Solution and Membrane-Bound Conformations of the Tandem C2A and C2B Domains of Synaptotagmin 1: Evidence for Bilayer Bridging

Synaptotagmin 1 (syt1) is a synaptic vesicle membrane protein that functions as the Ca²⁺ sensor in neuronal exocytosis. Here, site-directed spin labeling was used to generate models for the solution and membrane-bound structures of a soluble fragment of syt1 containing its two C2 domains, C2A and C2B. In solution, distance restraints between the two C2 domains of syt1 were measured using double electron-electron resonance and used in a simulated annealing routine to generate models for the structure of the tandem C2A-C2B fragment. The data indicate that the two C2 domains are flexibly linked and do not interact with each other in solution, with or without Ca²⁺. However, the favored orientation is one where the Ca²⁺-binding loops are oriented in opposite directions. A similar approach was taken for membrane-associated C2A-C2B, combining both distances and bilayer depth restraints with simulated annealing. The restraints can only be satisfied if the Ca²⁺ and membrane-binding surfaces of the domains are oriented in opposite directions so that C2A and C2B are docked to opposing bilayers. The result suggests that syt1 functions to bridge across the vesicle and plasma membrane surfaces in a Ca²⁺-dependent manner.     

Membrane-Binding Cooperativity and Coinsertion by C2AB Tandem Domains of Synaptotagmins 1 and 7

Synaptotagmin-1 (Syt-1) and synaptotagmin-7 (Syt-7) contain analogous tandem C2 domains, C2A and C2B, which together sense Ca²⁺ to bind membranes and promote the stabilization of exocytotic fusion pores. Syt-1 triggers fast release of neurotransmitters, whereas Syt-7 functions in processes that involve lower Ca²⁺ concentrations such as hormone secretion. Syt-1 C2 domains are reported to bind membranes cooperatively, based on the observation that they penetrate farther into membranes as the C2AB tandem than as individual C2 domains. In contrast, we previously suggested that the two C2 domains of Syt-7 bind membranes independently, based in part on measurements of their liposome dissociation kinetics. Here, we investigated C2A-C2B interdomain cooperativity with Syt-1 and Syt-7 using directly comparable measurements. Equilibrium Ca²⁺ titrations demonstrate that the Syt-7 C2AB tandem binds liposomes lacking phosphatidylinositol-4,5-bisphosphate (PIP₂) with greater Ca²⁺ sensitivity than either of its individual domains and binds to membranes containing PIP₂ even in the absence of Ca²⁺. Stopped-flow kinetic measurements show differences in cooperativity between Syt-1 and Syt-7: Syt-1 C2AB dissociates from PIP₂-free liposomes much more slowly than either of its individual C2 domains, indicating cooperativity, whereas the major population of Syt-7 C2AB has a dissociation rate comparable to its C2A domain, suggesting a lack of cooperativity. A minor subpopulation of Syt-7 C2AB dissociates at a slower rate, which could be due to a small cooperative component and/or liposome clustering. Measurements using an environment-sensitive fluorescent probe indicate that the Syt-7 C2B domain inserts deeply into membranes as part of the C2AB tandem, similar to the coinsertion previously reported for Syt-1. Overall, coinsertion of C2A and C2B domains is coupled to cooperative energetic effects in Syt-1 to a much greater extent than in Syt-7. The difference can be understood in terms of the relative contributions of C2A and C2B domains toward membrane binding in the two proteins.     

SNARE complex alters the interactions of the Ca2+ sensor synaptotagmin 1 with lipid bilayers

Release of neuronal transmitters from nerve terminals is triggered by the molecular Ca²⁺ sensor synaptotagmin 1 (Syt1). Syt1 is a transmembrane protein attached to the synaptic vesicle (SV), and its cytosolic region comprises two domains, C2A and C2B, which are thought to penetrate into lipid bilayers upon Ca²⁺ binding. Before fusion, SVs become attached to the presynaptic membrane (PM) by the four-helical SNARE complex, which is thought to bind the C2B domain in vivo. To understand how the interactions of Syt1 with lipid bilayers and the SNARE complex trigger fusion, we performed molecular dynamics (MD) simulations at a microsecond scale. We investigated how the isolated C2 modules and the C2AB tandem of Syt1 interact with membranes mimicking either SV or PM. The simulations showed that the C2AB tandem can either bridge SV and PM or insert into PM with its Ca²⁺-bound tips and that the latter configuration is more favorable. Surprisingly, C2 domains did not cooperate in penetrating into PM but instead mutually hindered their insertion into the bilayer. To test whether the interaction of Syt1 with lipid bilayers could be affected by the C2B-SNARE attachment, we performed systematic conformational analysis of the C2AB-SNARE complex. Notably, we found that the C2B-SNARE interface precludes the coupling of C2 domains and promotes their insertion into PM. We performed the MD simulations of the prefusion protein complex positioned between the lipid bilayers mimicking PM and SV, and our results demonstrated in silico that the presence of the Ca²⁺ bound C2AB tandem promotes lipid merging. Altogether, our MD simulations elucidated the role of the Syt1-SNARE interactions in the fusion process and produced the dynamic all-atom model of the prefusion protein-lipid complex.     

Quantitation of the Calcium and Membrane Binding Properties of the C2 Domains of Dysferlin

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca²⁺ sensitive, the Ca²⁺ binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca²⁺ and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca²⁺ with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with K_d values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar K_d value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca²⁺ enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca²⁺ albeit with varying affinity and stoichiometry.     

Quantitation of the Calcium and Membrane Binding Properties of the C2 Domains of Dysferlin

Dysferlin is a large membrane protein involved in calcium-triggered resealing of the sarcolemma after injury. Although it is generally accepted that dysferlin is Ca²⁺ sensitive, the Ca²⁺ binding properties of dysferlin have not been characterized. In this study, we report an analysis of the Ca²⁺ and membrane binding properties of all seven C2 domains of dysferlin as well as a multi-C2 domain construct. Isothermal titration calorimetry measurements indicate that all seven dysferlin C2 domains interact with Ca²⁺ with a wide range of binding affinities. The C2A and C2C domains were determined to be the most sensitive, with K_d values in the tens of micromolar, whereas the C2D domain was least sensitive, with a near millimolar K_d value. Mutagenesis of C2A demonstrates the requirement for negatively charged residues in the loop regions for divalent ion binding. Furthermore, dysferlin displayed significantly lower binding affinity for the divalent cations magnesium and strontium. Measurement of a multidomain construct indicates that the solution binding affinity does not change when C2 domains are linked. Finally, sedimentation assays suggest all seven C2 domains bind lipid membranes, and that Ca²⁺ enhances but is not required for interaction. This report reveals for the first time, to our knowledge, that all dysferlin domains bind Ca²⁺ albeit with varying affinity and stoichiometry.     

Partial Metal Ion Saturation of C2 Domains Primes Synaptotagmin 1-Membrane Interactions

Synaptotagmin 1 (Syt1) is an integral membrane protein whose phospholipid-binding tandem C2 domains, C2A and C2B, act as Ca²⁺ sensors of neurotransmitter release. Our objective was to understand the role of individual metal-ion binding sites of these domains in the membrane association process. We used Pb²⁺, a structural and functional surrogate of Ca²⁺, to generate the protein states with well-defined protein-metal ion stoichiometry. NMR experiments revealed that binding of one divalent metal ion per C2 domain results in loss of conformational plasticity of the loop regions, potentially pre-organizing them for additional metal-ion and membrane-binding events. In C2A, a divalent metal ion in site 1 is sufficient to drive its weak association with phosphatidylserine-containing membranes, whereas in C2B, it enhances the interactions with the signaling lipid phosphatidylinositol-4,5-bisphosphate. In full-length Syt1, both Pb²⁺-complexed C2 domains associate with phosphatidylserine-containing membranes. Electron paramagnetic resonance experiments show that the extent of membrane insertion correlates with the occupancy of the C2 metal ion sites. Together, our results indicate that upon partial metal ion saturation of the intra-loop region, Syt1 adopts a dynamic, partially membrane-bound state. The properties of this state, such as conformationally restricted loop regions and positioning of C2 domains in close proximity to anionic lipid headgroups, “prime” Syt1 for cooperative binding of a full complement of metal ions and deeper membrane insertion.     

A novel dual Ca2+ sensor system regulates Ca2+-dependent neurotransmitter release

Ca²⁺-dependent neurotransmitter release requires synaptotagmins as Ca²⁺ sensors to trigger synaptic vesicle (SV) exocytosis via binding of their tandem C2 domains—C2A and C2B—to Ca²⁺. We have previously demonstrated that SNT-1, a mouse synaptotagmin-1 (Syt1) homologue, functions as the fast Ca²⁺ sensor in Caenorhabditis elegans. Here, we report a new Ca²⁺ sensor, SNT-3, which triggers delayed Ca²⁺-dependent neurotransmitter release. snt-1;snt-3 double mutants abolish evoked synaptic transmission, demonstrating that C. elegans NMJs use a dual Ca²⁺ sensor system. SNT-3 possesses canonical aspartate residues in both C2 domains, but lacks an N-terminal transmembrane (TM) domain. Biochemical evidence demonstrates that SNT-3 binds both Ca²⁺ and the plasma membrane. Functional analysis shows that SNT-3 is activated when SNT-1 function is impaired, triggering SV release that is loosely coupled to Ca²⁺ entry. Compared with SNT-1, which is tethered to SVs, SNT-3 is not associated with SV. Eliminating the SV tethering of SNT-1 by removing the TM domain or the whole N terminus rescues fast release kinetics, demonstrating that cytoplasmic SNT-1 is still functional and triggers fast neurotransmitter release, but also exhibits decreased evoked amplitude and release probability. These results suggest that the fast and slow properties of SV release are determined by the intrinsically different C2 domains in SNT-1 and SNT-3, rather than their N-termini–mediated membrane tethering. Our findings therefore reveal a novel dual Ca²⁺ sensor system in C. elegans and provide significant insights into Ca²⁺-regulated exocytosis.     

A Highlights from MBoC Selection: Mutations that disrupt Ca2+-binding activity endow Doc2β with novel functional properties during synaptic transmission

Double C2-domain protein (Doc2) is a Ca²⁺-binding protein implicated in asynchronous and spontaneous neurotransmitter release. Here we demonstrate that each of its C2 domains senses Ca²⁺; moreover, the tethered tandem C2 domains display properties distinct from the isolated domains. We confirm that overexpression of a mutant form of Doc2β, in which two acidic Ca²⁺ ligands in the C2A domain and two in the C2B domain have been neutralized, results in markedly enhanced asynchronous release in synaptotagmin 1–knockout neurons. Unlike wild-type (wt) Doc2β, which translocates to the plasma membrane in response to increases in [Ca²⁺]_i, the quadruple Ca²⁺-ligand mutant does not bind Ca²⁺ but is constitutively associated with the plasma membrane; this effect is due to substitution of Ca²⁺ ligands in the C2A domain. When overexpressed in wt neurons, Doc2β affects only asynchronous release; in contrast, Doc2β Ca²⁺-ligand mutants that constitutively localize to the plasma membrane enhance both the fast and slow components of synaptic transmission by increasing the readily releasable vesicle pool size; these mutants also increase the frequency of spontaneous release events. Thus, mutations in the C2A domain of Doc2β that were intended to disrupt Ca²⁺ binding result in an anomalous enhancement of constitutive membrane-binding activity and endow Doc2β with novel functional properties.     

Roles of the NH2-terminal Domains of Cardiac Ryanodine Receptor in Ca2+ Release Activation and Termination

The NH₂-terminal region (residues 1–543) of the cardiac ryanodine receptor (RyR2) harbors a large number of mutations associated with cardiac arrhythmias and cardiomyopathies. Functional studies have revealed that the NH₂-terminal region is involved in the activation and termination of Ca²⁺ release. The three-dimensional structure of the NH₂-terminal region has recently been solved. It is composed of three domains (A, B, and C). However, the roles of these individual domains in Ca²⁺ release activation and termination are largely unknown. To understand the functional significance of each of these NH₂-terminal domains, we systematically deleted these domains and assessed their impact on caffeine- or Ca²⁺-induced Ca²⁺ release and store overload-induced Ca²⁺ release (SOICR) in HEK293 cells. We found that all deletion mutants were capable of forming caffeine- and ryanodine-sensitive functional channels, indicating that the NH₂-terminal region is not essential for channel gating. Ca²⁺ release measurements revealed that deleting domain A markedly reduced the threshold for SOICR termination but had no effect on caffeine or Ca²⁺ activation or the threshold for SOICR activation, whereas deleting domain B substantially enhanced caffeine and Ca²⁺ activation and lowered the threshold for SOICR activation and termination. Conversely, deleting domain C suppressed caffeine activation, abolished Ca²⁺ activation and SOICR, and diminished protein expression. These results suggest that domain A is involved in channel termination, domain B is involved in channel suppression, and domain C is critical for channel activation and expression. Our data shed new insights into the structure-function relationship of the NH₂-terminal domains of RyR2 and the action of NH₂-terminal disease mutations.     

Interaction of the C2 Ig-like Domain of Cardiac Myosin Binding Protein-C with F-actin

Cardiac muscle contraction depends on interactions between thick (myosin) and thin (actin) filaments (TFs). TFs are regulated by intracellular Ca²⁺ levels. Under activating conditions Ca²⁺ binds to the troponin complex and displaces tropomyosin from myosin binding sites on the TF surface to allow actomyosin interactions. Recent studies have shown that in addition to Ca²⁺, the first four N-terminal domains (NTDs) of cardiac myosin binding protein C (cMyBP-C) (e.g. C0, C1, M and C2), are potent modulators of the TF activity, but the mechanism of their collective action is poorly understood. Previously, we showed that C1 activates the TF at low Ca²⁺ and C0 stabilizes binding of C1 to the TF, but the ability of C2 to bind and/or affect the TF remains unknown. Here we obtained 7.5 Å resolution cryo-EM reconstruction of C2-decorated actin filaments to demonstrate that C2 binds to actin in a single structural mode that does not activate the TF unlike the polymorphic binding of C0 and C1 to actin. Comparison of amino acid sequences of C2 with either C0 or C1 shows low levels of identity between the residues involved in interactions with the TF but high levels of conservation for residues involved in Ig fold stabilization. This provides a structural basis for strikingly different interactions of structurally homologous C0, C1 and C2 with the TF. Our detailed analysis of the interaction of C2 with the actin filament provides crucial information required to model the collective action of cMyBP-C NTDs on the cardiac TF.     

Asp residues of the Glu-Glu-Asp-Asp pore filter contribute to ion permeation and selectivity of the Cav3.2 T-type channel

Voltage-activated Ca²⁺ channels are membrane protein machinery performing selective permeation of external calcium ions. The main Ca²⁺ selective filters of all high-voltage-activated Ca²⁺ channel isoforms are commonly composed of four Glu residues (EEEE), while those of low-voltage-activated T-type Ca²⁺ channel isoforms are made up of two Glu and two Asp residues (EEDD). We here investigate how the Asp residues at the pore loops of domains III and IV affect biophysical properties of the Ca_v3.2 channel. Electrophysiological characterization of the pore mutant channels in which the pore Asp residue(s) were replaced with Glu, showed that both Asp residues critically control the biophysical properties of Ca_v3.2, including relative permeability between Ba²⁺ and Ca²⁺, anomalous mole fraction effect (AMFE), voltage dependency of channel activation, Cd²⁺ blocking sensitivity, and pH effects, in distinctive ways.     

Calcium binding and conformational properties of calmodulin complexed with peptides derived from myristoylated alanine-rich C kinase substrate (MARCKS) and MARCKS-related protein (MRP)

The myristoylated alanine-rich C kinase substrate (MARCKS) and the MARCKS-related protein (MRP) are members of a distinct family of protein ki-nase C (PKC) substrates that bind calmodulin (CaM) in a manner regulated by Ca²⁺ and phosphorylation by PKC. The CaM binding region overlaps with the PKC phosphorylation sites, suggesting a potential coupling between Ca²⁺-CaM signalling and PKC-mediated phosphorylation cascades. We have studied Ca²⁺ binding of CaM complexed with CaM binding peptides from MARCKS and MRP using flow dialysis, NMR and circular dichroism (CD) spectroscopy. The wild-type MARCKS and MRP peptides induced significant increases in the Ca²⁺ affinity of CaM (pCa 6.1 and 5.8, respectively, compared to 5.2, for CaM in the absence of bound peptides), whereas a modified MARCKS peptide, in which the four serine residues susceptible to phosphorylation in the wild-type sequence have been replaced with aspartate residues to mimic phosphorylation, had smaller effect (pCa 5.6). These results are consistent with the notions that phosphorylation of MARCKS reduces its binding affinity for CaM and that the CaM binding affinity of the peptides is coupled to the Ca²⁺ affinity of CaM. All three MARCKS/MRP peptides perturbed the backbone NMR resonances of residues in both the N- and C-terminal domains of CaM and, in addition, the wild-type MARCKS and the MRP peptides induced strong positive cooperativity in Ca²⁺ binding by CaM, suggesting that the peptides interact with the amino- and carboxy-terminal domains of CaM simultaneously. NMR analysis of the Ca²⁺-CaM-MRP peptide complex, as well as CD measurements of Ca²⁺-CaM in the presence and absence of MARCKS/MRP peptides suggest that the peptide bound to CaM is non-helical, in contrast to the α-helical conformation found in the CaM binding regions of myosin light-chain kinase and CaM-dependent protein kinase II. The adaptation of the CaM molecule for binding the peptide requires disruption of its central helical linker between residues Lys-75 and Glu-82. Received: 26 September 1996 / 22 October 1996     

Role of the STIM1 C-terminal Domain in STIM1 Clustering

Store-operated Ca²⁺ entry (SOCE) represents a ubiquitous Ca²⁺ influx pathway activated by the filling state of intracellular Ca²⁺ stores. SOCE is mediated by coupling of STIM1, the endoplasmic reticulum Ca²⁺ sensor, to the Orai1 channel. SOCE inactivates during meiosis, partly because of the inability of STIM1 to cluster in response to store depletion. STIM1 has several functional domains, including the Orai1 interaction domain (STIM1 Orai Activating Region (SOAR) or CRAC Activation Domain (CAD)) and STIM1 homomerization domain. When Ca²⁺ stores are full, these domains are inactive to prevent constitutive Ca²⁺ entry. Here we show, using the Xenopus oocyte as an expression system, that the C-terminal 200 residues of STIM1 are important to maintain STIM1 in an inactive state when Ca²⁺ stores are full, through predicted intramolecular shielding of the active STIM1 domains (SOAR/CAD and STIM1 homomerization domain). Interestingly, our data argue that the C-terminal 200 residues accomplish this through a steric hindrance mechanism because they can be substituted by GFP or mCherry while maintaining all aspects of STIM1 function. We further show that STIM1 clustering inhibition during meiosis is independent of the C-terminal 200 residues.     

Interaction of synaptotagmin with lipid bilayers, analyzed by single-molecule force spectroscopy

Synaptotagmin I is the major Ca²⁺ sensor for membrane fusion during neurotransmitter release. The cytoplasmic domain of synaptotagmin consists of two C2 domains, C2A and C2B. On binding Ca²⁺, the tips of the two C2 domains rapidly and synchronously penetrate lipid bilayers. We investigated the forces of interaction between synaptotagmin and lipid bilayers using single-molecule force spectroscopy. Glutathione-S-transferase-tagged proteins were attached to an atomic force microscope cantilever via a glutathione-derivatized polyethylene glycol linker. With wild-type C2AB, the force profile for a bilayer containing phosphatidylserine had both Ca²⁺-dependent and Ca²⁺-independent components. No force was detected when the bilayer lacked phosphatidylserine, even in the presence of Ca²⁺. The binding characteristics of C2A and C2B indicated that the two C2 domains cooperate in binding synaptotagmin to the bilayer, and that the relatively weak Ca²⁺-independent force depends only on C2A. When the lysine residues K189-192 and K326, 327 were mutated to alanine, the strong Ca²⁺-dependent binding interaction was either absent or greatly reduced. We conclude that synaptotagmin binds to the bilayer via C2A even in absence of Ca²⁺, and also that positively charged regions of both C2A and C2B are essential for the strong Ca²⁺-dependent binding of synaptotagmin to the bilayer.     

Novel immune function for the TRPV1 channel in T lymphocytes

TRPM3 has been reported to play an important role in Ca²⁺ homeostasis, but its gating mechanisms and regulation via Ca²⁺ are unknown. Ca²⁺ binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that another Ca²⁺ binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P₂, interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.     

Free-energy linkage between folding and calcium binding in EF-hand proteins

Troponin is the singular Ca²⁺-sensitive protein in the contraction of vertebrate striated muscles. Troponin C (TnC), the Ca²⁺-binding subunit of the troponin complex, has two distinct domains, C and N, which have different properties despite their extensive structural homology. In this work, we analyzed the thermodynamic stability of the isolated N-domain of TnC using a fluorescent mutant with Phe 29 replaced by Trp (F29W/N-domain, residues 1-90). The complete unfolding of the N-domain of TnC in the absence or presence of Ca²⁺ was achieved by combining high hydrostatic pressure and urea, a maneuver that allowed us to calculate the thermodynamic parameters (ΔV and ΔG_atm). In this study, we propose that part of the affinity for Ca²⁺ is contributed by the free-energy change of folding of the N- and C-domains that takes place when Ca²⁺ binds. The importance of the free-energy change for the structural and regulatory functions of the TnC isolated domains was evaluated. Our results shed light on how the coupling between folding and ion binding contributes to the fine adjustment of the affinity for Ca²⁺ in EF-hand proteins, which is crucial to function.     

The crystal structure of Arabidopsis BON1 provides insights into the copine protein family

The Arabidopsis thaliana BON1 gene product is a member of the evolutionary conserved eukaryotic calcium‐dependent membrane‐binding protein family. The copine protein is composed of two C2 domains (C2A and C2B) followed by a vWA domain. The BON1 protein is localized on the plasma membrane, and is known to suppress the expression of immune receptor genes and to positively regulate stomatal closure. The first structure of this protein family has been determined to 2.5‐Å resolution and shows the structural features of the three conserved domains C2A, C2B and vWA. The structure reveals the third Ca²⁺‐binding region in C2A domain is longer than classical C2 domains and a novel Ca²⁺ binding site in the vWA domain. The structure of BON1 bound to Mn²⁺ is also presented. The binding of the C2 domains to phospholipid (PSF) has been modeled and provides an insight into the lipid‐binding mechanism of the copine proteins. Furthermore, the selectivity of the separate C2A and C2B domains and intact BON1 to bind to different phospholipids has been investigated, and we demonstrated that BON1 could mediate aggregation of liposomes in response to Ca²⁺. These studies have formed the basis of further investigations into the important role that the copine proteins play in vivo.     

PtdIns(4,5)P2 interacts with CaM binding domains on TRPM3 N-terminus

TRPM3 has been reported to play an important role in Ca²⁺ homeostasis, but its gating mechanisms and regulation via Ca²⁺ are unknown. Ca²⁺ binding proteins such as calmodulin (CaM) could be probable modulators of this ion channel. We have shown that this protein binds to two independent domains, A35-K124 and H291-G382 on the TRPM3 N-terminus, which contain conserved hydrophobic as well as positively charged residues in specific positions, and that these residues have a crucial impact on its binding. We also showed that another Ca²⁺ binding protein, S100A1, is able to bind to these regions and that CaM and S100A1 compete for these binding sites on the TRPM3 N-terminus. Moreover, our results suggest that another very important TRP channel activity modulator, PtdIns(4,5)P₂, interacts with the CaM/S100A1 binding sites on the TRPM3 N-terminus with high affinity.     

Identification of the divalent metal ion binding domain of myosin regulatory light chains using spin-labelling techniques

By the criterion of their primary structure myosin regulatory light chains belong to the ‘calcium binding protein’ family and are thought to contain domains related to the E-F hand structure found in parvalbumin. However, the presence of deletions and non-conservative substitutions in the regulatory light chains indicates that, of the four domains apparent in their structure, only the first is competent to bind Ca²⁺ or other divalent metal ions. Electron paramagnetic resonance studies were performed in an attempt to provide experimental verification of this hypothesis. The approach is based on the finding that the paramagnetic Mn²⁺ ion substitutes for Ca²⁺ at the divalent metal ion site and that different regulatory light-chain isotypes contain cysteine residues in different domains which may be spin-labelled with a nitroxide derivative. The electron spin interaction between these two paramagnetic centres is a function of the distance of their separation. Clam (Mercenaria mercenaria) regulatory light chain contains a single cysteine residue located near the first domain and, when spin-labelled, the intensity of the nitroxide signal is reduced by 25% on binding one mole of Mn²⁺. Rabbit skeletal regulatory light chain contains two cysteine residues located in the third and fourth domains and no (<5%) interaction is observed when Mn²⁺ binds to spin-labelled derivatives. Qualitatively, these results suggest that domain 1 is the most likely candidate for the Mn²⁺ binding site. A more quantitative evaluation using the Leigh (1970) theory for the dipolar coupling between rigid-lattice electron spins and various models for the regulatory light chain tertiary structure, including that predicted by Kretsinger &; Barry (1975) for the possibly isologous troponin C structure, substantiates this conclusion.     

Ca2+ releases E‐Syt1 autoinhibition to couple ER‐plasma membrane tethering with lipid transport

The extended synaptotagmins (E‐Syts) are endoplasmic reticulum (ER) proteins that bind the plasma membrane (PM) via C2 domains and transport lipids between them via SMP domains. E‐Syt1 tethers and transports lipids in a Ca²⁺‐dependent manner, but the role of Ca²⁺ in this regulation is unclear. Of the five C2 domains of E‐Syt1, only C2A and C2C contain Ca²⁺‐binding sites. Using liposome‐based assays, we show that Ca²⁺ binding to C2C promotes E‐Syt1‐mediated membrane tethering by releasing an inhibition that prevents C2E from interacting with PI(4,5)P₂‐rich membranes, as previously suggested by studies in semi‐permeabilized cells. Importantly, Ca²⁺ binding to C2A enables lipid transport by releasing a charge‐based autoinhibitory interaction between this domain and the SMP domain. Supporting these results, E‐Syt1 constructs defective in Ca²⁺ binding in either C2A or C2C failed to rescue two defects in PM lipid homeostasis observed in E‐Syts KO cells, delayed diacylglycerol clearance from the PM and impaired Ca²⁺‐triggered phosphatidylserine scrambling. Thus, a main effect of Ca²⁺ on E‐Syt1 is to reverse an autoinhibited state and to couple membrane tethering with lipid transport.