Saturated Calmodulin Research Articles

Chemical shift assignments of calmodulin bound to a cytosolic domain of GluN2A (residues 1004-1024) from the NMDA receptor.

N-methyl-D-aspartate receptors (NMDARs) consist of glycine-binding GluN1 and glutamate-binding GluN2 subunits that form tetrameric ion channels. NMDARs in the neuronal post-synaptic membrane are important for controlling neuroplasticity and synaptic transmission in the brain. Calmodulin (CaM) binds to the cytosolic C0 domains of both GluN1 (residues 841-865) and GluN2 (residues 1004-1024) that may play a role in the Ca2+-dependent desensitization of NMDAR channels. Mutations that disrupt Ca2+-dependent desensitization of NMDARs are linked to Alzheimer's disease, depression, stroke, epilepsy, and schizophrenia. NMR chemical shift assignments are reported here for Ca2+-saturated CaM bound to the GluN2A C0 domain of NMDAR (BMRB no. 51821).

Chemical shift assignments of calmodulin bound to the GluN1 C0 domain (residues 841–865) of the NMDA receptor

Neuroplasticity and synaptic transmission in the brain are regulated by N-methyl-D-aspartate receptors (NMDARs) that consist of hetero-tetrameric combinations of the glycine-binding GluN1 and glutamate-binding GluN2 subunits. Calmodulin (CaM) binds to the cytosolic C0 domain of GluN1 (residues 841–865) that may play a role in the Ca2+-dependent inactivation (CDI) of NMDAR channel activity. Dysregulation of NMDARs are linked to various neurological disorders, including Alzheimer’s disease, depression, stroke, epilepsy, and schizophrenia. Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the GluN1 C0 domain of the human NMDAR (BMRB no. 51715).

Half-calcified calmodulin promotes basal activity and inactivation of the L-type calcium channel CaV1.2

The L-type Ca2+ channel CaV1.2 controls gene expression, cardiac contraction, and neuronal activity. Calmodulin (CaM) governs CaV1.2 open probability (Po) and Ca2+-dependent inactivation (CDI) but the mechanisms remain unclear. Here, we present electrophysiological data that identify a half Ca2+-saturated CaM species (Ca2/CaM) with Ca2+ bound solely at the third and fourth EF-hands (EF3 and EF4) under resting Ca2+ concentrations (50-100nM) that constitutively preassociates with CaV1.2 to promote Po and CDI. We also present an NMR structure of a complex between the CaV1.2 IQ motif (residues 1644-1665) and Ca2/CaM12', a calmodulin mutant in which Ca2+ binding to EF1 and EF2 is completely disabled. We found that the CaM12' N-lobe does not interact with the IQ motif. The CaM12' C-lobe bound two Ca2+ ions and formed close contacts with IQ residues I1654 and Y1657. I1654A and Y1657D mutations impaired CaM binding, CDI, and Po, as did disabling Ca2+ binding to EF3 and EF4 in the CaM34 mutant when compared to WT CaM. Accordingly, a previously unappreciated Ca2/CaM species promotes CaV1.2 Po and CDI, identifying Ca2/CaM as an important mediator of Ca signaling.

Calmodulin promotes a Ca2+ -dependent conformational change in the C-terminal regulatory domain of CaV 1.2.

Calmodulin (CaM) binds to the membrane-proximal cytosolic C-terminal domain of CaV 1.2 (residues 1520-1669, CT(1520-1669)) and causes Ca2+ -induced conformational changes that promote Ca2+ -dependent channel inactivation (CDI). We report biophysical studies that probe the structural interaction between CT(1520-1669) and CaM. The recombinantly expressed CT(1520-1669) is insoluble, but can be solubilized in the presence of Ca2+ -saturated CaM (Ca4 /CaM), but not in the presence of Ca2+ -free CaM (apoCaM). We show that half-calcified CaM (Ca2 /CaM12 ) forms a complex with CT(1520-1669) that is less soluble than CT(1520-1669) bound to Ca4 /CaM. The NMR spectrum of CT(1520-1669) reveals spectral differences caused by the binding of Ca2 /CaM12 versus Ca4 /CaM, suggesting that the binding of Ca2+ to the CaM N-lobe may induce a conformational change in CT(1520-1669).

NMR Structures of Calmodulin Bound to Two Separate Regulatory Sites in the Retinal Cyclic Nucleotide-Gated Channel.

Retinal cyclic nucleotide-gated (CNG) channels (composed of three CNGA1 and one CNGB1 subunits) exhibit a Ca2+-induced reduction in channel open probability mediated by calmodulin (CaM). Defects in the Ca2+-dependent regulation of CNG channels may be linked to autosomal recessive retinitis pigmentosa and other inherited forms of blindness. Here, we report the NMR structure and binding analysis of CaM bound to two separate sites within CNGB1 (CaM1: residues 565-589 and CaM2: residues 1120-1147). Our binding studies reveal that CaM1 binds to the Ca2+-bound CaM N-lobe with at least fivefold higher affinity than it binds to the CaM C-lobe. By contrast, the CaM2 site binds to the Ca2+-bound CaM C-lobe with higher affinity than it binds to the N-lobe. CaM1 and CaM2 both exhibited very weak binding to Ca2+-free CaM. We present separate NMR structures of Ca2+-saturated CaM bound to CaM1 and CaM2 that define key intermolecular contacts: CaM1 residue F575 interacts with the CaM N-lobe while CaM2 residues L1129, L1132, and L1136 each make close contact with the CaM C-lobe. The CNGB1 mutation F575E abolishes CaM1 binding to the CaM N-lobe while L1132E and L1136E each abolish CaM2 binding to the CaM C-lobe. Thus, a single CaM can bind to both sites in CNGB1 in which the CaM N-lobe binds to CaM1 and the CaM C-lobe binds to CaM2. We propose a Ca2+-dependent conformational switch in the CNG channel caused by CaM binding, which may serve to attenuate cGMP binding to CNG channels at high cytosolic Ca2+ levels in dark-adapted photoreceptors.

Chemical shift assignments of calmodulin bound to a C-terminal site (residues 1120–1147) in the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

Retinal cyclic nucleotide-gated (CNG) channels consist of two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to two separate sites within the cytosolic region of CNGB1: CaM binding to an N-terminal site (human CNGB1 residues 565–587, called CaM1) decreases the open probability of CNG channels at elevated Ca2+ levels in dark-adapted photoreceptors, whereas CaM binding to a separate C-terminal site (CNGB1 residues 1120–1147, called CaM2) may increase channel open probability in light activated photoreceptors. We recently reported NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM1 site of CNGB1 (BMRB no. 51222). Here, we report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the C-terminal CaM2 site of CNGB1 (BMRB no. 51447).

Chemical shift assignments of calmodulin under standard conditions at neutral pH

The Ca2+ sensor protein, calmodulin (CaM) is ubiquitously expressed in all cells where it binds to hundreds of different target proteins, including dozens of enzymes, receptors, ion channels and numerous Ca2+ transporters. The only published NMR chemical shift assignments for Ca2+-bound CaM (in the absence of a target) have been determined under acidic conditions: at pH 6.5/310 K (BMRB 6541) and pH 6.3/320 K (BMRB 547). However, some CaM/target complexes are not soluble under these conditions. Also, amide chemical shifts are very sensitive to pH and temperature, which can cause large baseline errors when using the existing chemical shift assignments of free CaM to calculate chemical shift perturbations caused by target binding at neutral pH and physiological temperature. We report complete NMR chemical shift assignments of Ca2+-saturated CaM under a set of standard conditions at neutral pH and 308 K that will enable more accurate chemical shift comparison between free CaM and CaM/target complexes (BMRB 51289).

Chemical shift assignments of calmodulin bound to the β-subunit of a retinal cyclic nucleotide-gated channel (CNGB1)

Rod cyclic nucleotide-gated (CNG) channels are formed by two protein subunits (CNGA1 and CNGB1). Calmodulin (CaM) binds to the cytosolic regulatory domain of CNGB1 and decreases the open probability of CNGA1/CNGB1 channels. The CaM binding site within bovine CNGB1 (residues 679–702) binds tightly to Ca2+-bound CaM, which promotes Ca2+-induced inactivation of CNGA1/CNGB1 channels in retinal rods. We report complete NMR chemical shift assignments of Ca2+-saturated CaM bound to the CaM-binding domain of CNGB1 (BMRB no. 51222).

Ca2+-saturated calmodulin binds tightly to the N-terminal domain of A-type fibroblast growth factor homologous factors

Voltage-gated sodium channels (Navs) are tightly regulated by multiple conserved auxiliary proteins, including the four fibroblast growth factor homologous factors (FGFs), which bind the Nav EF-hand like domain (EFL), and calmodulin (CaM), a multifunctional messenger protein that binds the NaV IQ motif. The EFL domain and IQ motif are contiguous regions of NaV cytosolic C-terminal domains (CTD), placing CaM and FGF in close proximity. However, whether the FGFs and CaM act independently, directly associate, or operate through allosteric interactions to regulate channel function is unknown. Titrations monitored by steady-state fluorescence spectroscopy, structural studies with solution NMR, and computational modeling demonstrated for the first time that both domains of (Ca2+)4-CaM (but not apo CaM) directly bind two sites in the N-terminal domain (NTD) of A-type FGF splice variants (FGF11A, FGF12A, FGF13A, and FGF14A) with high affinity. The weaker of the (Ca2+)4-CaM-binding sites was known via electrophysiology to have a role in long-term inactivation of the channel but not known to bind CaM. FGF12A binding to a complex of CaM associated with a fragment of the NaV1.2 CTD increased the Ca2+-binding affinity of both CaM domains, consistent with (Ca2+)4-CaM interacting preferentially with its higher-affinity site in the FGF12A NTD. Thus, A-type FGFs can compete with NaV IQ motifs for (Ca2+)4-CaM. During spikes in the cytosolic Ca2+ concentration that accompany an action potential, CaM may translocate from the NaV IQ motif to the FGF NTD, or the A-type FGF NTD may recruit a second molecule of CaM to the channel.

Met872 is the key residue determining the novel binominal binding of metabotropic glutamate receptor 7 to calmodulin

Two mGluR7-derived peptides corresponding to residues 856 to 879 and 856 to 875 are known to bind to Ca2+-saturated calmodulin (Ca2+/CaM), and their binding manners are thought to differ. Met872 function is believed as one of the anchor residues for CaM-binding only in the shorter peptide. To uncover the role of Met872 in CaM-binding, we prepared a mutant of the long peptide, mGluR7 (M872A), in which Met872 was replaced with Ala. We used the mutant together with the two peptides to perform NMR-titration experiments to monitor interaction with stable isotope-labeled CaM. Interaction of Ca2+/CaM with mGluR7 (M872A) caused a spectrum that differed from that of Ca2+/CaM with the long peptide, suggesting that Met872 of mGluR7 could be involved in CaM-binding even in the long peptide. Analyses of all NMR data suggested that the binding between Ca2+/CaM and mGluR7 occurs in some conformational equilibrium manner. The unique CaM-binding properties caused by Met872 may be related to mGluR7’s function.

Inhibitor and peptide binding to calmodulin characterized by high pressure Fourier transform infrared spectroscopy and Förster resonance energy transfer

We compare the binding of an inhibitor with that of a natural peptide to Ca2+ saturated calmodulin (holo-CaM). As inhibitor we have chosen trifluoperazine (TFP) that is inducing a huge conformational change of holo-CaM from the open dumbbell-shaped to the closed globular conformation upon binding. On the other hand, melittin is used as model peptide, which is a well-known natural binding partner of holo-CaM. The experiments are carried out as a function of pressure to reveal the contribution of volume or packing effects to the stability of the calmodulin-ligand complexes. From high-pressure Fourier transform infrared (FTIR) spectroscopy, we find that the holo-CaM/TFP complex has a much higher pressure stability than the holo-CaM/melittin complex. Although the analysis of the secondary structure of holo-CaM (without and with ligand) indicates no major changes up to several kbar, pressure-induced exposure of α-helices to water is most pronounced for holo-CaM without ligand, followed by holo-CaM/melittin and then holo-CaM/TFP. Moreover, structural pressure resistance of the holo-CaM/TFP complex in comparison with the holo-CaM/melittin complex is also clearly visible by higher Ca2+ affinity. Förster resonance energy transfer (FRET) from the Tyr residues of holo-CaM to the Trp residue of melittin even suggests some partial dissociation of the complex under pressure which points to void volumes at the protein-ligand interface and to electrostatic binding. Thus, all results of this study show that the inhibitor TFP binds to holo-CaM with higher packing density than the peptide melittin enabling a favorable volume contribution to the inhibitor efficiency.

Charged Side Chain Mutations of CaMKII Peptide Alter Binding Affinity for CaM through Creation of Non-local Alternate Binding Contacts

Calmodulin (CaM) trapping is a phenomenon where the active and inactive states of calmodulin dependent protein kinase II (CaMKII) produce drastically different affinities for Ca2+ saturated CaM. The states of CaMKII are paramount in the scheme of calcium ion signaling, which is an essential biological function whose underlying mechanism is largely unknown. Experimentally, a set of peptides modeled after CaMKII's binding domain (293-312) were created through systematic mutations of charged residues to mimic the two distinct affinity states for CaM and probe the mechanisms responsible for the observed change in kinetics. Although a model was successfully created, the observed interactions could not be explained through current protein interaction models or electrostatic steering effects. We investigate the dynamics of this experiment through the use of all atom simulations, choosing three of the mutant peptides with a length of 20 amino acids. We refer to these peptides by the 296-298 residues, specifically RRK (wildtype), RAK (1-residue mutation) and AAA (3-residue mutation), and validate our simulation findings through comparison with circular dichroism (CD) data. We demonstrate that the large side chains present in the first 6 residues of each peptide interact with each other, and that the mutation of charged residues produce global changes in sidechain conformations and disordered regions. The specific ensemble of secondary structure/rotamer conformations present in RRK, that are not observed in AAA, are essential for high affinity binding between CaMKII and CaM.

CaM/BAG5/Hsc70 signaling complex dynamically regulates leaf senescence.

Calcium signaling plays an essential role in plant cell physiology, and chaperone-mediated protein folding directly regulates plant programmed cell death. The Arabidopsis thaliana protein AtBAG5 (Bcl-2-associated athanogene 5) is unique in that it contains both a BAG domain capable of binding Hsc70 (Heat shock cognate protein 70) and a characteristic IQ motif that is specific for Ca2+-free CaM (Calmodulin) binding and hence acts as a hub linking calcium signaling and the chaperone system. Here, we determined crystal structures of AtBAG5 alone and in complex with Ca2+-free CaM. Structural and biochemical studies revealed that Ca2+-free CaM and Hsc70 bind AtBAG5 independently, whereas Ca2+-saturated CaM and Hsc70 bind AtBAG5 with negative cooperativity. Further in vivo studies confirmed that AtBAG5 localizes to mitochondria and that its overexpression leads to leaf senescence symptoms including decreased chlorophyll retention and massive ROS production in dark-induced plants. Mutants interfering the CaM/AtBAG5/Hsc70 complex formation leads to different phenotype of leaf senescence. Collectively, we propose that the CaM/AtBAG5/Hsc70 signaling complex plays an important role in regulating plant senescence.

Calmodulin: a new partner for the α1A‐adrenergic receptor

In addition to its role in vasoconstriction, the α1A‐adrenergic receptor (α1A‐AR) is increasingly recognized as a rescue mechanism under circumstances where b‐ARs are insufficient to provide stimulus in the myocardium, such as in heart failure. Mechanisms controlling α1A‐AR function therefore have profound impact on cardiovascular functions. Calmodulin (CaM) is the ubiquitous transducer of Ca2+ signals, and has recently been demonstrated to interact with a number of G protein‐coupled receptors. We have observed that in fresh ventricular tissues, α1A‐AR forms a complex with calmodulin in resting condition as well as under α1A‐AR agonism. To identify and characterize the precise interaction domains between CaM and α1A‐AR, we developed novel biosensors that span multiple fragments of the four sub‐membrane domains (SMDs) of α1A‐AR. Responses of these biosensors to Ca2+‐saturated CaM reveals that α1A‐AR directly interacts with CaM at a number of locations with disparate affinities and Ca2+ sensitivities. These data implicate CaM as a novel partner for α1A‐AR and provide background for on‐going studies on the functional impact of these interactions in the control of cardiovascular functions.

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.

Capping of the N-terminus of PSD-95 by calmodulin triggers its postsynaptic release

Postsynaptic density protein-95 (PSD-95) is a central element of the postsynaptic architecture of glutamatergic synapses. PSD-95 mediates postsynaptic localization of AMPA receptors and NMDA receptors and plays an important role in synaptic plasticity. PSD-95 is released from postsynaptic membranes in response to Ca(2+) influx via NMDA receptors. Here, we show that Ca(2+)/calmodulin (CaM) binds at the N-terminus of PSD-95. Our NMR structure reveals that both lobes of CaM collapse onto a helical structure of PSD-95 formed at its N-terminus (residues 1-16). This N-terminal capping of PSD-95 by CaM blocks palmitoylation of C3 and C5, which is required for postsynaptic PSD-95 targeting and the binding of CDKL5, a kinase important for synapse stability. CaM forms extensive hydrophobic contacts with Y12 of PSD-95. The PSD-95 mutant Y12E strongly impairs binding to CaM and Ca(2+)-induced release of PSD-95 from the postsynaptic membrane in dendritic spines. Our data indicate that CaM binding to PSD-95 serves to block palmitoylation of PSD-95, which in turn promotes Ca(2+)-induced dissociation of PSD-95 from the postsynaptic membrane.

Biosensor‐based identification and characterization of three distinct calmodulin‐binding domains in the human angiotensin II receptor type 1A (LB569)

The angiotensin II receptor type 1A (AT1R) is responsible for many effects of angiotensin II (AngII). Calmodulin (CaM) is essential for many cell functions due to its interactions with many proteins. We tested the idea that signaling via AT1R involves CaM at the receptor level via multiple binding domains. AT1R coimmunoprecipitates with CaM in vascular smooth muscle under resting conditions, association that is enhanced by stimulation with AngII or thapsigargin. To identify all CaM‐binding domains in AT1R and characterize their binding properties, we generated FRET biosensors in which each the 4 sub‐membrane domains (SMDs) in AT1R is flanked by enhanced variants of yellow and cyan fluorescence protein. We named these biosensors BSAT1Rx, with x denoting the amino acid numbering of the SMD sequence. In response to purified Ca2+‐saturated CaM, BSAT1R125‐141, BSAT1R215‐242 and BSAT1R309‐327, corresponding to SMD2, SMD3 and the juxta membranous segment SMD4 in human AT1R, display drastic responses that are characteristic of conformational changes caused by direct binding of CaM. BSAT1R53‐64, corresponding to SMD1, does not response to Ca2+‐CaM. Titrations of biosensor responses with purified CaM yielded Kd values of 44.7±1.00, 0.36± 0.05, and 0.51±0.01 microM, respectively for SMD2, 3, and the juxta‐membranous segment of SMD4. The respective EC50(Ca2+) values of these interactions were 4.1±0.11, 0.13 ± 0.006, and 1.26 ± 0.09 microM, determined by monitoring concurrent responses of the respective BSAT1Rx and a suitable Ca2+ indicator. The entire SMD3 is involved in interaction with CaM, as a truncated domain (a.a. 215‐232) displays substantial decreases in affinity and Ca2+ sensitivity. These data demonstrate that CaM is involved in AngII signaling via direct interactions with multiple domains in AT1R and allow prediction of interactions between CaM and individual SMDs under distinct physiological scenarios, including resting and stimulated conditions.Grant Funding Source: National Institutes of Health

Phosphatidylinositol-4,5 bisphosphate (PIP2) inhibits apo-calmodulin binding to protein 4.1

Membrane skeletal protein 4.1R80 plays a key role in regulation of erythrocyte plasticity. Protein 4.1R80 interactions with transmembrane proteins, such as glycophorin C (GPC), are regulated by Ca2+-saturated calmodulin (Ca2+/CaM) through simultaneous binding to a short peptide (pep11; A264KKLWKVCVEHHTFFRL) and a serine residue (Ser185), both located in the N-terminal 30kDa FERM domain of 4.1R80 (H·R30). We have previously demonstrated that CaM binding to H·R30 is Ca2+-independent and that CaM binding to H·R30 is responsible for the maintenance of H·R30 β-sheet structure. However, the mechanisms responsible for the regulation of CaM binding to H·R30 are still unknown. To investigate this, we took advantage of similarities and differences in the structure of Coracle, the Drosophila sp. homologue of human 4.1R80, i.e. conservation of the pep11 sequence but substitution of the Ser185 residue with an alanine residue. We show that the H·R30 homologue domain of Coracle, Cor30, also binds to CaM in a Ca2+-independent manner and that the Ca2+/CaM complex does not affect Cor30 binding to the transmembrane protein GPC. We also document that both H·R30 and Cor30 bind to phosphatidylinositol-4,5 bisphosphate (PIP2) and other phospholipid species and that that PIP2 inhibits Ca2+-free CaM but not Ca2+-saturated CaM binding to Cor30. We conclude that PIP2 may play an important role as a modulator of apo-CaM binding to 4.1R80 throughout evolution.

4.1 Proteins: Ion Transporters in Check

The classical function of 4.1R in erythrocytes is to contribute to the mechanical properties of the membrane by promoting the interaction between spectrin and actin. It is now well recognized that 4.1R is required for the stable anchorage of numerous cell surface erythrocyte membrane proteins. 4.1R is the prototypical member of the family of 4.1 proteins, which are expressed in many cell types, besides erythrocytes. The other members of the protein 4.1 family include 4.1N, 4.1G, and 4.1B.NHE1 (Na+/H+ exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes, supporting a functional interaction between NHE1 and 4.1R. We recently demonstrated that 4.1R binds directly to the cytoplasmic domain of NHE1 (NHE1cd). This interaction involves an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain and two clusters of basic amino acids in the NHE1cd, K519R and R556FNKKYVKK, previously shown to mediate PIP2 (phosphatidylinositol 4,5-bisphosphate) binding. The affinity of this interaction is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of electrostatic nature. The binding affinity is also reduced upon binding of Ca2+/CaM (Ca2+-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity, through a direct protein-protein interaction that can be modulated by intracellular pH, as well as Na+ and Ca2+ concentrations. In this review, we discuss the increasing evidence for an important role for members of the protein 4.1 family of membrane skeletal proteins, in the regulation of various ion transporters in erythrocytes and in non-erythroid cells.

Characterization of cytoskeletal protein 4.1R interaction with NHE1 (Na+/H+ exchanger isoform 1)

NHE1 (Na(+)/H(+) exchanger isoform 1) has been reported to be hyperactive in 4.1R-null erythrocytes [Rivera, De Franceschi, Peters, Gascard, Mohandas and Brugnara (2006) Am. J. Physiol. Cell Physiol. 291, C880-C886], supporting a functional interaction between NHE1 and 4.1R. In the present paper we demonstrate that 4.1R binds directly to the NHE1cd (cytoplasmic domain of NHE1) through the interaction of an EED motif in the 4.1R FERM (4.1/ezrin/radixin/moesin) domain with two clusters of basic amino acids in the NHE1cd, K(519)R and R(556)FNKKYVKK, previously shown to mediate PIP(2) (phosphatidylinositol 4,5-bisphosphate) binding [Aharonovitz, Zaun, Balla, York, Orlowski and Grinstein (2000) J. Cell. Biol. 150, 213-224]. The affinity of this interaction (K(d) = 100-200 nM) is reduced in hypertonic and acidic conditions, demonstrating that this interaction is of an electrostatic nature. The binding affinity is also reduced upon binding of Ca(2+)/CaM (Ca(2+)-saturated calmodulin) to the 4.1R FERM domain. We propose that 4.1R regulates NHE1 activity through a direct protein-protein interaction that can be modulated by intracellular pH and Na(+) and Ca(2+) concentrations.