Fermentation product butane 2,3-diol induces Ca2+ transients in E. coli through activation of lanthanum-sensitive Ca2+ channels

The results here are the first demonstration of a physiological agonist opening Ca2+ channels in bacteria. Bacteria in the gut ferment glucose and other substrates, producing alcohols, diols, ketones and acids, that play a key role in lactose intolerance, through the activation of Ca2+ and other ion channels in host cells and neighbouring bacteria. Here we show butane 2,3-diol (5-200mM; half maximum 25mM) activates Ca2+ transients in E. coli, monitored by aequorin. Ca2+-transient magnitude depended on external Ca2+ (0.1-10mM). meso-Butane 2,3-diol was approximately twice as potent as 2R,3R (-) and 2S,3S (+) butane 2,3-diol. There were no detectable effects on cytosolic free Ca2+ of butane 1,3-diol, butane 1,4-diol and ethylene glycol. The glycerol fermentation product propane 1,3-diol only induced significant Ca2+ transients in 10mM external Ca2. Ca2+ butane 2,3-diol Ca2+ transients were due to activation of Ca2+ influx, followed by activation of Ca2+ efflux. The effect of butane 2,3-diol was abolished by La3+, and markedly reduced as a function of growth phase. These results were consistent with butane 2,3-diol activating a novel La3+-sensitive Ca2+ channel. They have important implications for the role of butane 2,3-diol and Ca2+ in bacterial-host cell signalling.     

Ca significantly enhanced development and salt-secretion rate of salt glands of Limonium bicolor under NaCl treatment

The divalent cation, Ca²⁺, plays crucial roles in plant growth, development and stress resistance. Limonium bicolor seedlings were treated with 200 mM NaCl combined with three levels of Ca²⁺ (0 mM, 5 mM and 20 mM) for 15 days to study the effects of Ca²⁺ on development and salt-secretion rates of salt glands. It was shown that the 4th leaf areas of L. bicolor seedlings under 20 mM Ca²⁺ treatment were significantly higher than those under 0 mM and 5 mM Ca²⁺ treatments. The total number and the densities of salt glands per leaf increased markedly with increased Ca²⁺ concentrations. The diameters of salt glands increased by 59% and 63% as Ca²⁺ concentration increased from zero to 5 mM and 20 mM, respectively. Under 20 mM Ca²⁺ treatment, the salt-secretion rate per leaf was obviously higher than that treated with 5 mM Ca²⁺, but there was no significant difference in the salt-secretion rates per salt gland between the two groups. Under 0 mM Ca²⁺ treatment, leaf-cell membrane permeability increased significantly, which led to serious leakage of ions and a significant increase in Na⁺ loss rate. The results showed that the increase of Ca²⁺ concentration markedly enhanced development and salt-secretion rates of salt glands in the leaves of L. bicolor, the increase of salt secretion per leaf is due to the efficiency of the secretion process per salt gland and the number of salt glands, the salt-secretion rates per salt gland have a relationship with the diameters of salt glands.     

K+ channel gating: C-type inactivation is enhanced by calcium or lanthanum outside

Many voltage-gated K⁺ channels exhibit C-type inactivation. This typically slow process has been hypothesized to result from dilation of the outer-most ring of the carbonyls in the selectivity filter, destroying this ring’s ability to bind K⁺ with high affinity. We report here strong enhancement of C-type inactivation upon extracellular addition of 10–40 mM Ca²⁺ or 5–50 µM La³⁺. These multivalent cations mildly increase the rate of C-type inactivation during depolarization and markedly promote inactivation and/or suppress recovery when membrane voltage (V_m) is at resting levels (−80 to −100 mV). At −80 mV with 40 mM Ca²⁺ and 0 mM K⁺ externally, ShBΔN channels with the mutation T449A inactivate almost completely within 2 min or less with no pulsing. This behavior is observed only in those mutants that show C-type inactivation on depolarization and is distinct from the effects of Ca²⁺ and La³⁺ on activation (opening and closing of the V_m-controlled gate), i.e., slower activation of K⁺ channels and a positive shift of the mid-voltage of activation. The Ca²⁺/La³⁺ effects on C-type inactivation are antagonized by extracellular K⁺ in the low millimolar range. This, together with the known ability of Ca²⁺ and La³⁺ to block inward current through K⁺ channels at negative voltage, strongly suggests that Ca²⁺/La³⁺ acts at the outer mouth of the selectivity filter. We propose that at −80 mV, Ca²⁺ or La³⁺ ions compete effectively with K⁺ at the channel’s outer mouth and prevent K⁺ from stabilizing the filter’s outer carbonyl ring.     

Octopaminergic modulation of the single Ca2+ channel currents in Kenyon cells isolated from the mushroom body of the cricket brain

Octopamine plays an important role in mediating reward signals in olfactory learning and memory formation in insect. However, its target molecules and signaling pathways are still unknown. In this study, we investigated the effects of octopamine on the voltage-activated Ca²⁺ channels expressed in native Kenyon cells isolated from the mushroom body of the cricket (Gryllus bimaculatus) brain. The cell-attached patch clamp recordings with 100 mM Ba²⁺ outside showed the presence of dihydropyridine (DHP) sensitive L-type Ca²⁺ channels with a single channel conductance of approximately 21 ± 2 pS (n = 12). The open probability (NPo) of single Ca²⁺ channel currents decreased by about 29 ± 7% (n = 6) by bath application of 10 μM octopamine. Octopamine-induced decrease in Po was imitated by bath application of 8-Br-cAMP, a membrane-permeable cAMP analog. Pre-treatment of Kenyon cells with the octopamine receptor antagonist phentolamine blocked the inhibitory effect of octopamine on Ca²⁺ channels. Pre-treatment of Kenyon cells with H-89, a selective inhibitor of cAMP-dependent protein kinase (PKA) attenuated the inhibitory effect of bath applied octopamine on Ca²⁺ channels. These results indicate that DHP-sensitive L-type Ca²⁺ channel is a target protein for octopamine and its modulation is mediated via cAMP and PKA-dependent signaling pathways in freshly isolated Kenyon cell in the cricket G. bimaculatus.     

Physiologic gating properties of unitary cardiac L-type Ca channels

The contraction of adult mammalian ventricular cardiomyocytes is triggered by the influx of Ca²⁺ ions through sarcolemmal L-type Ca²⁺ channels (LCCs). However, the gating properties of unitary LCCs under physiologic conditions have remained elusive. Towards this end, we investigated the voltage-dependence of the gating kinetics of unitary LCCs, with a physiologic concentration of Ca²⁺ ions permeating the channel. Unitary LCC currents were recorded with 2 mM external Ca²⁺ ions (in the absence of LCC agonists), using cell-attached patches on K-depolarized adult rat ventricular myocytes. The voltage-dependence of the peak probability of channel opening (Po vs. Vm) displayed a maximum value of 0.3, a midpoint of −12 mV, and a slope factor of 8.5. The maximum value for Po of the unitary LCC was significantly higher than previously assumed, under physiologic conditions. We also found that the mean open dwell time of the unitary LCC increased twofold with depolarization, ranging from 0.53 ± 0.02 ms at −30 mV to 1.08 ± 0.03 ms at 0 mV. The increase in mean LCC open time with depolarization counterbalanced the decrease in the single LCC current amplitude; the latter due to the decrease in driving force for Ca²⁺ ion entry. Thus, the average amount of Ca²⁺ ions entering through an individual LCC opening (∼300-400 ions) remained relatively constant over this range of potentials. These novel results establish the voltage-dependence of unitary LCC gating kinetics using a physiologic Ca²⁺ ion concentration. Moreover, they provide insight into local Ca²⁺-induced Ca²⁺ release and a more accurate basis for mathematical modeling of excitation-contraction coupling in cardiac myocytes.     

The relationship between single-channel and whole-cell conductance in the T-type Ca2+ channel CaV3.1

In T-type Ca²⁺ channels, macroscopic I_Ba is usually smaller than I_Ca, but at high Ca²⁺ and Ba²⁺, single-channel conductance (γ) is equal. We investigated γ as a function of divalent concentration and compared it to macroscopic currents using Ca_V3.1 channels studied under similar experimental conditions (TEA_o and K_i). Single-channel current-voltage relationships were nonlinear in a way similar to macroscopic open-channel I/Vs, so divalent γ was underestimated at depolarized voltages. To estimate divalent γ, concentration dependence, i_Div, was measured at voltages <−50 mV. Data were well described by Langmuir isotherms with γ_max(Ca²⁺) of 9.5 ± 0.4 pS and γ_max(Ba²⁺) of 10.3 ± 0.5 pS. Apparent K_M was lower for Ca²⁺ (2.3 ± 0.7 mM) than for Ba²⁺ (7.9 ± 1.3 mM). A subconductance state with an amplitude 70% that of the main state was observed, the relative occupancy of which increased with increasing Ca²⁺. As predicted by γ, macroscopic G_maxCa was larger than G_maxBa at 5 mM (G_maxCa²⁺/Ba:²⁺1.43 ± 0.14) and similar at 60 mM (G_maxCa²⁺/Ba:²⁺1.10 ± 0.02). However, over the range of activation, I_Ca was larger than I_Ba under both conditions. This was a consequence of the fact that V_rev was more negative for I_Ba than for I_Ca, so that the driving force determining I_Ba was smaller than that determining I_Ca over the range of potentials in standard current-voltage relationships.     

Coupled gating modifies the regulation of cardiac ryanodine receptors by luminal Ca

Cardiac ryanodine receptors (RYR2s) infrequently exhibit coupled gating that is manifested by synchronous opening and closing. To better characterize this phenomenon, we investigated the regulation of coupled RYR2 channels by luminal Ca^2 + focusing on effects that are likely mediated by the true luminal activation mechanism. By reconstituting an ion channel into a planar lipid bilayer and using substantially lower concentration of luminal Ba^2 + (8 mM, the virtual absence of Ca^2 +) and luminal Ca^2 + (8 mM), we show that response of coupled RYR2 channels to caffeine at a diastolic cytosolic Ca^2 + (90 nM) was affected by luminal Ca^2 + in a similar manner as for the single RYR2 channel except the gating behavior. Whereas, the single RYR2 channel responded to luminal Ca^2 + by prolongation in open and closed times, coupled RYR2 channels seemed to be resistant in this respect. In summary, we conclude that the class of Ca^2 + sites located on the luminal face of coupled RYR2 channels that is responsible for the channel potentiation by luminal Ca^2 + is functional and not structurally hindered by the channel coupling. Thus, the idea about non-functional luminal Ca^2 + sites as a source of the apparent gating resistance of coupled RYR2 channels to luminal Ca^2 + appears to be ruled out.     

Recruitment of the intracellular Ca by ultrashort electric stimuli: The impact of pulse duration

Nanosecond-duration electric stimuli are distinguished by the ability to permeabilize intracellular membranes and recruit Ca²⁺ from intracellular stores. We quantified this effect in non-excitable cells (CHO) using ratiometric Ca²⁺ imaging with Fura-2. In a Ca²⁺-free medium, 10-, 60-, and 300-ns stimuli evoked Ca²⁺ transients by mobilization of Ca²⁺ from the endoplasmic reticulum. With 2 mM external Ca²⁺, the transients included both extra- and intracellular components. The recruitment of intracellular Ca²⁺ increased as the stimulus duration decreased. At the threshold of 200–300 nM, the transients were amplified by calcium-induced calcium release. We conclude that nanosecond stimuli mimic Ca²⁺ signaling while bypassing the usual receptor- and channels-mediated cascades. The recruitment of the intracellular Ca²⁺ can be controlled by the duration of the stimulus.     

Characterization of Ca and phosphocholine interactions with C-reactive protein using a surface plasmon resonance biosensor

The interactions between Ca²⁺ and C-reactive protein (CRP) have been characterized using a surface plasmon resonance (SPR) biosensor. The protein was immobilized on a sensor chip, and increasing concentrations of Ca²⁺ or phosphocholine were injected. Binding of Ca²⁺ induced a 10-fold higher signal than expected from the molecular weight of Ca²⁺. It was interpreted to result from the conformational change that occurs on binding of Ca²⁺. Two sites with different characteristics were distinguished: a high-affinity site with K_D = 0.03 mM and a low-affinity site with K_D = 5.45 mM. The pH dependencies of the two Ca²⁺ interactions were different and enabled the assignment of the different sites in the three-dimensional structure of CRP. There was no evidence for cooperativity in the phosphocholine interaction, which had K_D = 5 μM at 10 mM Ca²⁺. SPR biosensors can clearly detect and quantify the binding of very small molecules or ions to immobilized proteins despite the theoretically very low signals expected on binding, provided that significant conformational changes are involved. Both the interactions and the conformational changes can be characterized. The data have important implications for the understanding of the function of CRP and suggest that Ca²⁺ is an efficient regulator under physiological conditions.     

Strong alkalinization of Chara cell surface in the area of cell wall incision as an early event in mechanoperception

Mechanical wounding of cell walls occurring in plants under the impact of pathogens or herbivores can be mimicked by cell wall incision with a glass micropipette. Measurements of pH at the surface of Chara corallina internodes following microperforation of cell wall revealed a rapid (10–30 s) localized alkalinization of the apoplast after a lag period of 10–20 s. The pH increase induced by incision could be as large as 3 pH units and relaxed slowly, with a halftime up to 20 min. The axial pH profile around the incision zone was bell-shaped and localized to a small area, extending over a distance of about 100 μm. The pH response was suppressed by lowering cell turgor upon the replacement of artificial pond water (APW) with APW containing 50 mM sorbitol. Stretching of the plasma membrane during its impression into the cell wall defect is likely to activate the Ca^2 + channels, as evidenced from sensitivity of the incision-induced alkalinization to the external calcium concentration and to the addition of Ca^2 +-channel blockers, such as La^3 +, Gd^3 +, and Zn^2 +. The maximal pH values attained at the incision site (~ 10.0) were close to pH in light-dependent alkaline zones of Chara cells. The involvement of cytoskeleton in the origin of alkaline patch was documented by observations that the incision-induced pH transients were suppressed by the inhibitors of microtubules (oryzalin and taxol) and, to a lesser extent, by the actin inhibitor (cytochalasin B). The results indicate that the localized increase in apoplastic pH is an early event in mechanoperception and depends on light, cytoskeleton, and intracellular calcium.     

Thermo-sensitive transient receptor potential vanilloid channel-1 regulates intracellular calcium and triggers chromogranin A secretion in pancreatic neuroendocrine BON-1 tumor cells

Transient receptor potential channels (TRPs) regulate tumor growth via calcium-dependent mechanisms. The (thermosensitive) capsaicin receptor TRPV1 is overexpressed in numerous highly aggressive cancers. TRPV1 has potent antiproliferative activity and is therefore potentially applicable in targeted therapy of malignancies. Recently, we characterized TRPM8 functions in pancreatic neuroendocrine tumors (NETs), however, the role of TRPV1 is unknown. Here, we studied the expression and the role of TRPV1 in regulating intracellular Ca²⁺ and chromogranin A (CgA) secretion in pancreatic NET BON-1 cell line and in primary NET cells (prNET). TRPV1 expression was detected by RT-PCR, Western blot and immunofluorescence. Intracellular free Ca²⁺ ([Ca²⁺]_i) was measured by fura-2; TRPV1 channel currents by the planar patch-clamp technique. Nonselective cation currents were analyzed by a color-coded plot method and CgA secretion by ELISA. Pancreatic BON-1 cells and NETs express TRPV1. Pharmacological blockade of TRPs by La³⁺ (100 μM) or by ruthenium-red (RuR) or by capsazepine (CPZ) (both at 10 μM) suppressed the capsaicin (CAP)- or heat-stimulated increase of [Ca²⁺]_i in NET cells. CAP (20 μM) also increased nonselective cation channel currents in BON-1 cells. Furthermore, CAP (10 μM) stimulated CgA secretion, which was inhibited by CPZ or by RuR (both 10 μM). La³⁺ potently reduced both stimulated and the basal CgA secretion. Our study shows for the first time that TRPV1 is expressed in pancreatic NETs. Activation of TRPV1 translates into changes of intracellular Ca²⁺, a known mechanism triggering the secretion of CgA. The clinical relevance of TRPV1 activation in NETs requires further investigations.     

Modeling local and global intracellular calcium responses mediated by diffusely distributed inositol 1,4,5-trisphosphate receptors

Considerable insight into intracellular Ca²⁺ responses has been obtained through the development of whole cell models that are based on molecular mechanisms, e.g., single channel kinetics of the inositol 1,4,5-trisphosphate (IP₃) receptor Ca²⁺ channel. However, a limitation of most whole cell models to date is the assumption that IP₃ receptor Ca²⁺ channels (IP₃Rs) are globally coupled by a “continuously stirred” bulk cytosolic [Ca²⁺], when in fact open IP₃Rs experience elevated “domain” Ca²⁺ concentrations. Here we present a 2N+2-compartment whole cell model of local and global Ca²⁺ responses mediated by N=100,000 diffusely distributed IP₃Rs, each represented by a four-state Markov chain. Two of these compartments correspond to bulk cytosolic and luminal Ca²⁺ concentrations, and the remaining 2N compartments represent time-dependent cytosolic and luminal Ca²⁺ domains associated with each IP₃R. Using this Monte Carlo model as a starting point, we present an alternative formulation that solves a system of advection-reaction equations for the probability density of cytosolic and luminal domain [Ca²⁺] jointly distributed with IP₃R state. When these equations are coupled to ordinary differential equations for the bulk cytosolic and luminal [Ca²⁺], a realistic but minimal model of whole cell Ca²⁺ dynamics is produced that accounts for the influence of local Ca²⁺ signaling on channel gating and global Ca²⁺ responses. The probability density approach is benchmarked and validated by comparison to Monte Carlo simulations, and the two methods are shown to agree when the number of Ca²⁺ channels is large (i.e., physiologically realistic). Using the probability density approach, we show that the time scale of Ca²⁺ domain formation and collapse (both cytosolic and luminal) may influence global Ca²⁺ oscillations, and we derive two reduced models of global Ca²⁺ dynamics that account for the influence of local Ca²⁺ signaling on global Ca²⁺ dynamics when there is a separation of time scales between the stochastic gating of IP₃Rs and the dynamics of domain Ca²⁺.     

The cardiac Ca2+-sensitive regulatory switch, a system in dynamic equilibrium

The Ca²⁺-sensitive regulatory switch of cardiac muscle is a paradigmatic example of protein assemblies that communicate ligand binding through allosteric change. The switch is a dimeric complex of troponin C (TnC), an allosteric sensor for Ca²⁺, and troponin I (TnI), an allosteric reporter. Time-resolved equilibrium Förster resonance energy transfer (FRET) measurements suggest that the switch activates in two steps: a TnI-independent Ca²⁺-priming step followed by TnI-dependent opening. To resolve the mechanistic role of TnI in activation we performed stopped-flow FRET measurements of activation after rapid addition of a lacking component (Ca²⁺ or TnI) and deactivation after rapid chelation of Ca²⁺. Time-resolved measurements, stopped-flow measurements, and Ca²⁺-titration measurements were globally analyzed in terms of a new quantitative dynamic model of TnC-TnI allostery. The analysis provided a mesoscopic parameterization of distance changes, free energy changes, and transition rates among the accessible coarse-grained states of the system. The results reveal that 1), the Ca²⁺-induced priming step, which precedes opening, is the rate-limiting step in activation; 2), closing is the rate-limiting step in de-activation; 3), TnI induces opening; 4), there is an incompletely deactivated population when regulatory Ca²⁺ is not bound, which generates an accessory pathway of activation; and 5), there is incomplete activation by Ca²⁺—when regulatory Ca²⁺ is bound, a 3:2 mixture of dynamically interconverting open (active) and primed-closed (partially active) conformers is observed (15°C). Temperature-dependent stopped-flow FRET experiments provide a near complete thermokinetic parameterization of opening: the enthalpy change (ΔH = −33.4 kJ/mol), entropy change (ΔS = −0.110 kJ/mol/K), heat capacity change (ΔC_p = −7.6 kJ/mol/K), the enthalpy of activation (δ^‡ = 10.6 kJ/mol) and the effective barrier crossing attempt frequency (ν_adj = 1.8 × 10⁴ s⁻¹).     

Measuring Ca binding to short chain fatty acids and gluconate with a Ca electrode: Role of the reference electrode

Many organic anions bind free Ca²⁺, the total concentration of which must be adjusted in experimental solutions. Because published values for the apparent dissociation constant (K_app) describing the Ca²⁺ affinity of short chain fatty acids (SCFAs) and gluconate are highly variable, Ca²⁺ electrodes coupled to either a 3 M KCl or a Na⁺ selective electrode were used to redetermine K_app. All solutions contained 130 mM Na⁺, whereas the concentration of the studied anion was varied from 15 to 120 mM, replacing Cl⁻ that was decreased concomitantly to maintain osmolarity. This induces changes in the liquid junction potential (LJP) at the 3 M KCl reference electrode, leading to a systematic underestimation of K_app if left uncorrected. Because the Na⁺ concentration in all solutions was constant, a Na⁺ electrode was used to directly measure the changes in the LJP at the 3 M KCl reference, which were under 5 mV but twice those predicted by the Henderson equation. Determination of K_app either after correction for these LJP changes or via direct reference to a Na⁺ electrode showed that SCFAs do not bind Ca²⁺ and that the K_app for the binding of Ca²⁺ to gluconate at pH 7.4, ionic strength 0.15 M, and 23 °C was 52.7 mM.     

Store-operated Ca entry and depolarization explain the anomalous behaviour of myometrial SR: Effects of SERCA inhibition on electrical activity,Ca and force

In the myometrium SR Ca²⁺ depletion promotes an increase in force but unlike several other smooth muscles, there is no Ca²⁺ sparks-STOCs coupling mechanism to explain this. Given the importance of the control of contractility for successful parturition, we have examined, in pregnant rat myometrium, the effects of SR Ca²⁺-ATPase (SERCA) inhibition on the temporal relationship between action potentials, Ca²⁺ transients and force. Simultaneous recording of electrical activity, calcium and force showed that SERCA inhibition, by cyclopiazonic acid (CPA 20 μM), caused time-dependent changes in excitability, most noticeably depolarization and elevations of baseline [Ca²⁺]_i and force. At the onset of these changes there was a prolongation of the bursts of action potentials and a corresponding series of Ca²⁺ spikes, which increased the amplitude and duration of contractions. As the rise of baseline Ca²⁺ and depolarization continued a point was reached when electrical and Ca²⁺ spikes and phasic contractions ceased, and a maintained, tonic force and Ca²⁺ was produced. Lanthanum, a non-selective blocker of store-operated Ca²⁺ entry, but not the L-type Ca²⁺ channel blocker nifedipine (1–10 μM), could abolish the maintained force and calcium. Application of the agonist, carbachol, produced similar effects to CPA, i.e. depolarization, elevation of force and calcium. A brief, high concentration of carbachol, to cause SR Ca²⁺ depletion without eliciting receptor-operated channel opening, also produced these results. The data obtained suggest that in pregnant rats SR Ca²⁺ release is coupled to marked Ca²⁺ entry, via store operated Ca²⁺ channels, leading to depolarization and enhanced electrical and mechanical activity.     

PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba²⁺ currents through voltage gated Ca²⁺ channels. Half maximal inhibitions occurred at 0.64 ± 0.07 mM and 0.86 ± 0.1 mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPβS (100 μM) together with staurosporine (200 nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca²⁺ channel blockers, volatile anesthetics blocked Ca²⁺ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an ‘initial block’. After the first 4–7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca²⁺ channel current whose amplitude was reduced to 41 ± 3.4% and to 57 ± 4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca²⁺ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer.     

How calcium controls microtubule anisotropic phase formation in the presence of microtubule-associated proteins in vitro

Here we show a new effect of Ca²⁺ on microtubule morphology: Ca²⁺ can cause smooth curving of microtubules in the presence of microtubule-associated proteins (MAPs). In vitro, microtubules self-organize, forming complex dissipative structures. Such structures may be strongly affected by relatively weak external factors. A factor such as Ca²⁺ potentially influences spatiotemporal patterns of microtubule assembly, but the dynamics are unclear. We tested Ca²⁺ effects on microtuble formation. Using EM, microtubule length, curvature, and alignment and were measured in two systems: 2 mg/ml microtubule protein containing MAPs and 1 mM EGTA with and without 1 mM Ca²⁺. The two systems were then tested using light scattering. In low Ca²⁺, a birefringent microtubular pattern is seen, increasing with polymerization. When 1 mM Ca²⁺ is added to the solution. anisotropic phase is prevented without microtubule disruption. This demonstrates an additional mechanism by which Ca²⁺ can alter the dynamics and morphology of microtules.     

Selectivity signatures of three isoforms of recombinant T-type Ca channels

Voltage-gated Ca²⁺ channels (VGCCs) are recognized for their superb ability for the preferred passage of Ca²⁺ over any other more abundant cation present in the physiological saline. Most of our knowledge about the mechanisms of selective Ca²⁺ permeation through VGCCs was derived from the studies on native and recombinant L-type representatives. However, the specifics of the selectivity and permeation of known recombinant T-type Ca²⁺-channel α1 subunits, Ca_v3.1, Ca_v3.2 and Ca_v3.3, are still poorly defined. In the present study we provide comparative analysis of the selectivity and permeation Ca_v3.1, Ca_v3.2, and Ca_v3.3 functionally expressed in Xenopus oocytes. Our data show that all Ca_v3 channels select Ca²⁺ over Na⁺ by affinity. Ca_v3.1 and Ca_v3.2 discriminate Ca²⁺, Sr²⁺ and Ba²⁺ based on the ion's effects on the open channel probability, whilst Ca_v3.3 discriminates based on the ion's intrapore binding affinity. All Ca_v3s were characterized by much smaller difference in the K_D values for Na⁺ current blockade by Ca²⁺ (K_D1 ∼ 6 μM) and for Ca²⁺ current saturation (K_D2 ∼ 2 mM) as compared to L-type channels. This enabled them to carry notable mixed Na⁺/Ca²⁺ current at close to physiological Ca²⁺ concentrations, which was the strongest for Ca_v3.3, smaller for Ca_v3.2 and the smallest for Ca_v3.1. In addition to intrapore Ca²⁺ binding site(s) Ca_v3.2, but not Ca_v3.1 and Ca_v3.3, is likely to possess an extracellular Ca²⁺ binding site that controls channel permeation. Our results provide novel functional tests for identifying subunits responsible for T-type Ca²⁺ current in native cells.     

Microglial calcium signal acts as a rapid sensor of single neuron damage in vivo

In the healthy adult brain microglia, the main immune-competent cells of the CNS, have a distinct (so-called resting or surveying) phenotype. Resting microglia can only be studied in vivo since any isolation of brain tissue inevitably triggers microglial activation. Here we used in vivo two-photon imaging to obtain a first insight into Ca²⁺ signaling in resting cortical microglia. The majority (80%) of microglial cells showed no spontaneous Ca²⁺ transients at rest and in conditions of strong neuronal activity. However, they reliably responded with large, generalized Ca²⁺ transients to damage of an individual neuron. These damage-induced responses had a short latency (0.4-4 s) and were localized to the immediate vicinity of the damaged neuron (< 50 μm cell body-to-cell body distance). They were occluded by the application of ATPγS as well as UDP and 2-MeSADP, the agonists of metabotropic P2Y receptors, and they required Ca²⁺ release from the intracellular Ca²⁺ stores. Thus, our in vivo data suggest that microglial Ca²⁺ signals occur mostly under pathological conditions and identify a Ca²⁺ store-operated signal, which represents a very sensitive, rapid, and highly localized response of microglial cells to brain damage. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Kinetics of the Ca, H, and Mg Interaction with the Ion-Binding Sites of the SR Ca-ATPase

Electrochromic styryl dyes were used to investigate mutually antagonistic effects of Ca²⁺ and H⁺ on binding of the other ion in the E₁ and P-E₂ states of the SR Ca-ATPase. On the cytoplasmic side of the protein in the absence of Mg²⁺ a strictly competitive binding sequence, H₂E₁?HE₁?E₁?CaE₁?Ca₂E₁, was found with two Ca²⁺ ions bound cooperatively. The apparent equilibrium dissociation constants were in the order of K_1/2(2 Ca) = 34 nM, K_1/2(H) = 1 nM and K_1/2(H₂) = 1.32 μM. Up to 2 Mg²⁺ ions were also able to enter the binding sites electrogenically and to compete with the transported substrate ions (K_1/2(Mg) = 165 μM, K_1/2(Mg₂) = 7.4 mM). In the P-E₂ state, with binding sites facing the lumen of the sarcoplasmatic reticulum, the measured concentration dependence of Ca²⁺ and H⁺ binding could be described satisfactorily only with a branched reaction scheme in which a mixed state, P-E₂CaH, exists. From numerical simulations, equilibrium dissociation constants could be determined for Ca²⁺ (0.4 mM and 25 mM) and H⁺ (2 μM and 10 μM). These simulations reproduced all observed antagonistic concentration dependences. The comparison of the dielectric ion binding in the E₁ and P-E₂ conformations indicates that the transition between both conformations is accompanied by a shift of their (dielectric) position.