FRET conformational analysis of calmodulin binding to nitric oxide synthase peptides and enzymes

Calmodulin (CaM) is a ubiquitous Ca (2+)-sensor protein that binds and activates the nitric oxide synthase (NOS) enzymes. We have used fluorescence resonance energy transfer (FRET) to examine the conformational transitions of CaM induced by its binding to synthetic nitric oxide synthase (NOS) CaM-binding domain peptides and full length heme-free constitutive NOS (cNOS) enzymes over a range of physiologically relevant free Ca (2+) concentrations. We demonstrate for the first time that the domains of CaM collapse when associated with Ca (2+)-independent inducible NOS CaM-binding domain, similar to the previously solved crystal structures of CaM bound to the Ca (2+)-dependent cNOS peptides. We show that the association of CaM is not detectable with the cNOS peptides at low free Ca (2+) concentrations (<40 nM). In contrast, we demonstrate that CaM associates with the cNOS holo-enzymes in the absence of Ca (2+) and that the Ca (2+)-dependent transition occurs at a lower free Ca (2+) concentration with the cNOS holo-enzymes. Our results suggest that other regions outside of the CaM-binding domain in the cNOS enzymes are involved in the recruitment and binding of CaM. We also demonstrate that CaM binds to the cNOS enzymes in a sequential manner with the Ca (2+)-replete C-lobe binding first followed by the Ca (2+)-replete N-lobe. This novel FRET study helps to clarify some of the observed similarities and differences between the Ca (2+)-dependent/independent interaction between CaM and the NOS isozymes.     

Binding and activation of nitric oxide synthase isozymes by calmodulin EF hand pairs

Calmodulin (CaM) is a cytosolic Ca(2+) signal-transducing protein that binds and activates many different cellular enzymes with physiological relevance, including the nitric oxide synthase (NOS) isozymes. CaM consists of two globular domains joined by a central linker; each domain contains an EF hand pair. Four different mutant CaM proteins were used to investigate the role of the two CaM EF hand pairs in the binding and activation of the mammalian inducible NOS (iNOS) and the constitutive NOS (cNOS) enzymes, endothelial NOS (eNOS) and neuronal NOS (nNOS). The role of the CaM EF hand pairs in different aspects of NOS enzymatic function was monitored using three assays that monitor electron transfer within a NOS homodimer. Gel filtration studies were used to determine the effect of Ca(2+) on the dimerization of iNOS when coexpressed with CaM and the mutant CaM proteins. Gel mobility shift assays were performed to determine binding stoichiometries of CaM proteins to synthetic NOS CaM-binding domain peptides. Our results show that the N-terminal EF hand pair of CaM contains important binding and activating elements for iNOS, whereas the N-terminal EF hand pair in conjunction with the central linker region is required for cNOS enzyme binding and activation. The iNOS enzyme must be coexpressed with wild-type CaM in vitro because of its propensity to aggregate when residues of the highly hydrophobic CaM-binding domain are exposed to an aqueous environment. A possible role for iNOS aggregation in vivo is also discussed.     

Ca(2+)-dependent and Ca(2+)-independent calmodulin binding sites in erythrocyte protein 4.1. Implications for regulation of protein 4.1 interactions with transmembrane proteins

In vitro protein binding assays identified two distinct calmodulin (CaM) binding sites within the NH(2)-terminal 30-kDa domain of erythrocyte protein 4.1 (4.1R): a Ca(2+)-independent binding site (A(264)KKLWKVCVEHHTFFRL) and a Ca(2+)-dependent binding site (A(181)KKLSMYGVDLHKAKDL). Synthetic peptides corresponding to these sequences bound CaM in vitro; conversely, deletion of these peptides from a 30-kDa construct reduced binding to CaM. Thus, 4.1R is a unique CaM-binding protein in that it has distinct Ca(2+)-dependent and Ca(2+)-independent high affinity CaM binding sites. CaM bound to 4.1R at a stoichiometry of 1:1 both in the presence and absence of Ca(2+), implying that one CaM molecule binds to two distinct sites in the same molecule of 4.1R. Interactions of 4.1R with membrane proteins such as band 3 is regulated by Ca(2+) and CaM. While the intrinsic affinity of the 30-kDa domain for the cytoplasmic tail of erythrocyte membrane band 3 was not altered by elimination of one or both CaM binding sites, the ability of Ca(2+)/CaM to down-regulate 4. 1R-band 3 interaction was abrogated by such deletions. Thus, regulation of protein 4.1 binding to membrane proteins by Ca(2+) and CaM requires binding of CaM to both Ca(2+)-independent and Ca(2+)-dependent sites in protein 4.1.     

Calcineurin B- and calmodulin-binding preferences identified with phage-displayed peptide libraries

Calcineurin B (CnB) and calmodulin (CaM) are two structurally similar but functionally distinct 'EF-hand' Ca2+-binding proteins. CnB is the regulatory subunit of the CaM-stimulated protein phosphatase, calcineurin. CaM is a unique multifunctional protein that interacts with and modulates the activity of many target proteins. CnB and CaM are both required for the full activation of the phosphatase activity of calcineurin and are not interchangeable. The two proteins recognize distinct binding sites on calcineurin A subunit (CnA) and perform different functions. Phage-displayed peptide libraries (pIII and pVIII libraries) were screened with CnB and CaM to isolate peptides that could then be compared to determine if there were binding preferences of the two proteins. The Ca2+-dependent binding of phage-displayed peptides to CnB and CaM is specifically blocked by synthetic peptides derived from the CnB-binding domain of CnA and the CaM-binding domain of myosin light chain kinase respectively. Both CnB- and CaM-binding peptides have a high content of tryptophan and leucine, but CnB-binding peptides are more hydrophobic than CaM-binding peptides. CnB-binding peptides are negatively charged with clusters of hydrophobic residues rich in phenylalanine, whereas the CaM-binding peptides are positively charged and often contain an Arg/Lys-Trp motif. The binding preferences identified with peptide libraries are consistent with the features of the CnB-binding domains of all CnA isoforms and the CaM-binding domains of CaM targets.     

Molecular and biochemical analysis of calmodulin interactions with the calmodulin-binding domain of plant glutamate decarboxylase.

We previously provided what to our knowledge is the first evidence that plant glutamate decarboxylase (GAD) is a calmodulin (CaM)-binding protein. Here, we studied the GAD CaM-binding domain in detail. A synthetic peptide of 26 amino acids corresponding to this domain forms a stable complex with Ca2+/CaM with a 1:1 stoichiometry, and amino acid substitutions suggest that tryptophan-485 has an indispensable role in CaM binding. Chemical cross-linking revealed specific CaM/GAD interactions even in the absence of Ca2+. However, increasing KCI concentrations or deletion of two carboxy-terminal lysines abolished these interactions but had a mild effect on CaM/GAD interactions in the presence of Ca2+. We conclude that in the presence of Ca(2+)-hydrophobic interactions involving tryptophan-485 and electrostatic interactions involving the carboxy-terminal lysines mediate CaM/GAD complex formation. By contrast, in the absence of Ca2+, CaM/GAD interactions are essentially electrostatic and involve the carboxy-terminal lysines. In addition, a tryptophan residue and carboxy-terminal lysines are present in the CaM-binding domain of an Arabidopsis GAD. Finally, we demonstrate that petunia GAD activity is stimulated in vitro by Ca2+/CaM. Our study provides a molecular basis for Ca(2+)-dependent CaM/GAD interactions and suggests the possible occurrence of Ca(2+)-independent CaM/GAD interactions.     

Characterization of a novel plant PP2C-like protein Ser/Thr phosphatase as a calmodulin-binding protein

Protein phosphatases regulated by calmodulin (CaM) mediate the action of intracellular Ca2+ and modulate functions of various target proteins by dephosphorylation. In plants, however, the role of Ca2+ in the regulation of protein dephosphorylation is not well understood due to a lack of information on characteristics of CaM-regulated protein phosphatases. Screening of a cDNA library of the moss Physcomitrella patens by using 35S-labeled calmodulin as a ligand resulted in identification of a gene, PCaMPP, that encodes a protein serine/threonine phosphatase with 373 amino acids. PCaMPP had a catalytic domain with sequence similarity to type 2C protein phosphatases (PP2Cs) with six conserved metal-associating amino acid residues and also had an extra C-terminal domain. Recombinant GST fusion proteins of PCaMPP exhibited Mn2+-dependent phosphatase activity, and the activity was inhibited by pyrophosphate and 1 mm Ca2+ but not by okadaic acid, orthovanadate, or beta-glycerophosphate. Furthermore, the PCaMPP activity was increased 1.7-fold by addition of CaM at nanomolar concentrations. CaM binding assays using deletion proteins and a synthetic peptide revealed that the CaM-binding region resides within the basic amphiphilic amino acid region 324-346 in the C-terminal domain. The CaM-binding region had sequence similarity to amino acids in one of three alpha-helices in the C-terminal domain of human PP2Calpha, suggesting a novel role of the C-terminal domains for the phosphatase activity. These results provide the first evidence showing possible regulation of PP2C-related phosphatases by Ca2+/CaM in plants. Genes similar to PCaMPP were found in genomes of various higher plant species, suggesting that PCaMPP-type protein phosphatases are conserved in land plants.     

Isolation and characterization of a novel calmodulin-binding protein from potato.

Tuberization in potato is controlled by hormonal and environmental signals. Ca(2+), an important intracellular messenger, and calmodulin (CaM), one of the primary Ca(2+) sensors, have been implicated in controlling diverse cellular processes in plants including tuberization. The regulation of cellular processes by CaM involves its interaction with other proteins. To understand the role of Ca(2+)/CaM in tuberization, we have screened an expression library prepared from developing tubers with biotinylated CaM. This screening resulted in isolation of a cDNA encoding a novel CaM-binding protein (potato calmodulin-binding protein (PCBP)). Ca(2+)-dependent binding of the cDNA-encoded protein to CaM is confirmed by (35)S-labeled CaM. The full-length cDNA is 5 kb long and encodes a protein of 1309 amino acids. The deduced amino acid sequence showed significant similarity with a hypothetical protein from another plant, Arabidopsis. However, no homologs of PCBP are found in nonplant systems, suggesting that it is likely to be specific to plants. Using truncated versions of the protein and a synthetic peptide in CaM binding assays we mapped the CaM-binding region to a 20-amino acid stretch (residues 1216-1237). The bacterially expressed protein containing the CaM-binding domain interacted with three CaM isoforms (CaM2, CaM4, and CaM6). PCBP is encoded by a single gene and is expressed differentially in the tissues tested. The expression of CaM, PCBP, and another CaM-binding protein is similar in different tissues and organs. The predicted protein contained seven putative nuclear localization signals and several strong PEST motifs. Fusion of the N-terminal region of the protein containing six of the seven nuclear localization signals to the reporter gene beta-glucuronidase targeted the reporter gene to the nucleus, suggesting a nuclear role for PCBP.     

Arabidopsis ubiquitin-specific protease 6 (AtUBP6) interacts with calmodulin

Calmodulin (CaM), a key Ca(2+) sensor in eukaryotes, regulates diverse cellular processes by interacting with many proteins. To identify Ca(2+)/CaM-mediated signaling components, we screened an Arabidopsis expression library with horseradish peroxidase-conjugated Arabidopsis calmodulin2 (AtCaM2) and isolated a homolog of the UBP6 deubiquitinating enzyme family (AtUBP6) containing a Ca(2+)-dependent CaM-binding domain (CaMBD). The CaM-binding activity of the AtUBP6 CaMBD was confirmed by CaM mobility shift assay, phosphodiesterase competition assay and site-directed mutagenesis. Furthermore, expression of AtUBP6 restored canavanine resistance to the Deltaubp6 yeast mutant. This is the first demonstration that Ca(2+) signaling via CaM is involved in ubiquitin-mediated protein degradation and/or stabilization in plants.     

Regulation of MAPK phosphatase 1 (AtMKP1) by calmodulin in Arabidopsis

The mitogen-activated protein kinases (MAPKs) are key signal transduction molecules, which respond to various external stimuli. The MAPK phosphatases (MKPs) are known to be negative regulators of MAPKs in eukaryotes. We screened an Arabidopsis cDNA library using horseradish peroxidase-conjugated calmodulin (CaM), and isolated AtMKP1 as a CaM-binding protein. Recently, tobacco NtMKP1 and rice OsMKP1, two orthologs of Arabidopsis AtMKP1, were reported to bind CaM via a single putative CaM binding domain (CaMBD). However, little is known about the regulation of phosphatase activity of plant MKP1s by CaM binding. In this study, we identified two Ca(2+)-dependent CaMBDs within AtMKP1. Specific binding of CaM to two different CaMBDs was verified using a gel mobility shift assay, a competition assay with a Ca(2+)/CaM-dependent enzyme, and a split-ubiquitin assay. The peptides for two CaMBDs, CaMBDI and CaMBDII, bound CaM in a Ca(2+)-dependent manner, and the binding affinity of CaMBDII was found to be higher than that of CaMBDI. CaM overlay assays using mutated CaMBDs showed that four amino acids, Trp(453) and Leu(456) in CaMBDI and Trp(678) and Ile(684) in CaMBDII, play a pivotal role in CaM binding. Moreover, the phosphatase activity of AtMKP1 was increased by CaM in a Ca(2+)-dependent manner. Our results suggest that two important signaling pathways, Ca(2+) signaling and the MAPK signaling cascade, are connected in plants via the regulation of AtMKP1 activity. To our knowledge, this is the first report to show that the biochemical activity of MKP1 in plants is regulated by CaM.     

Target-induced conformational adaptation of calmodulin revealed by the crystal structure of a complex with nematode Ca(2+)/calmodulin-dependent kinase kinase peptide.

Calmodulin (CaM) is a ubiquitous calcium (Ca(2+)) sensor which binds and regulates protein serine/threonine kinases along with many other proteins in a Ca(2+)-dependent manner. For this multi-functionality, conformational plasticity is essential; however, the nature and magnitude of CaM's plasticity still remains largely undetermined. Here, we present the 1.8 A resolution crystal structure of Ca(2+)/CaM, complexed with the 27-residue synthetic peptide corresponding to the CaM-binding domain of the nematode Caenorhabditis elegans Ca(2+)/CaM-dependent kinase kinase (CaMKK). The peptide bound in this crystal structure is a homologue of the previously NMR-derived complex with rat CaMKK, but benefits from improved structural resolution. Careful comparison of the present structure to previous crystal structures of CaM complexed with unrelated peptides derived from myosin light chain kinase and CaM kinase II, allow a quantitative analysis of the differences in the relative orientation of the N and C-terminal domains of CaM, defined as a screw axis rotation angle ranging from 156 degrees to 196 degrees. The principal differences in CaM interaction with various peptides are associated with the N-terminal domain of CaM. Unlike the C-terminal domain, which remains unchanged internally, the N-terminal domain of CaM displays significant differences in the EF-hand helix orientation between this and other CaM structures. Three hydrogen bonds between CaM and the peptide (E87-R336, E87-T339 and K75-T339) along with two salt bridges (E11-R349 and E114-K334) are the most probable determinants for the binding direction of the CaMKK peptide to CaM.     

Mlo, a modulator of plant defense and cell death, is a novel calmodulin-binding protein. Isolation and characterization of a rice Mlo homologue

Transient influx of Ca(2+) constitutes an early event in the signaling cascades that trigger plant defense responses. However, the downstream components of defense-associated Ca(2+) signaling are largely unknown. Because Ca(2+) signals are mediated by Ca(2+)-binding proteins, including calmodulin (CaM), identification and characterization of CaM-binding proteins elicited by pathogens should provide insights into the mechanism by which Ca(2+) regulates defense responses. In this study, we isolated a gene encoding rice Mlo (Oryza sativa Mlo; OsMlo) using a protein-protein interaction-based screening of a cDNA expression library constructed from pathogen-elicited rice suspension cells. OsMlo has a molecular mass of 62 kDa and shares 65% sequence identity and scaffold topology with barley Mlo, a heptahelical transmembrane protein known to function as a negative regulator of broad spectrum disease resistance and leaf cell death. By using gel overlay assays, we showed that OsMlo produced in Escherichia coli binds to soybean CaM isoform-1 (SCaM-1) in a Ca(2+)-dependent manner. We located a 20-amino acid CaM-binding domain (CaMBD) in the OsMlo C-terminal cytoplasmic tail that is necessary and sufficient for Ca(2+)-dependent CaM complex formation. Specific binding of the conserved CaMBD to CaM was corroborated by site-directed mutagenesis, a gel mobility shift assay, and a competition assay with a Ca(2+)/CaM-dependent enzyme. Expression of OsMlo was strongly induced by a fungal pathogen and by plant defense signaling molecules. We propose that binding of Ca(2+)-loaded CaM to the C-terminal tail may be a common feature of Mlo proteins.     

Activation of smooth muscle myosin light chain kinase by calmodulin. Role of LYS(30) and GLY(40).

Calmodulin (CaM)-dependent myosin light chain kinase (MLCK) plays a key role in activation of smooth muscle contraction. A soybean isoform of CaM, SCaM-4 (77% identical to human CaM) fails to activate MLCK, whereas SCaM-1 (90.5% identical to human CaM) is as effective as CaM. We exploited this difference to gain insights into the structural requirements in CaM for activation of MLCK. A chimera (domain I of SCaM-4 and domains II-IV of SCaM-1) behaved like SCaM4, and analysis of site-specific mutants of SCaM-1 indicated that K30E and G40D mutations were responsible for the reduction in activation of MLCK. Competition experiments showed that SCaM-4 binds to the CaM-binding site of MLCK with high affinity. Replacement of CaM in skinned smooth muscle by exogenous CaM or SCaM-1, but not SCaM-4, restored Ca(2+)-dependent contraction. K30E/M36I/G40D SCaM-1 was a poor activator of contraction, but site-specific mutants, K30E, M36I and G40D, each restored Ca(2+)-induced contraction to CaM-depleted skinned smooth muscle, consistent with their capacity to activate MLCK. Interpretation of these results in light of the high-resolution structures of (Ca(2+))(4)-CaM, free and complexed with the CaM-binding domain of MLCK, indicates that a surface domain containing Lys(30) and Gly(40) and residues from the C-terminal domain is created upon binding to MLCK, formation of which is required for activation of MLCK. Interactions between this activation domain and a region of MLCK distinct from the known CaM-binding domain are required for removal of the autoinhibitory domain from the active site, i.e., activation of MLCK, or this domain may be required to stabilize the conformation of (Ca(2+))(4)-CaM necessary for MLCK activation.     

Matrin 3 is a Ca2+/calmodulin-binding protein cleaved by caspases

Matrin 3 is a nuclear matrix protein that has been implicated in interacting with other nuclear proteins to anchor hyperedited RNAs to the nuclear matrix, in modulating the activity of proximal promoters, and as the main PKA substrate following NMDA receptor activation. In our proteome-wide selections for calmodulin (CaM) binding proteins and for caspase substrates using mRNA-displayed human proteome libraries, matrin 3 was identified as both a Ca(2+)-dependent CaM-binding protein and a downstream substrate of caspases. We report here, the in vitro characterization of the CaM-binding motif and the caspase cleavage site on matrin 3. Significantly, the Ca(2+)/CaM-binding motif is partially overlapped by the RRM of matrin 3 and is also very close to the bipartite NLS that is essential for its nuclear localization. The caspase cleavage site is downstream of the NLS but upstream of the second U1-like zinc finger. Our results suggest that the functions of matrin 3 could be regulated by both Ca(2+)-dependent interaction with CaM and caspase-mediated cleavage.     

Regulation of the ligand-dependent activation of the epidermal growth factor receptor by calmodulin

Calmodulin (CaM) is the major component of calcium signaling pathways mediating the action of various effectors. Transient increases in the intracellular calcium level triggered by a variety of stimuli lead to the formation of Ca(2+)/CaM complexes, which interact with and activate target proteins. In the present study the role of Ca(2+)/CaM in the regulation of the ligand-dependent activation of the epidermal growth factor receptor (EGFR) has been examined in living cells. We show that addition of different cell permeable CaM antagonists to cultured cells or loading cells with a Ca(2+) chelator inhibited ligand-dependent EGFR auto(trans)phosphorylation. This occurred also in the presence of inhibitors of protein kinase C, CaM-dependent protein kinase II and calcineurin, which are known Ca(2+)- and/or Ca(2+)/CaM-dependent EGFR regulators, pointing to a direct effect of Ca(2+)/CaM on the receptor. Furthermore, we demonstrate that down-regulation of CaM in conditional CaM knock out cells stably transfected with the human EGFR decreased its ligand-dependent phosphorylation. Substitution of six basic amino acid residues within the CaM-binding domain (CaM-BD) of the EGFR by alanine resulted in a decreased phosphorylation of the receptor and of its downstream substrate phospholipase Cγ1. These results support the hypothesis that Ca(2+)/CaM regulates the EGFR activity by directly interacting with the CaM-BD of the receptor located at its cytosolic juxtamembrane region.     

Calcium/calmodulin regulates ubiquitination of the ubiquitin-specific protease TRE17/USP6

The TRE17 (USP6/TRE-2) oncogene induces tumorigenesis in both humans and mice. However, little is known regarding its regulation or mechanism of transformation. TRE17 encodes a TBC (Tre-2/Bub2/Cdc16)/Rab GTPase-activating protein homology domain at its N terminus and a ubiquitin-specific protease at its C terminus. In the current study, we identified the ubiquitous calcium (Ca2+)-binding protein calmodulin (CaM) as a novel binding partner for TRE17. CaM bound directly to TRE17 in a Ca2+-dependent manner both in vitro and in vivo. The CaM-binding site was mapped to two hydrophobic motifs near the C terminus of the TBC domain. Point mutations within these motifs significantly reduced the interaction of TRE17 with CaM. We further found that TRE17 is monoubiquitinated and promotes its own deubiquitination in vivo. CaM binding-deficient mutants of TRE17 exhibited significantly reduced monoubiquitination, suggesting that binding of Ca2+/CaM to TRE17 promotes this modification. Consistent with this notion, treatment of cells with the CaM inhibitor W7 reduced levels of TRE17 monoubiquitination. Interestingly, the calcium ionophore A23187 induced accumulation of a polyubiquitinated TRE17 species. The effect of A23187 was attenuated in CaM binding-deficient mutants of TRE17. Taken together, these studies indicate a role for Ca2+/CaM in regulating ubiquitination through direct interaction with TRE17.     

The solution structure of a plant calmodulin and the CaM-binding domain of the vacuolar calcium-ATPase BCA1 reveals a new binding and activation mechanism

The type IIb class of plant Ca(2+)-ATPases contains a unique N-terminal extension that encompasses a calmodulin (CaM) binding domain and an auto-inhibitory domain. Binding of Ca(2+)-CaM to this region can release auto-inhibition and activates the calcium pump. Using multidimensional NMR spectroscopy, we have determined the solution structure of the complex of a plant CaM isoform with the CaM-binding domain of the well characterized Ca(2+)-ATPase BCA1 from cauliflower. The complex has a rather elongated structure in which the two lobes of CaM do not contact each other. The anchor residues Trp-23 and Ile-40 form a 1-8-18 interaction motif. Binding of Ca(2+)-CaM gives rise to the induction of two helical parts in this unique target peptide. The two helical portions are connected by a highly positively charged bend region, which represents a relatively fixed angle and positions the two lobes of CaM in an orientation that has not been seen before in any complex structure of calmodulin. The behavior of the complex was further characterized by heteronuclear NMR dynamics measurements of the isotope-labeled protein and peptide. These data suggest a unique calcium-driven activation mechanism for BCA1 and other plant Ca(2+)-ATPases that may also explain the action of calcium-CaM on some other target enzymes. Moreover, CaM activation of plant Ca(2+)-ATPases seems to occur in an organelle-specific manner.     

Identification of the calmodulin binding domain of connexin 43

Calmodulin (CaM) has been implicated in mediating the Ca(2+)-dependent regulation of gap junctions. This report identifies a CaM-binding motif comprising residues 136-158 in the intracellular loop of Cx43. A 23-mer peptide encompassing this CaM-binding motif was shown to bind Ca(2+)-CaM with 1:1 stoichiometry by using various biophysical approaches, including surface plasmon resonance, circular dichroism, fluorescence spectroscopy, and NMR. Far UV circular dichroism studies indicated that the Cx43-derived peptide increased its alpha-helical contents on CaM binding. Fluorescence and NMR studies revealed conformational changes of both the peptide and CaM following formation of the CaM-peptide complex. The apparent dissociation constant of the peptide binding to CaM in physiologic K(+) is in the range of 0.7-1 microM. Upon binding of the peptide to CaM, the apparent K(d) of Ca(2+) for CaM decreased from 2.9 +/- 0.1 to 1.6 +/- 0.1 microM, and the Hill coefficient n(H) increased from 2.1 +/- 0.1 to 3.3 +/- 0.5. Transient expression in HeLa cells of two different mutant Cx43-EYFP constructs without the putative Cx43 CaM-binding site eliminated the Ca(2+)-dependent inhibition of Cx43 gap junction permeability, confirming that residues 136-158 in the intracellular loop of Cx43 contain the CaM-binding site that mediates the Ca(2+)-dependent regulation of Cx43 gap junctions. Our results provide the first direct evidence that CaM binds to a specific region of the ubiquitous gap junction protein Cx43 in a Ca(2+)-dependent manner, providing a molecular basis for the well characterized Ca(2+)-dependent inhibition of Cx43-containing gap junctions.