IQ Domain Research Articles

Neuropsychological and dimensional behavioral trait profiles in Costa Rican ADHD sib pairs: Potential intermediate phenotypes for genetic studies.

Attention deficit hyperactivity disorder (ADHD) is associated with substantial functional impairment in children and in adults. Many individuals with ADHD have clear neurocognitive deficits, including problems with visual attention, processing speed, and set shifting. ADHD is etiologically complex, and although genetic factors play a role in its development, much of the genetic contribution to ADHD remains unidentified. We conducted clinical and neuropsychological assessments of 294 individuals (269 with ADHD) from 163 families (48 multigenerational families created using genealogical reconstruction, 78 affected sib pair families, and 37 trios) from the Central Valley of Costa Rica (CVCR). We used principal components analysis (PCA) to group neurocognitive and behavioral variables using the subscales of the Child Behavior Checklist (CBCL) and 15 neuropsychological measures, and created quantitative traits for heritability analyses. We identified seven cognitive and two behavioral domains. Individuals with ADHD were significantly more impaired than their unaffected siblings on most behavioral and cognitive domains. The verbal IQ domain had the highest heritability (92%), followed by auditory attention (87%), visual processing speed and problem solving (85%), and externalizing symptoms (81%). The quantitative traits identified here have high heritabilities, similar to the reported heritability of ADHD (70-90%), and may represent appropriate alternative phenotypes for genetic studies. The use of multigenerational families from a genetically isolated population may facilitate the identification of ADHD risk genes in the face of phenotypic and genetic heterogeneity.

Towards a Common Structure-Function Framework for Calmodulin Regulation of Calcium and Sodium Channels

The voltage-gated calcium (CaV) and sodium (NaV) channels represent two major ion-channel superfamilies with distinct biophysical properties that support diverse biological functions. One similarity between these channels is a homologous region within their carboxy tails, the Ca2+-inactivating (CI) region. This segment, composed of two vestigial EF hands and an IQ domain, may be a conserved module enabling vital Ca2+-dependent feedback regulation through interaction with calmodulin (Cell 157:1657). A single Ca2+-free calmodulin (apocalmodulin) preassociates with the CI region, and Ca2+ binding to this calmodulin triggers regulation in both channel families. Though this general regulatory scheme appears similar between these channel families, the structural likeness of the CI region has remained unclear. Here, we correspond the first atomic structure of apocalmodulin alone bound to the NaV1.5 CI domain (Gabelli et al (2014) Nature Communications in press) with functionally relevant apocalmodulin binding residues in CaV1.3. This comparison implicates an extraordinary structural resemblance between Nav and Cav channels. But does this resemblance support like functional sequelae? Apocalmodulin binding itself enhances peak open probability of Cav channels by severalfold (Adams et al (2014) Cell in press). We now find conservation of this same effect in NaV1.4 channels. NaV1.4 channels bind apocalmodulin with high affinity and exhibit a robust open probability ∼0.4. Substituting dual alanines for the ‘IQ’ residues in the IQ element significantly weakens apocalmodulin binding, and markedly diminishes peak open probability (∼0.1). Overexpressing apocalmodulin fully rescues open probability, demonstrating that the effects on opening reflect apocalmodulin binding per se. This extensive structural and functional similarity substantiates a striking conservation of calmodulin regulation across Nav and Cav channels, joint investigation of which now presents as a genuinely synergistic endeavor.

Loss of Calmodulin-Mediated Regulation of Na+ Channel Causes Remodeling of Electrical and Junctional Proteins; and Induces Dilated Cardiomyopathy in IQ/AA+/− Mice

Sodium channel mutations near the IQ and EFL motifs in the carboxyterminal (CT) domain have been linked to long QT (LQTS) and Brugada syndromes (BrS). IQ-calmodulin (CaM) interaction is important for regulation of cardiac Na channels. The aim of this study was to assess the role of Na+–Ca2+/CaM signaling via IQ motif of the Na+ channels in development and maturation of intercalated disc (ID). We studied transgenic mice with alanines knocked into IQ positions in the Nav1.5 CT. The homozygous mice are embryonic lethal and heterozygous mice (IQ/AA+/− mice), develop cardiomyopathy (DCM). We measured the signal and distribution of Nav1.5, syntrophin, Cx43 and ryanodine in 3 and 9 month old IQ/AA+/− mice. Results were compared to those obtained from age matched wild type mice. By immunohistochemistry we show that Nav1.5 protein in 9 month-old IQ/AA+/− mice is significantly reduced at the ID. Syntrophin that traffics Na channels to the membrane, is not altered. Cx43 which is co-located with Nav1.5 at the ID, is significantly reduced. The expression of these proteins were not altered in 3 month-old IQ/AA+/− mice. We also assessed the implication of IQ domain on the localization of Ca2+ handling protein such as ryanodine receptor and found that it was significantly altered in 9 month-old IQ/AA+/− mice. The data suggest that enhanced late INa,L in IQ/AA+/− mice contributes to DCM via remodeling of electrical and junctional proteins and demonstrate a dynamic interplay of Na+–Ca2+/CaM signaling via IQ motif of the Na+ channels in ID development and maturation. Our study highlights the importance of Ca2+/CaM-mediated regulation of Na+ channels in DCM and arrhythmia.

Ezrin interacts with the scaffold protein IQGAP1 and affects its cortical localization

The cortical cytoskeleton constitutes an important subcellular structure that determines cell shape and regulates cell migration as well as membrane traffic to and from the plasma membrane. Many components of the cortical cytoskeleton have been identified including structural and scaffolding proteins, membrane–cytoskeleton linker proteins and signaling intermediates. We describe here an association of the membrane-F-actin linker protein ezrin with the scaffolding protein IQGAP1 that serves as a hub for concentrating different signaling complexes. Both, ezrin and IQGAP1 bind in a Ca2+-dependent manner to the EF hand protein S100P and complexes consisting of Ca2+-bound S100P, IQGAP1 and ezrin can be isolated by immunoprecipitation. Ezrin and IQGAP1 also interact in the absence of Ca2+, thus independent of S100P. Direct ezrin–IQGAP1 interaction can be shown with the purified proteins. It is mediated via the N-terminal FERM domain of ezrin and the IQ domain of IQGAP1, respectively. Ezrin and IQGAP1 colocalize in the submembraneous cytoskeleton and in cellular protrusions of human epithelial cells and knockdown of ezrin reduces the cortical localization of IQGAP1. Thus, ezrin appears to participate in recruiting IQGAP1 to the cell cortex thereby establishing a close connection between membrane-F-actin contacts and actin regulators that can be assembled by IQGAP1. This article is part of a Special Issue entitled: 13th European Symposium on Calcium.

Domain Contributions to Signaling Specificity Differences Between Ras-Guanine Nucleotide Releasing Factor (Ras-GRF) 1 and Ras-GRF2

Ras-GRF1 (GRF1) and Ras-GRF2 (GRF2) constitute a family of similar calcium sensors that regulate synaptic plasticity. They are both guanine exchange factors that contain a very similar set of functional domains, including N-terminal pleckstrin homology, coiled-coil, and calmodulin-binding IQ domains and C-terminal Dbl homology Rac-activating domains, Ras-exchange motifs, and CDC25 Ras-activating domains. Nevertheless, they regulate different forms of synaptic plasticity. Although both GRF proteins transduce calcium signals emanating from NMDA-type glutamate receptors in the CA1 region of the hippocampus, GRF1 promotes LTD, whereas GRF2 promotes θ-burst stimulation-induced LTP (TBS-LTP). GRF1 can also mediate high frequency stimulation-induced LTP (HFS-LTP) in mice over 2-months of age, which involves calcium-permeable AMPA-type glutamate receptors. To add to our understanding of how proteins with similar domains can have different functions, WT and various chimeras between GRF1 and GRF2 proteins were tested for their abilities to reconstitute defective LTP and/or LTD in the CA1 hippocampus of Grf1/Grf2 double knock-out mice. These studies revealed a critical role for the GRF2 CDC25 domain in the induction of TBS-LTP by GRF proteins. In contrast, the N-terminal pleckstrin homology and/or coiled-coil domains of GRF1 are key to the induction of HFS-LTP by GRF proteins. Finally, the IQ motif of GRF1 determines whether a GRF protein can induce LTD. Overall, these findings show that for the three forms of synaptic plasticity that are regulated by GRF proteins in the CA1 hippocampus, specificity is encoded in only one or two domains, and a different set of domains for each form of synaptic plasticity.

Functional and molecular features of the calmodulin-interacting protein IQCG required for haematopoiesis in zebrafish.

We previously reported a fusion protein NUP98-IQCG in an acute leukaemia, which functions as an aberrant regulator of transcriptional expression, yet the structure and function of IQCG have not been characterized. Here we use zebrafish to investigate the role of iqcg in haematopoietic development, and find that the numbers of haematopoietic stem cells and multilineage-differentiated cells are reduced in iqcg-deficient embryos. Mechanistically, IQCG binds to calmodulin (CaM) and acts as a molecule upstream of CaM-dependent kinase IV (CaMKIV). Crystal structures of complexes between CaM and IQ domain of IQCG reveal dual CaM-binding footprints in this motif, and provide a structural basis for a higher CaM-IQCG affinity when deprived of calcium. The results collectively allow us to understand IQCG-mediated calcium signalling in haematopoiesis, and propose a model in which IQCG stores CaM at low cytoplasmic calcium concentrations, and releases CaM to activate CaMKIV when calcium level rises.

Neurogranin Alters the Structure and Calcium Binding Properties of Calmodulin

Neurogranin (Ng) is a member of the IQ motif class of calmodulin (CaM)-binding proteins, and interactions with CaM are its only known biological function. In this report we demonstrate that the binding affinity of Ng for CaM is weakened by Ca(2+) but to a lesser extent (2-3-fold) than that previously suggested from qualitative observations. We also show that Ng induced a >10-fold decrease in the affinity of Ca(2+) binding to the C-terminal domain of CaM with an associated increase in the Ca(2+) dissociation rate. We also discovered a modest, but potentially important, increase in the cooperativity in Ca(2+) binding to the C-lobe of CaM in the presence of Ng, thus sharpening the threshold for the C-domain to become Ca(2+)-saturated. Domain mapping using synthetic peptides indicated that the IQ motif of Ng is a poor mimetic of the intact protein and that the acidic sequence just N-terminal to the IQ motif plays an important role in reproducing Ng-mediated decreases in the Ca(2+) binding affinity of CaM. Using NMR, full-length Ng was shown to make contacts largely with residues in the C-domain of CaM, although contacts were also detected in residues in the N-terminal domain. Together, our results can be consolidated into a model where Ng contacts residues in the N- and C-lobes of both apo- and Ca(2+)-bound CaM and that although Ca(2+) binding weakens Ng interactions with CaM, the most dramatic biochemical effect is the impact of Ng on Ca(2+) binding to the C-terminal lobe of CaM.

IQGAP1 Binds to Estrogen Receptor-α and Modulates Its Function

The estrogen receptor (ER) is a steroid hormone receptor that acts as a transcription factor, modulating genes that regulate a vast range of cellular functions. IQGAP1 interacts with several signaling proteins, cytoskeletal components, and transmembrane receptors, thereby serving as a scaffold to integrate signaling pathways. Both ERα and IQGAP1 contribute to breast cancer. In this study, we report that IQGAP1 binds ERα and ERβ. In vitro analysis with pure proteins revealed a direct interaction between IQGAP1 and ERα. Investigation with multiple short fragments of each protein showed that ERα binds to the IQ domain of IQGAP1, whereas the hinge region of ERα is responsible for binding IQGAP1. In addition, IQGAP1 and ERα co-immunoprecipitated from cells, and the association was modulated by estradiol. The interaction has functional effects. Knockdown of endogenous IQGAP1 attenuated the ability of estradiol to induce transcription of the estrogen-responsive genes pS2, progesterone receptor, and cyclin D1. These data reveal that IQGAP1 binds to ERα and modulates its transcriptional function, suggesting that IQGAP1 might be a target for therapy in patients with breast carcinoma.

Class III Myosin Motor Activity Correlates with Localization in Actin Protrusions

Class III myosins (myo3A and myo3B) contain a N-terminal kinase domain, central motor domain, and an isoform specific C-terminal tail. Myo3A contains a longer tail and has an actin-binding motif at the C-terminus that is not present in the shorter tail of myo3B. Modifications to the myo3A gene result in late onset human deafness (DFNB30), suggesting myo3B may be able to compensate for myo3A only early in life. Thus, it is critical to understand differences in the motor properties of myo3A and myo3B. We investigated the in vitro motility of myo3A and myo3B, each with the kinase domain removed and containing two IQ domains and a C-terminal GFP fusion (myo3AΔK.GFP and myo3BΔK.GFP). The sliding velocity measured in the in vitro motility assay was ∼20% faster in myo3AΔK.GFP (76 nm/sec) compared to myo3BΔK.GFP (62 nm/sec). The sliding velocity correlated well with the maximum actin-activated ATPase activity (kcat = 1.0 sec−1 and 0.7 sec−1, respectively). Interestingly, myo3B.ΔK.GFP contained a 25-fold weaker actin affinity compared to myo3A.ΔK.GFP as assessed by the actin-dependence of the ATPase activity (KATPase = 3 and 76 µM, respectively). We transfected N-terminally GFP tagged myo3A.ΔK containing the full length tail domain into COS7 cells and examined the efficiency of localizing to the filopodia tips by examining the tip to cell body ratio. Since the tail actin binding motif is required for localization to filopodia tips, we generated a chimera that contained the myo3B motor and the actin binding motif of the myo3A tail. The tip localization efficiency was approximately 17% (p<0.05) reduced in myo3B compared to myo3A. Thus myo3B is a slightly slower motor with a weaker actin affinity compared to myo3A, which may explain why it is unable to compensate for the loss of myo3A in DFNB30.

Meta-Analysis of Associations Between Human Brain Volume And Intelligence Differences: How Strong Are They and What Do They Mean?

Positive associations between human intelligence and brain size have been suspected for more than 150 years. Nowadays, modern non-invasive measures of in vivo brain volume (Magnetic Resonance Imaging) make it possible to reliably assess associations with IQ. By means of a systematic review of published studies and unpublished results obtained by personal communications with researchers, we identified 88 studies examining effect sizes of 148 healthy and clinical mixed-sex samples (> 8,000 individuals). Our results showed significant positive associations of brain volume and IQ (r = .24, R2 = .06) that generalize over age (children vs. adults), IQ domain (full-scale, performance, and verbal IQ), sample type (clinical vs. healthy sample), and sex. Application of a number of methods for detection of publication bias indicates that strong and positive correlation coefficients have been reported frequently in the literature whilst small and non-significant associations appear to have been often omitted from reports. We show that although the positive association of brain volume and IQ seems to be robust, its strength has been overestimated in the literature. While it is tempting to interpret this association in the context of human cognitive evolution and species differences in brain size and cognitive ability, we show that it is not warranted to interpret brain size as a necessary cause or isomorphic proxy of human intelligence differences.

Disordered binding regions of Ewing’s sarcoma fusion proteins

A relationship was found between the Amino acid (AA) composition, Intrinsic Protein Disorder (IPD) and Protein Binding Regions (PBRs) of the functional regions of Ewing's sarcoma protein (EWS) and oncogenic EWS fusion proteins (EFPs). EWS has high IPD and 64% predicted Disordered Binding Regions (DBRs) by ANCHOR. The native Transcription Factors, fused to EWS Activation Domain (EAD) in EFPs, show high DBRs in N-terminal domain and relatively low in C-terminal domain. EFPs oncogenic function is related to IPD and PBRs probabilities, high around breakpoint and decreased in the fused Transcription Factor. The increased IPD in EAD around (AA 82), and the small RBRs around (AAs (50-60) and 100) are consistent with the reported physical interactions with RNA Polymerase II subunits. The AAs (228-264) of EWS, interacting with ZFM1 (SF1), correspond to two peaks of DBRs by Anchor and high IPD by IUPred. The IQ domain of EAD (AAs 258-280) that is phosphorylated by PKC and interacts with calmodulin, has high IPD and DBRs probability. The Ser266, conserved site of PKC phosphorylation, is situated in DBR and IPD region with about 100% probability. The small PBRs found in the EAD correspond to important physical protein-protein interactions, confirmed by experimental data. Thus regions of EWS and EFPs, included in functional interactions with other partners, are enriched of Protein Binding Regions by ANCHOR. The development of IPD- and PBRs-related, EWS-FLI1-directed specific therapies will help the design of antitumor agents against ESFT because of high patient mortality in cases of meta- static disease.

IQ Domain GTPase-Activating Protein 1 is Involved in Shear Stress-Induced Progenitor-Derived Endothelial Cell Alignment

Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m2) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This “switch” was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.

IQGAP1 Mediates Angiotensin II-Induced Apoptosis of Podocytes via the ERK1/2 MAPK Signaling Pathway

Background/Aims: The mechanism underlying angiotensin II (AngII)-promoted podocyte apoptosis has not been established. IQ domain GTPase-activating protein 1 (IQGAP1) is a scaffolding protein of the mitogen-activated protein kinases (MAPK) signaling pathway, and plays a significant role in apoptosis. The present study evaluates the role of IQGAP1 in AngII-induced podocyte apoptosis. Methods: We randomly assigned 36 male Wistar rats to a normal saline-infused group, an AngII-infused group, or a normal control group, and measured podocyte apoptosis by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and transmission electron microscopic analysis. In addition, we exposed differentiated mouse podocytes to AngII and then assessed apoptosis by flow cytometry and Hoechst-33258 staining. Expression of IQGAP1 was measured by Western blotting, real-time PCR and immunofluorescence assay in vivo and in vitro. IQGAP1 siRNA and MAPK pathway inhibitors were further introduced to investigate the role of IQGAP1 and MAPK signaling in the process. Coimmunoprecipitation was used to evaluate the interaction between ERK1/2 and IQGAP1. Results: AngII promoted podocyte apoptosis in vivo and in vitro. IQGAP1 had a linear distribution along the capillary loops of glomeruli in vivo, and was in the cellular membrane and cytoplasm of cultured podocytes. AngII stimulated IQGAP1 expression and increased phosphorylation of P38, JNK, and ERK1/2. Knockdown of IQGAP1 with siRNA prevented AngII-induced apoptosis of podocytes and reduced AngII-induced phosphorylation of ERK1/2, but not that of P38, JNK. This was accompanied by a reduced interaction between ERK1/2 and IQGAP1. Conclusion: IQGAP1 contributes to AngII-induced apoptosis of podocytes by interacting with the ERK1/2 signaling protein.

IQGAP1 is a novel phosphatidylinositol 4,5 bisphosphate effector in regulation of directional cell migration

Phosphatidylinositol 4,5 bisphosphate (PIP₂) is a key lipid messenger for regulation of cell migration. PIP₂ modulates many effectors, but the specificity of PIP₂ signalling can be defined by interactions of PIP₂-generating enzymes with PIP₂ effectors. Here, we show that type Iγ phosphatidylinositol 4-phosphate 5-kinase (PIPKIγ) interacts with the cytoskeleton regulator, IQGAP1, and modulates IQGAP1 function in migration. We reveal that PIPKIγ is required for IQGAP1 recruitment to the leading edge membrane in response to integrin or growth factor receptor activation. Moreover, IQGAP1 is a PIP₂ effector that directly binds PIP₂ through a polybasic motif and PIP₂ binding activates IQGAP1, facilitating actin polymerization. IQGAP1 mutants that lack PIPKIγ or PIP₂ binding lose the ability to control directional cell migration. Collectively, these data reveal a synergy between PIPKIγ and IQGAP1 in the control of cell migration.

The genetic basis of fruit morphology in horticultural crops: lessons from tomato and melon

Fruits represent an important part of the human diet and show extensive variation in size and shape between and within cultivated species. The genetic basis of such variation has been studied most extensively in tomato, where currently six quantitative trait loci (QTLs) involving these traits have been fine-mapped and the genes underlying the QTLs identified. The genes responsible for the cloned QTLs belong to families with a few to many members. FASCIATED is encoded by a member of the YABBY family, CNR/FW2.2 by a member of the Cell Number Regulator family, SlKLUH/FW3.2 by a cytochrome P450 of the 78A class (CYP78A), LOCULE NUMBER by a member of the WOX family including WUSCHEL, OVATE by a member of the Ovate Family Proteins (OFP), and SUN by a member of the IQ domain family. A high portion of the history and current diversity in fruit morphology among tomato cultivars can be explained by modifications at four of these cloned QTLs. In melon, a number of QTLs involved in fruit morphology have been mapped, but the molecular basis for these QTLs is unknown. In the present review, we examine the current knowledge on the molecular basis of fruit morphology in tomato and transfer that information in order to define candidate genes of melon fruit shape and size QTLs. We hypothesize that different members of the gene families identified in tomato may have a role in the regulation of fruit morphology in other species. We anchored the published melon QTL map on the genome sequence and identified the melon family members of the six cloned tomato QTLs in the genome. We investigated the co-localization of melon fruit morphology QTLs and the candidate genes. We found that QTLs for fruit weight co-localized frequently with members of the CNR/FW2.2 and KLUH/FW3.2 families, as well as co-localizations between OFP family members and fruit-shape QTLs, making this family the most suitable to explain fruit shape variation among melon accessions.

The Ras GTPase-activating-like Protein IQGAP1 Mediates Nrf2 Protein Activation via the Mitogen-activated Protein Kinase/Extracellular Signal-regulated Kinase (ERK) Kinase (MEK)-ERK Pathway

Nrf2 plays a critical role in the regulation of cellular oxidative stress. MEK-ERK activation has been shown to be one of the major pathways resulting in the activation of Nrf2 and induction of Nrf2 downstream targets, including phase II detoxifying/antioxidant genes in response to oxidative stress and xenobiotics. In this study, IQGAP1 (IQ motif-containing GTPase-activating protein 1), a new Nrf2 interaction partner that we have published previously, was found to modulate MEK-ERK-mediated Nrf2 activation and induction of phase II detoxifying/antioxidant genes. Nrf2 binds directly to the IQ domain (amino acids 699-905) of IQGAP1. Knockdown of IQGAP1 significantly attenuated phenethyl isothiocyanate- or MEK-mediated activation of the MEK-ERK-Nrf2 pathway. Knockdown of IQGAP1 also attenuated MEK-mediated increased stability of Nrf2, which in turn was associated with a decrease in the nuclear translocation of Nrf2 and a decrease in the expression of phase II detoxifying/antioxidant genes. In the aggregate, these results suggest that IQGAP1 may play an important role in the MEK-ERK-Nrf2 signaling pathway.

Pore directions for the expression of a Ca2+‐activated chloride channel

Pore directions for the expression of a Ca²⁺‐activated chloride channel

Apo States of Calmodulin and CaBP1 Control CaV1 Voltage-Gated Calcium Channel Function through Direct Competition for the IQ Domain

In neurons, binding of calmodulin (CaM) or calcium-binding protein 1 (CaBP1) to the CaV1 (L-type) voltage-gated calcium channel IQ domain endows the channel with diametrically opposed properties. CaM causes calcium-dependent inactivation and limits calcium entry, whereas CaBP1 blocks calcium-dependent inactivation (CDI) and allows sustained calcium influx. Here, we combine isothermal titration calorimetry with cell-based functional measurements and mathematical modeling to show that these calcium sensors behave in a competitive manner that is explained quantitatively by their apo-state binding affinities for the IQ domain. This competition can be completely blocked by covalent tethering of CaM to the channel. Further, we show that Ca2+/CaM has a sub-picomolar affinity for the IQ domain that is achieved without drastic alteration of calcium-binding properties. The observation that the apo forms of CaM and CaBP1 compete with each other demonstrates a simple mechanism for direct modulation of CaV1 function and suggests a means by which excitable cells may dynamically tune CaV activity.

Mind the IQGAP

The scaffold protein IQGAP1 regulates cell signaling through the RAF/MEK/ERK pathway. Recent data show that cancer cells in which the RAF/MEK/ERK pathway is activated are particularly sensitive to the disruption of IQGAP1 function. IQGAP drugs may be particularly effective in tumors that develop resistance to existing pathway drugs.

Competitive and Non-competitive Regulation of Calcium-dependent Inactivation in CaV1.2 L-type Ca2+ Channels by Calmodulin and Ca2+-binding Protein 1

CaV1.2 interacts with the Ca(2+) sensor proteins, calmodulin (CaM) and calcium-binding protein 1 (CaBP1), via multiple, partially overlapping sites in the main subunit of CaV1.2, α1C. Ca(2+)/CaM mediates a negative feedback regulation of Cav1.2 by incoming Ca(2+) ions (Ca(2+)-dependent inactivation (CDI)). CaBP1 eliminates this action of CaM through a poorly understood mechanism. We examined the hypothesis that CaBP1 acts by competing with CaM for common interaction sites in the α1C- subunit using Förster resonance energy transfer (FRET) and recording of Cav1.2 currents in Xenopus oocytes. FRET detected interactions between fluorescently labeled CaM or CaBP1 with the membrane-attached proximal C terminus (pCT) and the N terminus (NT) of α1C. However, mutual overexpression of CaM and CaBP1 proved inadequate to quantitatively assess competition between these proteins for α1C. Therefore, we utilized titrated injection of purified CaM and CaBP1 to analyze their mutual effects. CaM reduced FRET between CaBP1 and pCT, but not NT, suggesting competition between CaBP1 and CaM for pCT only. Titrated injection of CaBP1 and CaM altered the kinetics of CDI, allowing analysis of their opposite regulation of CaV1.2. The CaBP1-induced slowing of CDI was largely eliminated by CaM, corroborating a competition mechanism, but 15-20% of the effect of CaBP1 was CaM-resistant. Both components of CaBP1 action were present in a truncated α1C where N-terminal CaM- and CaBP1-binding sites have been deleted, suggesting that the NT is not essential for the functional effects of CaBP1. We propose that CaBP1 acts via interaction(s) with the pCT and possibly additional sites in α1C.