μM Free Ca2 Research Articles

Molecular mechanism of the severe MH/CCD mutation Y522S in skeletal ryanodine receptor (RyR1) by cryo-EM

Ryanodine receptors (RyRs) are main regulators of intracellular Ca2+ release and muscle contraction. The Y522S mutation of RyR1 causes central core disease, a weakening myopathy, and malignant hyperthermia, a sudden and potentially fatal response to anesthetics or heat. Y522 is in the core of the N-terminal subdomain C of RyR1 and the mechanism of how this mutation orchestrates malfunction is unpredictable for this 2-MDa ion channel, which has four identical subunits composed of 15 distinct cytoplasmic domains each. We expressed and purified the RyR1 rabbit homolog, Y523S, from HEK293 cells and reconstituted it in nanodiscs under closed and open states. The high-resolution cryogenic electron microscopic (cryo-EM) three-dimensional (3D) structures show that the phenyl ring of Tyr functions in a manner analogous to a "spacer" within an α-helical bundle. Mutation to the much smaller Ser alters the hydrophobic network within the bundle, triggering rearrangement of its α-helices with repercussions in the orientation of most cytoplasmic domains. Examining the mutation-induced readjustments exposed a series of connected α-helices acting as an ∼100 Å-long lever: One end protrudes toward the dihydropyridine receptor, its molecular activator (akin to an antenna), while the other end reaches the Ca2+ activation site. The Y523S mutation elicits channel preactivation in the absence of any activator and full opening at 1.5 µM free Ca2+, increasing by ∼20-fold the potency of Ca2+ to activate the channel compared with RyR1 wild type (WT). This study identified a preactivated pathological state of RyR1 and a long-range lever that may work as a molecular switch to open the channel.

Ca2+-dependent inhibition of branched-chain α-ketoacid dehydrogenase kinase by thiamine pyrophosphate

Catabolism of the branched-chain amino acids (BCAAs: leucine, isoleucine, and valine) is regulated by the branched-chain α-ketoacid dehydrogenase (BCKDH) complex, which in turn is regulated by phosphorylation catalyzed by BCKDH kinase (BDK). Thiamine pyrophosphate (TPP) is required as a coenzyme for the E1 component of the BCKDH complex and can also bring about activation of the complex by inhibiting BDK. The present study shows that free Ca2+ in the physiological range greatly increases the sensitivity of BDK to inhibition by TPP (IC50 of 2.5 μM in the presence of 1 μM free Ca2+). This novel mechanism may be responsible for the stimulation of BCAA oxidation by conditions that increase mitochondrial free Ca2+ levels, e.g. in skeletal muscle during exercise.

Ca2+ Regulation of Cav3.3 T-type Ca2+ Channel Is Mediated by Calmodulin

Calcium-dependent inactivation of high voltage-activated Ca2+ channels plays a crucial role in limiting rises in intracellular calcium (Ca2+i). A key mediator of these effects is calmodulin, which has been found to bind the C-terminus of the pore-forming α subunit. In contrast, little is known about how Ca2+i can regulate low voltage-activated T-type Ca2+ channels. Using whole cell patch clamp, we examined the biophysical properties of Ca2+ current through the three T-type Ca2+ channel isoforms, Cav3.1, Cav3.2, or Cav3.3, comparing internal solutions containing 27 nM and l μM free Ca2+ Both activation and inactivation kinetics of Cav3.3 current in l μM Ca2+i solution were more rapid than those in 27 nM Ca2+i solution. In addition, both activation and steady-state inactivation curves of Cav3.3 were negatively shifted in the higher Ca2+i solution. In contrast, the biophysical properties of Cav3.1 and Cav3.2 isoforms were not significantly different between the two internal solutions. Overexpression of CaM1234 (a calmodulin mutant that doesn't bind Ca2+) occluded the effects of l μM Ca2+i on Cav3.3, implying that CaM is involved in the Ca2+i regulation effects on Cav3.3. Yeast two-hybrid screening and co-immunoprecipitation experiments revealed a direct interaction of CaM with the carboxyl terminus of Cav3.3. Taken together, our results suggest that Cav3.3 T-type channel is potently regulated by Ca2+i via interaction of Ca2+/CaM with the carboxyl terminus of Cav3.3.

Modulation by CGP-37157 (CGP) Analogs of the Sarcoplasmic Reticulum Calcium ATPase SERCA)

We have reported that CGP, a 4-1 benzothiazepine (BZT) associated with cardioprotection in ischemia, is a SERCA inhibitor. CGP blocking action differs from that of thapsigargin because is Ca2+ dependent; i.e., CGP blocking potency increases when Ca2+ levels decrease. We utilized purified sarcoplasmic reticulum microsomal membranes (SRM; from rabbit skeletal muscle and pig ventricle) to test the inhibitory actions of various novel CGP analogs on ATPase function (estimated by measurements of Ca2+ loading and ATPase activity). The 4,1-benzothiazepin-2-one structural arrangement seems to be important for SERCA blocking action since function is lost within compounds where: the Sulfur is substituted by Nitrogen (as seen in benzodiazepines); the Oxygen is replaced by an alkyl group; or the arrangement between Nitrogen, Oxygen, and Sulfur in the BZT ring is reorganized (as seen in Diltiazem). Removal of Chlorine from position 7 in the BZT ring did not affect SERCA blocking potency. A combination of substitutions: 3H (benzylaminomethyl-carboxymethyl), alkylation of the Nitrogen (neopentyl) and replacement of the position 1 of benzyl group (naphtyl), significantly increased potency 10-fold (IC50 ∼ 1.5 μM, in presence of 2 μM free Ca2+). We determined (using SRM Ca2+ leak assay, [3H] ryanodine binding and channel studies in bilayers) CGP analogs can also activate ryanodine receptors (RyRs), albeit with low potency. In summary, we have identified structural features that may confer Ca2+-dependent SERCA blocking in 4,1 BZT by utilizing CGP-like molecules. We also identified ph000995, a potent blocker, which may have potential for assessing SERCA roles in cells as well as being a potential drug-template for therapeutic intervention in diseases of abnormal Ca2+ homeostasis, such as ischemia. (Supported by American Heart Association, Eskridge Foundation and APS Frontiers in Physiology Research Program).

The Prostacyclin Analog Treprostinil Inhibits Ano1-Encoded Ca2+-Activated Cl− Channels and Mouse Pulmonary Arterial Tone Through Stimulation of cAMP-Dependent Signaling Pathway

Ca2+-activated Cl- channels (CaCCs) encoded by the gene Tmem16a or Anoctamin1 (ANO1) produce membrane depolarization and contraction of vascular smooth muscle cells (VSMCs) when stimulated by endogenous vasoconstrictors acting on Gq-protein coupled receptors. Treprostinil (TPL) is one of several prostacyclin analogs clinically used to treat patients diagnosed with pulmonary arterial hypertension (PAH). TPL reduces pulmonary arterial tone in PAH patients by binding to the IP prostanoid receptor. Activation of this Gs-coupled receptor leads to elevation in cAMP levels causing vasorelaxation by reducing intracellular [Ca2+]i. In this study, we examined whether TPL could exert vasorelaxation of mouse pulmonary arteries (mPA) by inhibiting, at least in part, ANO1-encoded CaCCs. TPL dose-dependently inhibited the contraction of mPA elicited by 10 μM 5-HT (IC50 ≈ 500 nM). The specific ANO1 inhibitor T16Ainh-A01 also dose-dependently inhibited the 5-HT-induced contraction of mPA but had no effect on the KCl-induced contraction (85.4 mM) at concentrations ≤ 10 μM. 200 nM TPL produced a rightward shift of the dose-response curve to T16AInh-A01, suggesting that TPL may inhibit CaCCs. Consistent with this hypothesis, Ca2+-activated Cl- currents (elicited by 1 μM free Ca2+ in the pipette solution) recorded in HEK-293 cells transfected with mouse ANO1 were significantly reduced by TPL (1 μM), or the adenylate cyclase activator Forskolin (10 μM). These preliminary results suggest that elevation of cAMP levels (perhaps through a phosphorylation step involving Protein Kinase A) may down-regulate ANO1-encoded CaCCs and attenuate the depolarization and vasoconstriction triggered by 5-HT. Our findings also suggest that part of the beneficial effects of TPL in PAH patients might be attributable to an interaction with ANO1 channels.

Nav1.5 C-Terminal Domains Influence Calcium Regulation of Fast Inactivation Separately from Calmodulin Interaction

The cardiac sodium channel (Nav1.5) has a complex ‘intracellular Ca2+ sensing apparatus’ within its C-terminal domain (CTD) consisting of a partial EF-hand domain (CTD-EF) and a calmodulin (CaM) binding IQ motif (CTD-IQ). There are additional CaM binding motifs within the DIII-DIV linker. Variation in intracellular Ca2+ concentration influences the voltage-dependence of steady-state fast inactivation (SSI) by an unclear mechanism. Here, we mutated 16 key residues implicated in the interactions between CTD-EF and CTD-IQ, CTD-IQ and CaM, or DIII-DIV and CaM and explored their impact on NaV1.5 function and biochemistry. We used quantitative yeast-two-hybrid assays to measure effects of mutations on the interaction between the full length CTD on CaM, and evaluated SSI in high (1 µM free Ca2+) and low [Ca2+]i conditions. Using either BAPTA or HEDTA as chelator, 1 µM free [Ca2+]i was sufficient to shift SSI (elicited by 50 ms prepulses) towards depolarized potentials. We observed that 3 of 5 CTD-IQ mutations (F1912A, A1924T and IQ/AA) strongly reduced the interaction between the CTD and CaM. However, these mutations did not affect the [Ca2+]i effect on SSI suggesting that SSI [Ca2+]i sensitivity does not depend on CaM binding to the CTD-IQ. Unexpectedly, single and combination mutations of the CTD-EF (L1786A, F1791A, Q1807A, L1862A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) diminished the CTD-CaM interaction, and some of these mutations (L1786A, E1788A-D1790A-D1792A-E1799A, E1804A-D1802A) also suppressed the [Ca2+]i effect on SSI. Indeed, the only mutations we studied that blunted the [Ca2+]i effect on SSI were within the CTD-EF. These results suggest that the CTD-EF influences NaV1.5 [Ca2+]i sensitivity and mutations in this domain can also alter the interaction of the CTD with CaM, but CaM interaction with the CTD-IQ is not required for the effect of intracellular Ca2+ on inactivation.

Stimulation of oxidative phosphorylation by calcium in cardiac mitochondria is not influenced by cAMP and PKA activity

Cardiac oxidative ATP generation is finely tuned to match several-fold increases in energy demand. Calcium has been proposed to play a role in the activation of ATP production via PKA phosphorylation in response to intramitochondrial cAMP generation. We evaluated the effect of cAMP, its membrane permeable analogs (dibutyryl-cAMP, 8-bromo-cAMP), and the PKA inhibitor H89 on respiration of isolated pig heart mitochondria. cAMP analogs did not stimulate State 3 respiration of Ca2+-depleted mitochondria (82.2±3.6% of control), in contrast to the 2-fold activation induced by 0.95μM free Ca2+, which was unaffected by H89. Using fluorescence and integrating sphere spectroscopy, we determined that Ca2+ increased the reduction of NADH (8%), and of cytochromes bH (3%), c1 (3%), c (4%), and a (2%), together with a doubling of conductances for Complex I+III and Complex IV. None of these changes were induced by cAMP analogs nor abolished by H89. In Ca2+-undepleted mitochondria, we observed only slight changes in State 3 respiration rates upon addition of 50μM cAMP (85±9.9%), dibutyryl-cAMP (80.1±5.2%), 8-bromo-cAMP (88.6±3.3%), or 1μM H89 (89.7±19.9%) with respect to controls. Similar results were obtained when measuring respiration in heart homogenates. Addition of exogenous PKA with dibutyryl-cAMP or the constitutively active catalytic subunit of PKA to isolated mitochondria decreased State 3 respiration by only 5–15%. These functional studies suggest that alterations in mitochondrial cAMP and PKA activity do not contribute significantly to the acute Ca2+ stimulation of oxidative phosphorylation.

A Canonical EF‐Loop Directs Ca2+‐Sensitivity in Phospholipase C‐η2

Phospholipase C-η (PLCη) enzymes are a class of phosphatidylinositol 4,5-bisphosphate-hydrolyzing enzymes involved in intracellular signaling. PLCη2 can sense Ca(2+) (stimulated by ∼1 µM free Ca(2+) ) suggesting that it can amplify transient Ca(2+) signals. PLCη enzymes possess an EF-hand domain composed of two EF-loops; a canonical 12-residue loop (EF-loop 1) and a non-canonical 13-residue loop (EF-loop 2). Ca(2+) -binding to synthetic peptides corresponding to EF-loops 1 and 2 of PLCη2 and EF-loop 1 of calmodulin (as a control) was examined by 2D-[(1) H,(1) H] TOCSY NMR. Both PLCη2 EF-loop peptides bound Ca(2+) in a similar manner to that of the canonical calmodulin EF-loop 1, particularly at their N-terminus. A molecular model of the PLCη2 EF-hand domain, constructed based upon the structure of calmodulin, suggested both EF-loops may participate in Ca(2+) -binding. To determine whether the EF-hand is responsible for Ca(2+) -sensing, inositol phosphate accumulation was measured in COS7 cells transiently expressing wild-type or mutant PLCη2 proteins. Addition of 70 µM monensin (a Na(+) /H(+) antiporter that increases intracellular Ca(2+) ) induced a 4- to 7-fold increase in wild-type PLCη2 activity. In permeabilized cells, PLCη2 exhibited a ∼4-fold increase in activity in the presence of 1 µM free Ca(2+) . The D256A (EF-loop1) mutant exhibited a ∼10-fold reduction in Ca(2+) -sensitivity and was not activated by monensin, highlighting the involvement of EF-loop 1 in Ca(2+) -sensing. Involvement of EF-loop 2 was examined using D292A, H296A, Q297A, and E304A mutants. Interestingly, the monensin responses and Ca(2+) -sensitivities were largely unaffected by the mutations, indicating that the non-canonical EF-loop 2 is not involved in Ca(2+) -sensing.

Evidence for the Interaction of Endophilin 3 with the SK3 Channels in PC12 Cells

Small conductance calcium-activated (SK) channels play an important role by controlling the after-hyperpolarization of excitable cells. The level of expression and density of these channels is an essential factor for controlling different cellular functions. Several studies showed a co-localization of SK3 channels and Endophilin 3 in different tissues. Using the yeast two hybrid system and the GST pull down assay we demonstrated that Endophilin 3 interact with the N-terminus of SK3 channels. We studied the impact of this interaction on channel activity by patch clamp measurements in PC12 cells expressing endogenous SK3 channels. SK3 currents were activated by using pipette solutions containing 1 μM free Ca2+. Whole-cell measurements of PC12 cells transfected with SH3GL3 showed a reduction of SK3 specific Cs+-currents. SK3 currents could be increased with DC-EBIO (30 μM), an SK-channel activator. These data implicate that Endophilin 3 did not significantly remove SK3 channels from the membrane and that the interaction of Endophilin 3 with SK3 channels presumably caused a change in the Ca2+-sensitivity of the channel which could be overcome by DC-EBIO. This hypothesis needs to be demonstrated in future calcium imaging experiments.Supported by a grant from the DFG (Gr848/16-1).

The Maguk Scaffolding Protein CASK Regulates TMEM16A Channel Function by Phosphorylation

Ca-activated Cl channels (CaCCs) encoded by TMEM16A (Ano1) play essential roles in many physiological processes including epithelial fluid secretion, gut motility, and smooth muscle tone. Using a quantitative SILAC proteomics approach, we discovered that CASK, a unique MAGUK protein containing an N-terminal CaM-kinase homology domain, is very highly enriched in the TMEM16A interactome. CASK contains multiple protein-protein interaction domains linking receptors and signaling molecules at membrane-cytoskeletal interfaces and has unique protein kinase activity that is inhibited by Mg2+. Here we show that CASK co-immunoprecipitates with TMEM16A and regulates the voltage-dependence of TMEM16A in Mg2+-sensitive manner. In the presence of 1 μM free intracellular Ca2+, decreasing intracellular Mg2+ from 2 mM to 0 mM shifts the conductance-voltage (G-V) relationship 98.4 mV in a hyperpolarizing direction without changing GMAX or Ca2+ sensitivity. The Mg2+-dependent shift in the G-V curve is dependent on CASK protein kinase activity because shRNA-knockdown of CASK or replacement of ATP with AMP-PNP eliminates the shift. Mutation of S639 in TMEM16A identifies this amino acid is the site phosphorylated by CASK. The G-V curve of the S639A mutant is unaffected by Mg2+ or ATP and has a V0.5 value similar to WT in the presence of 2 mM Mg2+ [WT(+Mg2+) 98.6 mV; S639A(0Mg2+) 78.8 mV]. The G-V curve of the phosphomimetic S639E mutant is unaffected by Mg2+ or ATP, but the V0.5 value is similar to WT in the absence of Mg2+ [WT(0 Mg2+) −3.5 mV; S639A(2 Mg2+) 7.7 mV]. Inhibitory autocamtide-2 (AIP-2), a CaMKII inhibitory substrate that also interferes with the catalytic activity of CASK in the internal solution efficiently abolishes the effect of Mg2+ on TMEM16A currents. These data suggest that CASK may regulate TMEM16A channels, although the physiological significance in cells remains to be determined.

16 Does Inorganic Phosphate inhibit Cardiac Myofibrillar Force Production at 37°C?

In the healthy heart, inorganic phosphate (Pi) is present in millimolar concentrations, rising to 20 millimolar or more in ischaemic heart. Since acute myocardial ischaemia leads to heart failure, there is need to gain better functional understanding of cardiac preparations in the presence of added Pi. Studies with skinned cardiac myocytes over the range 15°C–23°C have shown that Pi suppresses myofibrillar force generation. However, in skinned skeletal fibres a similar inhibitory action of Pi at low temperatures is reduced or abolished at temperatures closer to physiological. We therefore compared the effects of Pi on force development in skinned single cardiomyocytes at 15°C and 37°C. Permeabilised single cardiomyocytes from human ventricular myocardium (either from healthy, unused donor or from failing hearts) were maximally activated (30 μM free Ca 2+ ) in the presence of Pi (0–20 mM added). A rapid 1% stretch was applied during isometric force development to additionally measure crossbridge kinetics. Maximum Ca 2+ activated force decreased exponentially as [Pi] was raised. The sensitivity to Pi was similar for the two temperatures, e.g. 20 mM added Pi decreased force by ~87% at 15°C and ~78% at 37°C (n = 4, P = 0.74). The rates of stretch-induced cross-bridge detachment (krel) and the subsequent force re-development (kdf) increased exponentially as a function of Pi. These findings suggest that the inhibitory effect of Pi on myofibrillar force development is substantial at both 15°C and 37°C. This deleterious effect is therefore likely to contribute to the contractile dysfunction induced by cardiac ischaemia in vivo .

The Intermediate Conductance Calcium-activated Potassium Channel KCa3.1 Regulates Vascular Smooth Muscle Cell Proliferation via Controlling Calcium-dependent Signaling

The intermediate conductance calcium-activated potassium channel KCa3.1 contributes to a variety of cell activation processes in pathologies such as inflammation, carcinogenesis, and vascular remodeling. We examined the electrophysiological and transcriptional mechanisms by which KCa3.1 regulates vascular smooth muscle cell (VSMC) proliferation. Platelet-derived growth factor-BB (PDGF)-induced proliferation of human coronary artery VSMCs was attenuated by lowering intracellular Ca(2+) concentration ([Ca(2+)]i) and was enhanced by elevating [Ca(2+)]i. KCa3.1 blockade or knockdown inhibited proliferation by suppressing the rise in [Ca(2+)]i and attenuating the expression of phosphorylated cAMP-response element-binding protein (CREB), c-Fos, and neuron-derived orphan receptor-1 (NOR-1). This antiproliferative effect was abolished by elevating [Ca(2+)]i. KCa3.1 overexpression induced VSMC proliferation, and potentiated PDGF-induced proliferation, by inducing CREB phosphorylation, c-Fos, and NOR-1. Pharmacological stimulation of KCa3.1 unexpectedly suppressed proliferation by abolishing the expression and activity of KCa3.1 and PDGF β-receptors and inhibiting the rise in [Ca(2+)]i. The stimulation also attenuated the levels of phosphorylated CREB, c-Fos, and cyclin expression. After KCa3.1 blockade, the characteristic round shape of VSMCs expressing high l-caldesmon and low calponin-1 (dedifferentiation state) was maintained, whereas KCa3.1 stimulation induced a spindle-shaped cellular appearance, with low l-caldesmon and high calponin-1. In conclusion, KCa3.1 plays an important role in VSMC proliferation via controlling Ca(2+)-dependent signaling pathways, and its modulation may therefore constitute a new therapeutic target for cell proliferative diseases such as atherosclerosis.

Structural Studies of the Apo and Ca2+-Bound States of the Human BK (SLO1) Channel Gating Ring in Solution

The gating ring (GR) regulates the activity of large-conductance voltage- and Ca^(2+)-activated K^+ channels (BK) by interacting with intracellular signaling molecules. To understand the operation of this biological sensor under physiological conditions, we performed Small-Angle X-ray Scattering (SAXS) analysis, at beamline 4-2 at the Stanford Synchrotron Radiation laboratory. SAXS measurements of the purified GR were performed in the absence or in the presence of 35 μM free Ca^(2+), found to be a saturating concentration in previous work. The quality of the circularly-averaged scattering data was evaluated with Guinier analysis, while the ATSAS software suite was used to derive structural information. The radius of gyration (R_g) and maximum interparticle distance (D_(max)) of the apo GR were 48.65±1.372 A and 185 A, respectively. These values are comparable to data obtained from crystal structure of GR (3NAF), where the envelope R_g, calculated with CRYSOL, is 45.55 A, and its diameter 155.6 A. Ca^(2+)-bound GR shows a decrease in R_g to 42.77±1.058 A and D_(max) to 160 A, demonstrating the structural response of GR to Ca^(2+). Low-resolution structural models of the GR were generated from the experimental scattering pattern using DAMMIN. The Ca^(2+)-bound GR revealed notable changes in both flexible and assembly interfaces of the superstructure's constituent RCK1 (Regulator of Conductance for K^+) and RCK2 domains. Since the structural changes are resolved under physiologically-relevant conditions, we speculate that they represent the molecular transitions that initiate the Ca^(2+)-induced activation of human BK channels.

Alternative Pathways for Association and Dissociation of the Calmodulin-binding Domain of Plasma Membrane Ca2+-ATPase Isoform 4b (PMCA4b)

The calmodulin (CaM)-binding domain of isoform 4b of the plasma membrane Ca(2+) -ATPase (PMCA) pump is represented by peptide C28. CaM binds to either PMCA or C28 by a mechanism in which the primary anchor residue Trp-1093 binds to the C-terminal lobe of the extended CaM molecule, followed by collapse of CaM with the N-terminal lobe binding to the secondary anchor Phe-1110 (Juranic, N., Atanasova, E., Filoteo, A. G., Macura, S., Prendergast, F. G., Penniston, J. T., and Strehler, E. E. (2010) J. Biol. Chem. 285, 4015-4024). This is a relatively rapid reaction, with an apparent half-time of ~1 s. The dissociation of CaM from PMCA4b or C28 is much slower, with an overall half-time of ~10 min. Using targeted molecular dynamics, we now show that dissociation of Ca(2+)-CaM from C28 may occur by a pathway in which Trp-1093, although deeply embedded in a pocket in the C-terminal lobe of CaM, leaves first. The dissociation begins by relatively rapid release of Trp-1093, followed by very slow release of Phe-1110, removal of C28, and return of CaM to its conformation in the free state. Fluorescence measurements and molecular dynamics calculations concur in showing that this alternative path of release of the PMCA4b CaM-binding domain is quite different from that of binding. The intermediate of dissociation with exposed Trp-1093 has a long lifetime (minutes) and may keep the PMCA primed for activation.

NMR chemical shift assignments for androcam, a testis-specific myosin VI light chain in D. melanogaster

Androcam is a D. melanogaster calmodulin-like protein expressed exclusively in the testis that interacts with myosin VI and is critical to spermatogenesis. At micromolar free Ca(2+), androcam binds two calcium ions using its C-terminal lobe but its N-terminal lobe is Ca(2+)-free. We are pursuing structural studies on androcam at physiological (10 μM) and high (10 mM) calcium. Here we report the (1)H, (15)N, and (13)C chemical shifts of androcam at 10 μM free Ca(2+) determined using multi-dimensional NMR experiments.

Calcineurin Selectively Docks with the Dynamin Ixb Splice Variant to Regulate Activity-dependent Bulk Endocytosis

Depolarization of nerve terminals stimulates rapid dephosphorylation of two isoforms of dynamin I (dynI), mediated by the calcium-dependent phosphatase calcineurin (CaN). Dephosphorylation at the major phosphorylation sites Ser-774/778 promotes a dynI-syndapin I interaction for a specific mode of synaptic vesicle endocytosis called activity-dependent bulk endocytosis (ADBE). DynI has two main splice variants at its extreme C terminus, long or short (dynIxa and dynIxb) varying only by 20 (xa) or 7 (xb) residues. Recombinant GST fusion proteins of dynIxa and dynIxb proline-rich domains (PRDs) were used to pull down interacting proteins from rat brain nerve terminals. Both bound equally to syndapin, but dynIxb PRD exclusively bound to the catalytic subunit of CaNA, which recruited CaNB. Binding of CaN was increased in the presence of calcium and was accompanied by further recruitment of calmodulin. Point mutations showed that the entire C terminus of dynIxb is a CaN docking site related to a conserved CaN docking motif (PXIXI(T/S)). This sequence is unique to dynIxb among all other dynamin variants or genes. Peptide mimetics of the dynIxb tail blocked CaN binding in vitro and selectively inhibited depolarization-evoked dynI dephosphorylation in nerve terminals but not of other dephosphins. Therefore, docking to dynIxb is required for the regulation of both dynI splice variants, yet it does not regulate the phosphorylation cycle of other dephosphins. The peptide blocked ADBE, but not clathrin-mediated endocytosis of synaptic vesicles. Our results indicate that Ca(2+) influx regulates assembly of a fully active CaN-calmodulin complex selectively on the tail of dynIxb and that the complex is recruited to sites of ADBE in nerve terminals.

Metal-driven Operation of the Human Large-conductance Voltage- and Ca2+-dependent Potassium Channel (BK) Gating Ring Apparatus

Large-conductance voltage- and Ca(2+)-dependent K(+) (BK, also known as MaxiK) channels are homo-tetrameric proteins with a broad expression pattern that potently regulate cellular excitability and Ca(2+) homeostasis. Their activation results from the complex synergy between the transmembrane voltage sensors and a large (>300 kDa) C-terminal, cytoplasmic complex (the "gating ring"), which confers sensitivity to intracellular Ca(2+) and other ligands. However, the molecular and biophysical operation of the gating ring remains unclear. We have used spectroscopic and particle-scale optical approaches to probe the metal-sensing properties of the human BK gating ring under physiologically relevant conditions. This functional molecular sensor undergoes Ca(2+)- and Mg(2+)-dependent conformational changes at physiologically relevant concentrations, detected by time-resolved and steady-state fluorescence spectroscopy. The lack of detectable Ba(2+)-evoked structural changes defined the metal selectivity of the gating ring. Neutralization of a high-affinity Ca(2+)-binding site (the "calcium bowl") reduced the Ca(2+) and abolished the Mg(2+) dependence of structural rearrangements. In congruence with electrophysiological investigations, these findings provide biochemical evidence that the gating ring possesses an additional high-affinity Ca(2+)-binding site and that Mg(2+) can bind to the calcium bowl with less affinity than Ca(2+). Dynamic light scattering analysis revealed a reversible Ca(2+)-dependent decrease of the hydrodynamic radius of the gating ring, consistent with a more compact overall shape. These structural changes, resolved under physiologically relevant conditions, likely represent the molecular transitions that initiate the ligand-induced activation of the human BK channel.

Signal mass and Ca2+ kinetics in local calcium events: a modeling study

We use a detailed modeling formalism based on numerical simulations of local calcium release events where the blurring of the image, the presence of diffusional barriers provided by large organelles situated close to the release site, as well as the variable position of the scan line with respect to the release site are taken into consideration. We have investigated the effect of the fluorescence noise fluctuations on the accuracy in computing the signal mass from linescan recordings and obtained a quantitative description of both the signal mass and the local increase in the free Ca(2+) level as a function of the release current, the release duration and the orientation of the scan line, for three different levels of noise magnitudes. The model could provide a very good fit to a wide set of available experimental data regarding the signal mass of puffs visualized by fluorescence microscopy in the Xenopus oocyte loaded with 40 μM Oregon Green-1 in the absence of the calcium chelator EGTA. Numerical simulations also predict the amplitude and the kinetics of calcium signals evolving in the absence of the indicator, and indicate that sub-maximal activation of IP(3) receptors could produce in average levels of about 2 μM and 0.4 μM free Ca(2+) close to a release site located in the animal or in the vegetal hemisphere, respectively, whereas the maximal levels reached in more rare events could be 11 μM and 4 μM, respectively.

Lobe-specific Functions of Ca2+·Calmodulin in αCa2+·Calmodulin-dependent Protein Kinase II Activation

N-Methyl-d-aspartic acid receptor-dependent long term potentiation (LTP), a model of memory formation, requires Ca2+·calmodulin-dependent protein kinase II (αCaMKII) activity and Thr286 autophosphorylation via both global and local Ca2+ signaling, but the mechanisms of signal transduction are not understood. We tested the hypothesis that the Ca2+-binding activator protein calmodulin (CaM) is the primary decoder of Ca2+ signals, thereby determining the output, e.g. LTP. Thus, we investigated the function of CaM mutants, deficient in Ca2+ binding at sites 1 and 2 of the N-terminal lobe or sites 3 and 4 of the C-terminal CaM lobe, in the activation of αCaMKII. Occupancy of CaM Ca2+ binding sites 1, 3, and 4 is necessary and sufficient for full activation. Moreover, the N- and C-terminal CaM lobes have distinct functions. Ca2+ binding to N lobe Ca2+ binding site 1 increases the turnover rate of the enzyme 5-fold, whereas the C lobe plays a dual role; it is required for full activity, but in addition, via Ca2+ binding site 3, it stabilizes ATP binding to αCaMKII 4-fold. Thr286 autophosphorylation is also dependent on Ca2+ binding sites on both the N and the C lobes of CaM. As the CaM C lobe sites are populated by low amplitude/low frequency (global) Ca2+ signals, but occupancy of N lobe site 1 and thus activation of αCaMKII requires high amplitude/high frequency (local) Ca2+ signals, lobe-specific sensing of Ca2+-signaling patterns by CaM is proposed to explain the requirement for both global and local Ca2+ signaling in the induction of LTP via αCaMKII.

On the Properties of the RCK1 Domain of the Human BK (SLO1) Channel

In BK channels, four RCK1 and four RCK2 domains assemble into a cytosolic ligand-sensing superstructure known as the gating ring. While electrophysiological data suggest that both RCK1 and RCK2 contain high affinity Ca2+-sensing sites, recent crystallographic data (Wu et al., 2010) has revealed Ca2+ binding only within the RCK2 domain. Does the RCK1 domain bind Ca2+? What is the role of the high-affinity Ca2+ sensing residues D362/D367 (Xia, et al 2002) located in RCK1 domain? To address these questions, we expressed and purified the region of the human BK channel C-terminus corresponding to the amino acid sequence 322IIE¼HDP667. We have probed the structure and Ca2+ sensing properties of the RCK1 domain in solution, under physiologically relevant conditions. In addition to the α/β fold shared with its bacterial counterparts and human BK RCK2 domain, the RCK1 domain preferentially self-assembles into a homo-octameric structure. We recently reported that RCK1 undergoes Ca2+-dependent conformational changes similarly to the RCK2 domain (Yusifov et al., 2008 and 2010). The neutralization of residues D362/D367 altered the secondary and quaternary structure of the RCK1 domain and prevented Ca2+-induced structural transitions in RCK1. 45Ca2+ overlay assay suggested that the neutralization of D362/367 did not abolish the Ca2+-binding activity of RCK1 in the range of 2.1-115 µM free Ca2+. While it cannot be excluded that D362/D367 are elements of a Ca2+ binding site with multiple coordinating residues, our results favor the view that D362 /D367 have a predominantly structural role. Possibly, these mutations set RCK1 domains in a conformational state that hampers the propagation of structural changes caused by ligand binding.