EF hands at the N-lobe of calmodulin are required for both SK channel gating and stable SK–calmodulin interaction

Small conductance calcium-activated potassium (SK) channels respond to intracellular Ca²⁺ via constitutively associated calmodulin (CaM). Previous studies have proposed a modular design for the interaction between CaM and SK channels. The C-lobe and the linker of CaM are thought to regulate the constitutive binding, whereas the N-lobe binds Ca²⁺ and gates SK channels. However, we found that coexpression of mutant CaM (E/Q) where the N-lobe has only one functional EF hand leads to rapid rundown of SK channel activity, which can be recovered with exogenously applied wild-type (WT), but not mutant, CaM. Our results suggest that the mutation at the N-lobe EF hand disrupts the stable interaction between CaM and SK channel subunits, such that mutant CaM dissociates from the channel complex when the inside of the membrane is exposed to CaM-free solution. The disruption of the stable interaction does not directly result from the loss of Ca²⁺-binding capacity because SK channels and WT CaM can stably interact in the absence of Ca²⁺. These findings question a previous conclusion that CaM where the N-lobe has only one functional EF hand can stably support the gating of SK channels. They cannot be explained by the current model of modular interaction between CaM and SK channels, and they imply a role for N-lobe EF hand residues in binding to the channel subunits. Additionally, we found that a potent enhancer for SK channels, 3-oxime-6,7-dichloro-1H-indole-2,3-dione (NS309), enables the recovery of channel activity with CaM (E/Q), suggesting that NS309 stabilizes the interaction between CaM and SK channels. CaM (E/Q) can regulate Ca²⁺-dependent gating of SK channels in the presence of NS309, but with a lower apparent Ca²⁺ affinity than WT CaM.     

Decalmodulation of Cav1 channels by CaBPs

Ca²⁺-dependent inactivation (CDI) is a negative feedback regulation of voltage-gated Ca_v1 and Ca_v2 channels that is mediated by the Ca²⁺ sensing protein, calmodulin (CaM), binding to the pore-forming Ca_v α₁ subunit. David Yue and his colleagues made seminal contributions to our understanding of this process, as well as factors that regulate CDI. Important in this regard are members of a family of Ca²⁺ binding proteins (CaBPs) that are related to calmodulin. CaBPs are expressed mainly in neural tissues and can antagonize CaM-dependent CDI for Ca_v1 L-type channels. This review will focus on the roles of CaBPs as Ca_v1-interacting proteins, and the significance of these interactions for vision, hearing, and neuronal Ca²⁺ signaling events.     

Activity of the yeast vacuolar TRP channel TRPY1 is inhibited by Ca2+–calmodulin binding

Transient receptor potential (TRP) cation channels, which are conserved across mammals, flies, fish, sea squirts, worms, and fungi, essentially contribute to cellular Ca²⁺ signaling. The activity of the unique TRP channel in yeast, TRP yeast channel 1 (TRPY1), relies on the vacuolar and cytoplasmic Ca²⁺ concentration. However, the mechanism(s) of Ca²⁺-dependent regulation of TRPY1 and possible contribution(s) of Ca²⁺-binding proteins are yet not well understood. Our results demonstrate a Ca²⁺-dependent binding of yeast calmodulin (CaM) to TRPY1. TRPY1 activity was increased in the cmd1–6 yeast strain, carrying a non–Ca²⁺-binding CaM mutant, compared with the parent strain expressing wt CaM (Cmd1). Expression of Cmd1 in cmd1–6 yeast rescued the wt phenotype. In addition, in human embryonic kidney 293 cells, hypertonic shock-induced TRPY1-dependent Ca²⁺ influx and Ca²⁺ release were increased by the CaM antagonist ophiobolin A. We found that coexpression of mammalian CaM impeded the activity of TRPY1 by reinforcing effects of endogenous CaM. Finally, inhibition of TRPY1 by Ca²⁺–CaM required the cytoplasmic amino acid stretch E₃₃–Y₉₂. In summary, our results show that TRPY1 is under inhibitory control of Ca²⁺–CaM and that mammalian CaM can replace yeast CaM for this inhibition. These findings add TRPY1 to the innumerable cellular proteins, which include a variety of ion channels, that use CaM as a constitutive or dissociable Ca²⁺-sensing subunit, and contribute to a better understanding of the modulatory mechanisms of Ca²⁺–CaM.     

Calcium-dependent stoichiometries of the KCa2.2 (SK) intracellular domain/calmodulin complex in solution

Ca²⁺ activates SK Ca²⁺-activated K⁺ channels through the protein Ca²⁺ sensor, calmodulin (CaM). To understand how SK channels operate, it is necessary to determine how Ca²⁺ regulates CaM binding to its target on SK. Tagless, recombinant SK peptide (SKp), was purified for binding studies with CaM at low and high Ca²⁺ concentrations. Composition gradient multi-angle light scattering accurately measures the molar mass, stoichiometry, and affinity of protein complexes. In 2 mM Ca²⁺, SKp and CaM bind with three different stoichiometries that depend on the molar ratio of SKp:CaM in solution. These complexes include 28 kD 1SKp/1CaM, 39 kD 2SKp/1CaM, and 44 kD 1SKp/2CaM. A 2SKp/2CaM complex, observed in prior crystallographic studies, is absent. At <5 nM Ca²⁺, 1SKp/1CaM and 2SKp/1CaM were observed; however, 1SKp/2CaM was absent. Analytical ultracentrifugation was used to characterize the physical properties of the three SKp/CaM stoichiometries. In high Ca²⁺, the sedimentation coefficient is smaller for a 1SKp:1CaM solution than it is for either 2SKp:1CaM or 1SKp:2CaM. At low Ca²⁺ and at >100 µM protein concentrations, a molar excess of SKp over CaM causes aggregation. Aggregation is not observed in Ca²⁺ or with CaM in molar excess. In low Ca²⁺ both 1SKp:1CaM and 1SKp:2CaM solutions have similar sedimentation coefficients, which is consistent with the absence of a 1SKp/2CaM complex in low Ca²⁺. These results suggest that complexes with stoichiometries other than 2SKp/2CaM are important in gating.     

Ca-dependent binding of calcium-binding protein 1 to presynaptic group III metabotropic glutamate receptors and blockage by phosphorylation of the receptors

Presynaptic group III metabotropic glutamate receptors (mGluRs) and Ca²⁺ channels are the main neuronal activity-dependent regulators of synaptic vesicle release, and they use common molecules in their signaling cascades. Among these, calmodulin (CaM) and the related EF-hand Ca²⁺-binding proteins are of particular importance as sensors of presynaptic Ca²⁺, and a multiple of them are indeed utilized in the signaling of Ca²⁺ channels. However, despite its conserved structure, CaM is the only known EF-hand Ca²⁺-binding protein for signaling by presynaptic group III mGluRs. Because the mGluRs and Ca²⁺ channels reciprocally regulate each other and functionally converge on the regulation of synaptic vesicle release, the mGluRs would be expected to utilize more EF-hand Ca²⁺-binding proteins in their signaling. Here I show that calcium-binding protein 1 (CaBP1) bound to presynaptic group III mGluRs competitively with CaM in a Ca²⁺-dependent manner and that this binding was blocked by protein kinase C (PKC)-mediated phosphorylation of these receptors. As previously shown for CaM, these results indicate the importance of CaBP1 in signal cross talk at presynaptic group III mGluRs, which includes many molecules such as cAMP, Ca²⁺, PKC, G protein, and Munc18-1. However, because the functional diversity of EF-hand calcium-binding proteins is extraordinary, as exemplified by the regulation of Ca²⁺ channels, CaBP1 would provide a distinct way by which presynaptic group III mGluRs fine-tune synaptic transmission.     

Experimental and computational approaches for the study of calmodulin interactions

Ca²⁺, a universal messenger in eukaryotes, plays a major role in signaling pathways that control many growth and developmental processes in plants as well as their responses to various biotic and abiotic stresses. Cellular changes in Ca²⁺ in response to diverse signals are recognized by protein sensors that either have their activity modulated or that interact with other proteins and modulate their activity. Calmodulins (CaMs) and CaM-like proteins (CMLs) are Ca²⁺ sensors that have no enzymatic activity of their own but upon binding Ca²⁺ interact and modulate the activity of other proteins involved in a large number of plant processes. Protein-protein interactions play a key role in Ca²⁺/CaM-mediated in signaling pathways. In this review, using CaM as an example, we discuss various experimental approaches and computational tools to identify protein-protein interactions. During the last two decades hundreds of CaM-binding proteins in plants have been identified using a variety of approaches ranging from simple screening of expression libraries with labeled CaM to high-throughput screens using protein chips. However, the high-throughput methods have not been applied to the entire proteome of any plant system. Nevertheless, the data provided by these screens allows the development of computational tools to predict CaM-interacting proteins. Using all known binding sites of CaM, we developed a computational method that predicted over 700 high confidence CaM interactors in the Arabidopsis proteome. Most (>600) of these are not known to bind calmodulin, suggesting that there are likely many more CaM targets than previously known. Functional analyses of some of the experimentally identified Ca²⁺ sensor target proteins have uncovered their precise role in Ca²⁺-mediated processes. Further studies on identifying novel targets of CaM and CMLs and generating their interaction network - “calcium sensor interactome” - will help us in understanding how Ca²⁺ regulates a myriad of cellular and physiological processes.     

Fluorescence polarization assay for calmodulin binding to plasma membrane Ca-ATPase: Dependence on enzyme and Ca concentrations

Calmodulin (CaM) is a Ca²⁺ signaling protein that binds to a wide variety of target proteins, and it is important to establish methods for rapid characterization of these interactions. Here we report the use of fluorescence polarization (FP) to measure the K_d for the interaction of CaM with the plasma membrane Ca²⁺-ATPase (PMCA), a Ca²⁺ pump regulated by binding of CaM. Previous assays of PMCA-CaM interactions were indirect, based on activity or kinetics measurements. We also investigated the Ca²⁺ dependence of CaM binding to PMCA. FP assays directly detect CaM-target interactions and are rapid, sensitive, and suitable for high-throughput screening assay formats. Values for the dissociation constant K_d in the nanomolar range are readily measured. We measured the changes in anisotropy of CaM labeled with Oregon Green 488 on titration with PMCA, yielding a K_d value of CaM with PMCA (5.8 ± 0.5 nM) consistent with previous indirect measurements. We also report the binding affinity of CaM with oxidatively modified PMCA (K_d = 9.8 ± 2.0 nM), indicating that the previously reported loss in CaM-stimulated activity for oxidatively modified PMCA is not a result of reduced CaM binding. The Ca²⁺ dependence follows a simple Hill plot demonstrating cooperative binding of Ca²⁺ to the binding sites in CaM.     

Pull-down of Calmodulin-binding Proteins

Calcium (Ca²⁺) is an ion vital in regulating cellular function through a variety of mechanisms. Much of Ca²⁺ signaling is mediated through the calcium-binding protein known as calmodulin (CaM)^1,2. CaM is involved at multiple levels in almost all cellular processes, including apoptosis, metabolism, smooth muscle contraction, synaptic plasticity, nerve growth, inflammation and the immune response. A number of proteins help regulate these pathways through their interaction with CaM. Many of these interactions depend on the conformation of CaM, which is distinctly different when bound to Ca²⁺ (Ca²⁺-CaM) as opposed to its Ca²⁺-free state (ApoCaM)³.While most target proteins bind Ca²⁺-CaM, certain proteins only bind to ApoCaM. Some bind CaM through their IQ-domain, including neuromodulin⁴, neurogranin (Ng)⁵, and certain myosins⁶. These proteins have been shown to play important roles in presynaptic function⁷, postsynaptic function⁸, and muscle contraction⁹, respectively. Their ability to bind and release CaM in the absence or presence of Ca²⁺ is pivotal in their function. In contrast, many proteins only bind Ca²⁺-CaM and require this binding for their activation. Examples include myosin light chain kinase¹⁰, Ca²⁺/CaM-dependent kinases (CaMKs)¹¹ and phosphatases (e.g. calcineurin)¹², and spectrin kinase¹³, which have a variety of direct and downstream effects¹⁴.The effects of these proteins on cellular function are often dependent on their ability to bind to CaM in a Ca²⁺-dependent manner. For example, we tested the relevance of Ng-CaM binding in synaptic function and how different mutations affect this binding. We generated a GFP-tagged Ng construct with specific mutations in the IQ-domain that would change the ability of Ng to bind CaM in a Ca²⁺-dependent manner. The study of these different mutations gave us great insight into important processes involved in synaptic function^8,15. However, in such studies, it is essential to demonstrate that the mutated proteins have the expected altered binding to CaM.Here, we present a method for testing the ability of proteins to bind to CaM in the presence or absence of Ca²⁺, using CaMKII and Ng as examples. This method is a form of affinity chromatography referred to as a CaM pull-down assay. It uses CaM-Sepharose beads to test proteins that bind to CaM and the influence of Ca²⁺ on this binding. It is considerably more time efficient and requires less protein relative to column chromatography and other assays. Altogether, this provides a valuable tool to explore Ca²⁺/CaM signaling and proteins that interact with CaM.     

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 30 kDa 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.     

Ca2+-independent Activation of Ca2+/Calmodulin-dependent Protein Kinase II Bound to the C-terminal Domain of CaV2.1 Calcium Channels

Ca²⁺/calmodulin-dependent protein kinase II (CaMKII) forms a major component of the postsynaptic density where its functions in synaptic plasticity are well established, but its presynaptic actions are poorly defined. Here we show that CaMKII binds directly to the C-terminal domain of Ca_V2.1 channels. Binding is enhanced by autophosphorylation, and the kinase-channel signaling complex persists after dephosphorylation and removal of the Ca²⁺/CaM stimulus. Autophosphorylated CaMKII can bind the Ca_V2.1 channel and synapsin-1 simultaneously. CaMKII binding to Ca_V2.1 channels induces Ca²⁺-independent activity of the kinase, which phosphorylates the enzyme itself as well as the neuronal substrate synapsin-1. Facilitation and inactivation of Ca_V2.1 channels by binding of Ca²⁺/CaM mediates short term synaptic plasticity in transfected superior cervical ganglion neurons, and these regulatory effects are prevented by a competing peptide and the endogenous brain inhibitor CaMKIIN, which blocks binding of CaMKII to Ca_V2.1 channels. These results define the functional properties of a signaling complex of CaMKII and Ca_V2.1 channels in which both binding partners are persistently activated by their association, and they further suggest that this complex is important in presynaptic terminals in regulating protein phosphorylation and short term synaptic plasticity.     

Ca-Calmodulin Increases RyR2 Open Probability yet Reduces Ryanoid Association with RyR2

We have shown that physiological levels of Ca²⁺-calmodulin (Ca²⁺CaM; 50-100 nM) activate cardiac ryanodine receptors (RyR2) incorporated into bilayers and increase the frequency of Ca²⁺ sparks and waves in cardiac cells. In contrast, it is well known that Ca²⁺CaM inhibits [³H]ryanodine binding to cardiac sarcoplasmic reticulum. Since the [³H]ryanodine binding technique does not reflect the effects of Ca²⁺CaM on RyR2 open probability (Po), we have investigated, using the reversible ryanoid, ryanodol, whether Ca²⁺CaM can directly influence the binding of ryanoids to single RyR2 channels independently of Po. We demonstrate that Ca²⁺CaM reduces the rate of ryanodol association to RyR2 without affecting the rate of dissociation. We also find that ryanodol-bound channels fluctuate between at least two distinct subconductance states, M₁ and M₂, in a voltage-dependent manner. Ca²⁺CaM significantly alters the equilibrium between these two states. The results suggest that Ca²⁺CaM binding to RyR2 causes a conformation change to regions of the channel that include the ryanoid binding site, thereby leading to a decrease in ryanoid association rate and modulation of gating within the ryanoid/RyR2 bound state. Our data provide a possible explanation for why the effects of Ca²⁺CaM at the single-channel level are not mirrored by [³H]ryanodine binding studies.     

Functional interaction of calmodulin with a plant cyclic nucleotide gated cation channel

A family of plant ligand gated nonselective cation channels (cngcs) can be activated by direct, and reversible binding of cyclic nucleotide. These proteins have a cytoplasm-localized cyclic nucleotide binding domain (CNBD) at the carboxy-terminus of the polypeptide. A portion of the cngc CNBD also acts as a calmodulin (CaM) binding domain (CaMBD). The objective of this work is to further characterize interaction of cyclic nucleotide and CaM in gating plant cngc currents. The three-dimensional structure of an Arabidopsis thaliana cngc (Atcngc2) CNBD was modeled, indicating cAMP binding to the Atcngc2 CNBD in a pocket formed by a β barrel structure appressing a shortened (relative to animal cngc CNBDs) αC helix. The Atcngc2 CaMBD was expressed as a fusion peptide linking blue and green fluorescent proteins, and used to quantify CaM (A. thaliana CaM isoform 4) binding. CaM bound the fusion protein in a Ca²⁺–dependent manner with a K_d of 7.6 nM and a Ca²⁺ binding K_d of 200 nM. Functional characterization (voltage clamp analysis) of Atcngc2 was undertaken by expression in human embryonic kidney cells. CaM reversed cAMP activation of Atcngc2 currents. This functional interaction was dependent on free cytosolic Ca²⁺. Increasing cytosolic Ca²⁺ was found to inhibit cAMP activation of the channel in the absence of added CaM. We conclude that the physical interaction of Ca²⁺/CaM with plant cngcs blocks cyclic nucleotide activation of these channels. Thus, the cytosolic secondary messengers CaM, cAMP, and Ca²⁺ can act in an integrated fashion to gate currents through these plant ion channels.     

Current view on regulation of voltage‐gated sodium channels by calcium and auxiliary proteins

In cardiac and skeletal myocytes, and in most neurons, the opening of voltage‐gated Na⁺ channels (Na_V channels) triggers action potentials, a process that is regulated via the interactions of the channels’ intercellular C‐termini with auxiliary proteins and/or Ca²⁺. The molecular and structural details for how Ca²⁺ and/or auxiliary proteins modulate Na_V channel function, however, have eluded a concise mechanistic explanation and details have been shrouded for the last decade behind controversy about whether Ca²⁺ acts directly upon the Na_V channel or through interacting proteins, such as the Ca²⁺ binding protein calmodulin (CaM). Here, we review recent advances in defining the structure of Na_V intracellular C‐termini and associated proteins such as CaM or fibroblast growth factor homologous factors (FHFs) to reveal new insights into how Ca²⁺ affects Na_V function, and how altered Ca²⁺‐dependent or FHF‐mediated regulation of Na_V channels is perturbed in various disease states through mutations that disrupt CaM or FHF interaction.     

Calmodulin regulation of TMEM16A and 16B Ca2+-activated chloride channels

Ca²⁺-activated chloride channels encoded by TMEM16A and 16B are important for regulating epithelial mucus secretion, cardiac and neuronal excitability, smooth muscle contraction, olfactory transduction, and cell proliferation. Whether and how the ubiquitous Ca²⁺ sensor calmodulin (CaM) regulates the activity of TMEM16A and 16B channels has been controversial and the subject of an ongoing debate. Recently, using a bioengineering approach termed ChIMP (Channel Inactivation induced by Membrane-tethering of an associated Protein) we argued that Ca²⁺-free CaM (apoCaM) is pre-associated with functioning TMEM16A and 16B channel complexes in live cells. Further, the pre-associated apoCaM mediates Ca²⁺-dependent sensitization of activation (CDSA) and Ca²⁺-dependent inactivation (CDI) of some TMEM16A splice variants. In this review, we discuss these findings in the context of previous and recent results relating to Ca²⁺-dependent regulation of TMEM16A/16B channels and the putative role of CaM. We further discuss potential future directions for these nascent ideas on apoCaM regulation of TMEM16A/16B channels, noting that such future efforts will benefit greatly from the pioneering work of Dr. David T. Yue and colleagues on CaM regulation of voltage-dependent calcium channels.     

The Calmodulin Regulator Protein,PEP-19, Sensitizes ATP-induced Ca2+ Release

PEP-19 is a small, intrinsically disordered protein that binds to the C-domain of calmodulin (CaM) via an IQ motif and tunes its Ca²⁺ binding properties via an acidic sequence. We show here that the acidic sequence of PEP-19 has intrinsic Ca²⁺ binding activity, which may modulate Ca²⁺ binding to CaM by stabilizing an initial Ca²⁺-CaM complex or by electrostatically steering Ca²⁺ to and from CaM. Because PEP-19 is expressed in cells that exhibit highly active Ca²⁺ dynamics, we tested the hypothesis that it influences ligand-dependent Ca²⁺ release. We show that PEP-19 increases the sensitivity of HeLa cells to ATP-induced Ca²⁺ release to greatly increase the percentage of cells responding to sub-saturating doses of ATP and increases the frequency of Ca²⁺ oscillations. Mutations in the acidic sequence of PEP-19 that inhibit or prevent it from modulating Ca²⁺ binding to CaM greatly inhibit its effect on ATP-induced Ca²⁺ release. Thus, this cellular effect of PEP-19 does not depend simply on binding to CaM via the IQ motif but requires its acidic metal binding domain. Tuning the activities of Ca²⁺ mobilization pathways places PEP-19 at the top of CaM signaling cascades, with great potential to exert broad effects on downstream CaM targets, thus expanding the biological significance of this small regulator of CaM signaling.     

Lipid rafts/caveolae as microdomains of calcium signaling

Ca²⁺ is a major signaling molecule in both excitable and non-excitable cells, where it serves critical functions ranging from cell growth to differentiation to cell death. The physiological functions of these cells are tightly regulated in response to changes in cytosolic Ca²⁺ that is achieved by the activation of several plasma membrane (PM) Ca²⁺ channels as well as release of Ca²⁺ from the internal stores. One such channel is referred to as store-operated Ca²⁺ channel that is activated by the release of endoplasmic reticulum (ER) Ca²⁺ which initiates store-operated Ca²⁺ entry (SOCE). Recent advances in the field suggest that some members of TRPCs and Orai channels function as SOCE channels. However, the molecular mechanisms that regulate channel activity and the exact nature of where these channels are assembled and regulated remain elusive. Research from several laboratories has demonstrated that key proteins involved in Ca²⁺ signaling are localized in discrete PM lipid rafts/caveolar microdomains. Lipid rafts are cholesterol and sphingolipid-enriched microdomains that function as unique signal transduction platforms. In addition lipid rafts are dynamic in nature which tends to scaffold certain signaling molecules while excluding others. By such spatial segregation, lipid rafts not only provide a favorable environment for intra-molecular cross-talk but also aid to expedite the signal relay. Importantly, Ca²⁺ signaling is shown to initiate from these lipid raft microdomains. Clustering of Ca²⁺ channels and their regulators in such microdomains can provide an exquisite spatiotemporal regulation of Ca²⁺-mediated cellular function. Thus in this review we discuss PM lipid rafts and caveolae as Ca²⁺-signaling microdomains and highlight their importance in organizing and regulating SOCE channels.     

Calcium/Calmodulin Inhibits the Binding of Specific [125I]Omega-Conotoxin GVIA to Chick Brain Membranes

The effect of Ca²⁺/calmodulin (CaM) on the specific binding of [¹²⁵I]omega-conotoxin GVIA (¹²⁵I--CTX) to crude membranes from chick brain was investigated. When we examined the effects of the activation of various endogenous protein kinases on specific [¹²⁵I]-CTX binding to crude membranes, we observed that Ca²⁺/CaM had an inhibitory effect regardless of whether or not the standard medium contained ATP (0.5 mM). Ca²⁺/CaM also had an inhibitory effect in a simple binding-assay medium containing HEPES-HCl buffer, BSA, Ca²⁺ and CaM, and this effect was dependent on the concentration of Ca²⁺. The effect of Ca²⁺/CaM was attenuated by the CaM antagonists W-7 and CaM-kinase II fragment (290–309). An experiment with modified ELISA using purified anti -CTX antibody indicated that Ca²⁺/CaM did not affect the direct binding of [¹²⁵I]-CTX and CaM. These results suggest that Ca²⁺/CaM either directly or indirectly affects specific [¹²⁵I]-CTX binding sites, probably N-type Ca²⁺ channels in crude membranes from chick whole brain.     

Properties of the apamin-sensitive component of Ca2+-dependent K+ current in smooth muscle cells of the guinea pigtaenia coli

With the help of a standard voltage-clamp technique, we investigated transmembrane ion currents in isolated smooth muscle cells of the guinea pigtaenia coli. In Ca²⁺-dependent K⁺ current, we identified and studied the properties of an apamin-sensitive voltage-independent component carried through the channels of low conductance (in many publications called small conductance,I _SK(Ca)). This component did not show the temporal inactivation;I _SK(Ca) was insensitive to the action of 4 mM tetraethylammonium, but was completely blocked by 500 nM of apamin. It was shown thatI _SK(Ca) is very sensitive to changes in the intracellular Ca²⁺ concentration ([Ca²⁺] _i ): a decrease in [Ca²⁺] _i up to 50 nM resulted in the almost complete blockade of the current. The entry of Ca ions into a cell from the external solution through the voltage-operated Ca²⁺ channels of L-type was not an obligatory condition for activation ofI _SK(Ca). The current-voltage relationship forI _SK(Ca) had a maximum within the voltage range of +20 to +50 mV. Neirofiziologiya/Neurophysiology, Vol. 32, No. 2, pp. 87–94, March–April, 2000.     

Gap junction regulation by calmodulin

Intracellular Ca²⁺ activated calmodulin (CaM) inhibits gap junction channels in the low nanomolar to high micromolar range of [Ca²⁺]_i. This regulation plays an essential role in numerous cellular processes that include hearing, lens transparency, and synchronized contractions of the heart. Previous studies have indicated that gap junction mediated cell-to-cell communication was inhibited by CaM antagonists. More recent evidence indicates a direct role of CaM in regulating several members of the connexin family. Since the intracellular loop and carboxyl termini of connexins are largely “invisible” in electron microscopy and X-ray crystallographic structures due to disorder in these domains, peptide models encompassing the putative CaM binding sites of several intracellular domains of connexins have been used to identify the Ca²⁺-dependent CaM binding sites of these proteins. This approach has been used to determine the CaM binding affinities of peptides derived from a number of different connexin-subfamilies.