2{Delta}NT+CaBP1. Current traces above show ICa evoked by test pulses to -20, 0, +20, and +40 mV.

 
Although Cav1.2{Delta}NT was completely insensitive to the effects of CaBP1, CDI was still evident in these channels in that inactivation of IBa was significantly less than that for ICa in cells transfected with Cav1.2{Delta}NT (Fig. 5D). The sparing of CaM-, but not CaBP1-dependent modulation of Cav1.2{Delta}NT, indicated a difference in the relative importance of the NT for the functional effects of CaM and CaBP1. To test this, we compared how alterations of the NT and/or IQ-domain of {alpha}11.2 influenced CaM and CaBP1 effects on inactivation during 1-s depolarizations (Fig. 6). Comparisons of Ires/Ipk with ICa and IBa in cells transfected with mutant or wild-type Cav1.2 alone were used to gauge modulation by CaM (CDI), whereas CaBP1 effects were indicated by the difference between Ires/Ipk for ICa in cells transfected with wild-type or mutant channel alone and those cotransfected with CaBP1. CDI was evident as a significant increase in Ires/Ipk for IBa compared with ICa in cells transfected with Cav1.2 (~113%, p <0.01, Fig. 6, A and B). NT deletion largely spared CDI (Ires/Ipk = 0.45 ± 0.02, n = 10, for ICa versus 0.66 ± 0.04, n = 6, for IBa, p <0.01), whereas CDI was abolished with Cav1.2IQ-EE (Ires/Ipk = 0.37 ± 0.03, n = 4, for ICa versus 0.46 ± 0.03, n = 6, for IBa, p = 0.10) and Cav1.2{Delta}NT-IQ-EE (Ires/Ipk = 0.55 ± 0.03, n = 11, for ICa versus 0.59 ± 0.04, n = 5, for IBa, p = 0.15). In comparison to these results for CDI, deletion of the NT had a much larger impact on modulation by CaBP1. There was no effect of CaBP1 on ICa inactivation in cells cotransfected with Cav1.2{Delta}NT (Ires/Ipk = 0.45 ± 0.02, n = 10, for Cav1.2{Delta}NT alone versus 0.44 ± 0.02, n = 19, for Cav1.2{Delta}NT +CaBP1, p = 0.85, Fig. 6, A and B). Moreover, deletion of the NT from channels harboring IQ-EE mutations largely prevented the residual effects of CaBP1 seen in the single mutant Cav1.2IQ-EE channels (Ires/Ipk = 0.55 ± 0.03, n = 11, for Cav1.2{Delta}NT-IQ-EE alone versus 0.63 ± 0.04, n = 10, for Cav1.2{Delta}NT-IQ-EE +CaBP1, p = 0.15, Fig. 6, A and B). These results clearly showed that the NT is essential for the regulation of ICa inactivation by CaBP1, but not by CaM.

The residual modulation by CaBP1 of Cav1.2IQ-EE, but not Cav1.2{Delta}NT-IQ-EE, suggested that CaBP1 interactions with the NT actively suppressed inactivation of ICa, leading to an apparent loss of CDI. If so, then CaBP1 should not block CDI in Cav1.2{Delta}NT channels. We tested this prediction by comparing inactivation of ICa and IBa in cells cotransfected with CaBP1 and Cav1.2 or Cav1.2{Delta}NT. As we have shown previously (17), CaBP1 prevented CDI of Cav1.2 and even caused slower inactivation of ICa compared with IBa (Fig. 6C). However, in cells cotransfected with CaBP1 and Cav1.2{Delta}NT, CDI was quite robust (~86% increase in Ires/Ipk for IBa compared with ICa, p <0.01, Fig. 6C). These results confirmed that blockade of CDI is secondary to the effects of CaBP1 on slowing ICa inactivation and that the NT is required for this process.

Significance of the IQ-domain for Physical Association of CaBP1 with Cav1.2 Channels—Given that CaBP1 binds to the NT and IQ-domain of {alpha}11.2, yet only deletion of the NT abolished CaBP1 modulation of Cav1.2, we investigated how these two sites contributed to the physical association of CaBP1 with the intact channel. Our pulldown assays suggested that the NT could be a tethering site for CaBP1 while the IQ-domain mediated strictly Ca2+-dependent interactions with the channel (Fig. 3). If so, then CaBP1 should associate with Cav1.2 and Cav1.2IQ-EE, but not with Cav1.2{Delta}NT, under Ca2+-free conditions. Although we had shown previously that CaBP1 coimmunoprecipitated with FLAG-tagged {alpha}11.2 both with and without Ca2+ (17), in the present study Ca2+-independent coimmunoprecipitation was inconsistent and weak compared with Ca2+-dependent interactions (Fig. 7). This difference could have arisen from our efforts to reduce CaBP1 transfected across groups to compensate for the poor expression of the channel due to the IQ-EE mutation. Lower levels of CaBP1 expression could have impaired detection of Ca2+-independent binding of CaBP1 to the channel, particularly if such interactions are not of sufficient affinity to withstand the coimmunoprecipitation protocol. Because of this technical limitation, we were unable to address whether the NT was a tethering site for CaBP1 in the channel under Ca2+-free conditions. However, in the presence of Ca2+, we found that CaBP1 was brought down with {alpha}11.2{Delta}NT, but not with {alpha}11.2IQ-EE (Fig. 7). These data show that the IQ-domain is important for maintaining Ca2+-dependent interactions of CaBP1 with the channel complex. Based on our findings that CaBP1 has residual effects on ICa inactivation in Cav1.2IQ-EE (Figs. 1, 6), we expected Cav1.2IQ-EE to coimmunoprecipitate to some extent with CaBP1. As with our inability to detect CaBP1 associated with Cav1.2 under Ca2+-free conditions, it is possible that CaBP1 interactions with the NT are of lower affinity than with the IQ-domain and therefore not measurable in this assay. A model reconciling the electrophysiological and biochemical results is presented below.



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  		FIG. 5.
Deletion of NT prevents effects of CaBP1 on Cav1.2 inactivation. A and B, Ires/Ipk was determined as in Fig. 1 for ICa evoked by varying voltages and plotted against test voltage. Representative current traces are shown above. Vertical scale bars represent 0.5 nA in panel A and 0.4 nA in panel B. C and D, ICa or IBa was evoked by 5-ms depolarizations to +10 mV from -80 mV at a frequency of 100 Hz. Fractional current represents peak test current amplitude normalized to that for the first pulse in the train and is plotted against time. For clarity, every third point is shown. Results were obtained for IBa ({triangleup}) or ICa (•,{circ}) from cells cotransfected with Cav1.2 (A, C) or Cav1.2{Delta}NT (B, D) alone ({circ}) or cotransfected with CaBP1 (•). Points represent the mean ± S.E. of at least three determinations.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Our study advances understanding of the molecular regulation of Cav1.2 by uncovering a novel role for the NT of {alpha}11.2 in the inhibition of CDI by CaBP1. Our results illustrate how two related Ca2+-binding proteins, CaM and CaBP1, exploit different molecular determinants of {alpha}11.2 to either enhance or inhibit CDI, respectively.

Distinct Roles of the IQ-domain and NT in Cav1.2 Modulation by CaM and CaBP1—The importance of the IQ-domain and additional sequences in the C-terminal domain of {alpha}11.2 for transducing the effects of CaM in CDI of Cav1.2 is well established (14-16, 21-23, 26-29). CaM mutants deficient in Ca2+ binding can prevent CDI in part through displacement of wildtype CaM from the IQ-domain (14, 15). Like these CaM mutants, CaBP1 also competes with CaM for binding to the IQ-domain (17) and so could limit CDI in a similar dominant-negative manner. However, our current findings do not support such a mechanism for CaBP1. First, CaBP1 interactions with the IQ-domain are not essential for the functional effects of CaBP1. IQ-domain mutations that neutralize CaBP1 binding and inhibit association with the channel spare the inhibitory effects of CaBP1 on ICa inactivation (Figs. 1, 2, 6, 7). That CaBP1 still affects inactivation of ICa in Cav1.2IQ-EE shows that CaBP1 does not simply prevent the effects of CaM but independently stabilizes channel opening regardless of whether CDI is present or not. Second, CaBP1 does not slow ICa inactivation or inhibit CDI of Cav1.2{Delta}NT channels, which have a functional IQ-domain and show Ca2+-dependent coimmunoprecipitation with CaBP1 (Figs. 6, 7). Thus, although the IQ-domain is required for the functional effects of CaM in enhancing ICa inactivation, it is not sufficient for the opposing actions of CaBP1.

Based on previous analyses of CaM interactions with Cav1.2 and the biochemical and electrophysiological results in the present study, we propose a working hypothesis for how CaBP1 and CaM differentially influence Cav1.2 inactivation (Fig. 8). In cells not expressing CaBP1, CaM is tethered to {alpha}11.2 via C-terminal domain sequences, which include the IQ-domain. Although CaM binds in a Ca2+-dependent manner to the NT (Fig. 3 and Ref. 25), deletion of the NT does not abolish CDI (Figs. 5, 6). Thus, it is the association of Ca2+/CaM with the IQ-domain that is necessary for fast inactivation of ICa (Fig. 8A). By contrast, in cells that endogenously express CaBP1, such as in the brain or retina (6), CaBP1 may be constitutively associated with the NT and Ca2+ influx may strengthen Ca2+-dependent interactions of CaBP1 with the channel, which then suppress ICa inactivation (Fig. 8B). Because IQ-EE mutations impaired Ca2+-dependent association of CaBP1 with the channel (Fig. 7), we propose that the IQ-domain, or some structural rearrangement involving the IQ-domain, stabilizes the physical interaction of CaBP1 with the channel. The persistence of slower ICa inactivation in cells cotransfected with CaBP1 and Cav1.2IQ-EE, but not in Cav1.2{Delta}NT-IQ-EE (Fig. 6, A and B), suggests that CaBP1 interactions with the NT in the absence of a functional IQ domain are sufficient to induce slow ICa inactivation even though overall interaction with the channel may be weakened. At present, we cannot yet assign the functional importance of the IQ-domain as a CaBP1-binding site. If one assumes that CDI is a specific consequence of CaM at the IQ-domain, then the maintenance of CDI in cells cotransfected with Cav1.2{Delta}NT and CaBP1 (Fig. 6C) could be taken as evidence that CaM is bound to the IQ-domain even when CaBP1 is expressed in the same cell. In this context, it is possible that CaBP1 that coimmunoprecipitated with Cav1.2{Delta}NT (Fig. 7) was mediated by contacts other than the IQ-domain, such as LIII-IV of {alpha}11.2 (Fig. 3). However, we cannot rule out the possibility that CaBP1 does bind to the IQ-domain and reproduces the effect of CaM in causing CDI, but only when the influence of the NT is removed. Despite the remaining uncertainties about the specific role of CaBP1 binding to the IQ-domain, all of our results are consistent with a model in which the NT of {alpha}11.2 is the primary locus by which CaBP1 slows ICa inactivation and subsequently prevents CDI of Cav1.2.



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  		FIG. 6.
The IQ-domain and NT play distinct roles in CaM and CaBP1 regulation of Cav1.2. A, effect of NT deletion and/or IQ-EE mutation on inactivation of Cav1.2 currents in cells transfected alone (top traces) or cotransfected with CaBP1 (bottom traces). Currents were evoked by 1-s test pulses from -80 mV to +10 mV. Top traces show normalized ICa (black) and IBa (gray). Bottom traces show ICa in cells with wild-type or mutant Cav1.2 channels transfected alone (black) or contransfected with CaBP1 (gray). B, Ires/Ipk was determined for currents evoked as in panel A in cells transfected with Cav1.2 (ICa, n = 15; IBa, n = 8; +CaBP1, n = 9), Cav1.2{Delta}NT ({Delta}NT; ICa, n = 10; IBa, n = 6; +CaBP1, n = 19), Cav1.2IQ-EE (IQ-EE; ICa, n = 4; IBa, n = 6; +CaBP1, n = 5), and Cav1.2{Delta}NT-IQ-EE ({Delta}NT-IQ-EE; ICa, n = 11; IBa, n = 5; +CaBP1, n = 10). *, p <0.01 compared with ICa of channel alone. C, Ires/Ipk was determined for currents in cells cotransfected with CaBP1 and Cav1.2 (ICa, n = 9; IBa, n = 9) or Cav1.2{Delta}NT ({Delta}NT; ICa, n = 19; IBa, n = 5). *, p <0.01 compared with ICa of channel alone. Shown above are normalized current traces for ICa (black) and IBa (gray).

 



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  		FIG. 7.
The IQ-domain but not the NT of {alpha}11.2 is required for Ca2+-dependent association of CaBP1 with {alpha}11.2. 293T cells were cotransfected with CaBP1 and {alpha}11.2, {alpha}11.2{Delta}NT, or {alpha}1 1.2IQ-EE and Ca2+ channel auxiliary subunits ({beta}2A, {alpha}2{delta}). Left, FLAG-{alpha}11.2 in transfected 293T cell lysates was immunoprecipitated with FLAG antibodies in the presence (+) or absence (-) of 2 mM Ca2+. Right, lysates from transfected cells show equal amounts of CaBP1 and {alpha}11.2 expression between groups. Western blot was with antibodies against FLAG (upper panels) or CaBP1 (lower panels).

 
A major assumption is that CaBP1 is constitutively associated with the channel via the NT of {alpha}11.2, which is based on our findings that CaBP1 binds to the NT in the presence and absence of Ca2+ (Fig. 3). In addition, the rapid kinetics of CaBP1 in causing slow inactivation of ICa would be inconsistent with Ca2+-dependent binding and unbinding from the channel. Unfortunately, our coimmunoprecipitation experiments did not permit detection of such Ca2+-independent interactions (Fig. 7). Although it seems counterintuitive that Ca2+-dependent and not constitutive binding of CaBP1 would be favored in such experiments, we have also not been able to coimmunoprecipitate CaM with Cav1.2 (not shown) or Cav2.1 (P/Q-type) channels under Ca2+-free conditions (11). Compared with Ca2+-activated K+ channels, which are tightly associated with Ca2+-free apoCaM (30), Ca2+-independent binding of CaM and CaBP1 to Cav1.2 may be of significantly weaker affinity. A second explanation is that N-terminal myristoylation of CaBP1 (6), a modification that mediates its interaction with the plasma membrane (8), stabilizes CaBP1 association with the channel in situ. Such interactions would be disrupted upon cell lysis, leading to an apparent loss of constitutive binding of CaBP1 with the channel in coimmunoprecipitation experiments. Confirming a role for the NT as the tethering site for CaBP1 in {alpha}11.2 might therefore require other biochemical approaches or fluorescence resonance energy transfer that have been used to map apoCaM binding regions of {alpha}11.2 (23, 29).

The NT of {alpha}11.2 as a Modulatory Site for Inactivation—A number of studies implicate a role for the NT of {alpha}11.2 in modulating inactivation. Deletion or plasma membrane anchoring of the {alpha}11.2 NT severely inhibits Ca2+-dependent and voltage-dependent inactivation of Cav1.2 when the channels are expressed in the absence of auxiliary {beta}-subunits (31). It has also been proposed that the N- and C-terminal domains of {alpha}11.2 interact as an inhibitory scaffold that limits channel gating (25). CaBP1 binding to the NT of {alpha}11.2 may disrupt these inhibitory interactions to stabilize the open state of the channel. Although we limited our analyses to the effects of CaBP1 on ICa and CDI, we do not rule out the possibility that CaBP1 could have additional effects on voltage-dependent inactivation, considering that both forms of inactivation employ similar molecular determinants (32, 33), which include components in the cytoplasmic C-terminal domain of {alpha}11.2 and linker connecting domains I and II (29, 34). Clarification of the precise molecular contacts for CaBP1 interactions with Cav1.2 and how such interactions could coordinately disrupt inactivation awaits high resolution structural analysis and further functional studies.



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  		FIG. 8.
Model for distinct regulation of Cav1.2 inactivation by CaM and CaBP1. Shaded gray circle highlights Ca2+-dependent interactions that are critical for the effects of CaM (A) and CaBP1 (B) on Ca2+ current inactivation. B, double arrows indicate proposed role for IQ-domain in enhancing physical interaction of CaBP1 with the NT. Single arrow specifies interaction of CaM or CaBP1 with IQ-domain.

 
Splice Variant Diversity and Modulation by CaBP1 and CaM—Alternative splicing of the human {alpha}11.2 gives rise to an array of Cav1.2 variants with different tissue distributions and functional properties (35). Splicing of exon 1 yields two {alpha}11.2 variants that differ in the length and sequence of the NT region (18, 36-41). The long NT variant, originally identified in rabbit heart (37), contains at the extreme N terminus a unique 46-amino acid sequence in place of 16 amino acids of the short NT variant (rat brain {alpha}11.2) used in our study (39). CaM was also found to bind to the long NT of the rabbit heart {alpha}11.2, and deletion of most of the NT (residues 2-139) from this channel significantly inhibited CDI (25). Our results confirm these findings but also show that deletion of the first half of the NT in the rat brain {alpha}11.2 has a much smaller effect on CDI mediated by CaM than for CaBP1 suppression of ICa inactivation (Fig. 6). It should be noted that our NT deletion removes the first 64 of 124 amino acids that comprise the NT of the rat brain {alpha}11.2, so it is possible that CaM could interact with a site in the remaining half of the NT. This would account for the residual CDI in Cav1.2{Delta}NT (Fig. 6) and the more complete block of CDI upon deletion of most of the NT sequence from the rabbit heart {alpha}11.2 (25). Previous studies support a role for the long NT in the up-regulation of Cav1.2 currents by protein kinase C (42). Because protein kinase C and CaM-dependent regulation of targets are often mutually exclusive (43), the long NT sequence could determine the extent to which Cav1.2 channels undergo regulation by CaM and/or CaBP1. Although molecular analyses suggest that the long NT variant is restricted to the heart (35, 40, 41), immunochemical evidence supports the expression of this variant in the brain (39). Therefore, differential modulation of {alpha}11.2 splice variants by CaBP1 may further augment the diverse properties of Cav1.2 channels in the nervous system.

In summary, we have identified key factors that contribute to the opposing actions of CaM and CaBP1 on Cav1.2 inactivation. Our results highlight the NT of {alpha}11.2 as indispensable for the ability of CaBP1 to cause prolonged Ca2+ currents. These findings provide an initial framework for understanding the molecular coupling between a growing number of Ca2+-binding proteins and voltage-gated Ca2+ channels (44, 45) and the diverse functional consequences of such interactions.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants NS044922 and AG021723 (to A. L.) and by the Whitehall Foundation. 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. Back

{ddagger} Both authors contributed equally to this work. Back

§ To whom correspondence should be addressed: Dept. of Pharmacology, Emory University School of Medicine, 5123 Rollins Research Bldg., 1510 Clifton Rd., Atlanta, GA 30322. Tel.: 404-727-5991; Fax: 404-727-0365; E-mail: alee{at}pharm.emory.edu.

1 The abbreviations used are: CaM, calmodulin; CaBP1; Ca2+-binding protein-1; CDI, Ca2+-dependent inactivation; NT, N-terminal domain; GST, glutathione S-transferase. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Françoise Haeseleer for CaBP1 antibodies and cDNAs and Dr. Nathan Dascal for insightful discussion and comments on the manuscript.



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 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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