TPC2 mediates new mechanisms of platelet dense granule membrane dynamics through regulation of Ca2+ release

Platelet dense granules (PDGs) are acidic calcium stores essential for normal hemostasis. They develop from late endosomal compartments upon receiving PDG-specific proteins through vesicular trafficking, but their maturation process is not well understood. Here we show that two-pore channel 2 (TPC2) is a component of the PDG membrane that regulates PDG luminal pH and the pool of releasable Ca²⁺. Using a genetically encoded Ca²⁺ biosensor and a pore mutant TPC2, we establish the function of TPC2 in Ca²⁺ release from PDGs and the formation of perigranular Ca²⁺ nanodomains. For the first time, Ca²⁺ spikes around PDGs—or any organelle of the endolysosome family—are visualized in real time and revealed to precisely mark organelle “kiss-and-run” events. Further, the presence of membranous tubules transiently connecting PDGs is revealed and shown to be dramatically enhanced by TPC2 in a mechanism that requires ion flux through TPC2. “Kiss-and-run” events and tubule connections mediate transfer of membrane proteins and luminal content between PDGs. The results show that PDGs use previously unknown mechanisms of membrane dynamics and content exchange that are regulated by TPC2.     

Distinct roles for CaV1.1’s voltage-sensing domains

Study reveals how a slowly activating calcium channel is able to control rapid excitation–contraction coupling in skeletal muscle.

Skeletal muscle contraction is initiated by action potentials that depolarize the muscle fiber and trigger the rapid release of Ca²⁺ from the SR via RYR1 channels. This process of excitation–contraction coupling depends on voltage-gated Ca_V1.1 channels in the plasma membrane, or sarcolemma, of muscle fibers. But Ca_V1.1 channels are only slowly activated by changes in the sarcolemma membrane potential, and it is therefore unclear how they are able to trigger the much faster activation of RYR1 channels. In this issue of JGP, Savalli et al. reveal that this paradox can be explained by the fact that each of Ca_V1.1’s four voltage-sensing domains (VSDs) have distinct biophysical properties (1).Nicoletta Savalli (left), Riccardo Olcese (center), and colleagues reveal the distinct physical properties of the Ca_V1.1 channel’s four voltage-sensing domains (VSD I–IV, right). VSD-I shows slow activation kinetics and is the main contributor to the opening of Ca_V1.1. The other VSDs activate much faster and may therefore be coupled to RYR1 to mediate the rapid release of Ca²⁺ from the SR during skeletal muscle contraction.RYR1 channels have no voltage-sensing machinery of their own and therefore rely on a physical connection to Ca_V1.1 channels to release Ca²⁺ and initiate muscle contraction in response to muscle fiber depolarization. But RYR1 channels open ∼25 times faster than Ca_V1.1 channels. “So, how can these slowly activating Ca_V1.1 channels trigger the rapid release of Ca²⁺ from the SR?” asks Riccardo Olcese, a professor at the David Geffen School of Medicine, UCLA.Olcese and colleagues, including Assistant Project Scientist Nicoletta Savalli, suspected that the answer might lie in the fact that, like many other voltage-gated ion channels, Ca_V1.1 has four VSDs that alter their conformation in response to voltage changes. These domains are similar, but not identical, to each other, potentially enabling them to have distinct biophysical properties and perform distinct functions. Indeed, Olcese and colleagues previously demonstrated that, in the closely related channel Ca_V1.2, only VSDs II and III are involved in pore opening (2, 3).Savalli et al. used voltage-clamp fluorometry to compare the properties of Ca_V1.1’s VSDs, expressing the channel in Xenopus oocytes and labeling each of its VSDs in turn with an environmentally sensitive fluorophore to report voltage-dependent changes in their conformation (1). “We found that the four VSDs were very heterogenous in both their kinetics and voltage dependencies,” says Olcese. “VSD-I had very slow kinetics, compatible with the slow activation of the Ca_V1.1 pore. The other three VSDs had much faster kinetics and could, therefore, be good candidates to be the voltage sensors for RYR1 activation.”Olcese and colleagues confirmed the importance of VSD-I for Ca_V1.1 activation by analyzing a naturally occurring, charge-neutralizing mutation in this domain, R174W, that is linked to malignant hyperthermia (4). The team found that this mutation reduced the voltage-sensitivity of VSD-I and abolished the ability of Ca_V1.1 to conduct Ca²⁺ at physiological membrane potentials, but had no effect on the behavior of the other three VSDs.Finally, Savalli et al. applied their data on both the wild-type and mutant VSDs to an allosteric model of Ca_V activation (2, 3), which predicted that VSD-I contributes most of the energy required to stabilize the open state of Ca_V1.1, while the other VSDs contribute little to nothing.Thus, Ca_V1.1 activation is mainly driven by a single VSD—a mechanism that hasn’t been seen in any other voltage-gated ion channel—leaving the other VSDs free to perform other functions, such as the rapid activation of RYR1. Olcese and colleagues now want to pinpoint exactly which VSD(s) are coupled to RYR1 and determine how they trigger rapid Ca²⁺ release from the SR.     

Elevations of intracellular calcium reflect normal voltage-dependent behavior,and not constitutive activity,of voltage-dependent calcium channels in gastrointestinal and vascular smooth muscle

In smooth muscle, the gating of dihydropyridine-sensitive Ca²⁺ channels may either be stochastic and voltage dependent or coordinated among channels and constitutively active. Each form of gating has been proposed to be largely responsible for Ca²⁺ influx and determining the bulk average cytoplasmic Ca²⁺ concentration. Here, the contribution of voltage-dependent and constitutively active channel behavior to Ca²⁺ signaling has been studied in voltage-clamped single vascular and gastrointestinal smooth muscle cells using wide-field epifluorescence with near simultaneous total internal reflection fluorescence microscopy. Depolarization (−70 to +10 mV) activated a dihydropyridine-sensitive voltage-dependent Ca²⁺ current (I_Ca) and evoked a rise in [Ca²⁺] in each of the subplasma membrane space and bulk cytoplasm. In various regions of the bulk cytoplasm the [Ca²⁺] increase ([Ca²⁺]_c) was approximately uniform, whereas that of the subplasma membrane space ([Ca²⁺]_PM) had a wide range of amplitudes and time courses. The variations that occurred in the subplasma membrane space presumably reflected an uneven distribution of active Ca²⁺ channels (clusters) across the sarcolemma, and their activation appeared consistent with normal voltage-dependent behavior. Indeed, in the present study, dihydropyridine-sensitive Ca²⁺ channels were not normally constitutively active. The repetitive localized [Ca²⁺]_PM rises (“persistent Ca²⁺ sparklets”) that characterize constitutively active channels were observed rarely (2 of 306 cells). Neither did dihydropyridine-sensitive constitutively active Ca²⁺ channels regulate the bulk average [Ca²⁺]_c. A dihydropyridine blocker of Ca²⁺ channels, nimodipine, which blocked I_Ca and accompanying [Ca²⁺]_c rise, reduced neither the resting bulk average [Ca²⁺]_c (at −70 mV) nor the rise in [Ca²⁺]_c, which accompanied an increased electrochemical driving force on the ion by hyperpolarization (−130 mV). Activation of protein kinase C with indolactam-V did not induce constitutive channel activity. Thus, although voltage-dependent Ca²⁺ channels appear clustered in certain regions of the plasma membrane, constitutive activity is unlikely to play a major role in [Ca²⁺]_c regulation. The stochastic, voltage-dependent activity of the channel provides the major mechanism to generate rises in [Ca²⁺].     

Dual Effect of Phosphate Transport on Mitochondrial Ca2+ Dynamics

The large inner membrane electrochemical driving force and restricted volume of the matrix confer unique constraints on mitochondrial ion transport. Cation uptake along with anion and water movement induces swelling if not compensated by other processes. For mitochondrial Ca²⁺ uptake, these include activation of countertransporters (Na⁺/Ca²⁺ exchanger and Na⁺/H⁺ exchanger) coupled to the proton gradient, ultimately maintained by the proton pumps of the respiratory chain, and Ca²⁺ binding to matrix buffers. Inorganic phosphate (P_i) is known to affect both the Ca²⁺ uptake rate and the buffering reaction, but the role of anion transport in determining mitochondrial Ca²⁺ dynamics is poorly understood. Here we simultaneously monitor extra- and intra-mitochondrial Ca²⁺ and mitochondrial membrane potential (ΔΨ_m) to examine the effects of anion transport on mitochondrial Ca²⁺ flux and buffering in P_i-depleted guinea pig cardiac mitochondria. Mitochondrial Ca²⁺ uptake proceeded slowly in the absence of P_i but matrix free Ca²⁺ ([Ca²⁺]_mito) still rose to ∼50 μm. P_i (0.001–1 mm) accelerated Ca²⁺ uptake but decreased [Ca²⁺]_mito by almost 50% while restoring ΔΨ_m. P_i-dependent effects on Ca²⁺ were blocked by inhibiting the phosphate carrier. Mitochondrial Ca²⁺ uptake rate was also increased by vanadate (V_i), acetate, ATP, or a non-hydrolyzable ATP analog (AMP-PNP), with differential effects on matrix Ca²⁺ buffering and ΔΨ_m recovery. Interestingly, ATP or AMP-PNP prevented the effects of P_i on Ca²⁺ uptake. The results show that anion transport imposes an upper limit on mitochondrial Ca²⁺ uptake and modifies the [Ca²⁺]_mito response in a complex manner.     

Divergence of Ca2+ selectivity and equilibrium Ca2+ blockade in a Ca2+ release-activated Ca2+ channel

Prevailing models postulate that high Ca²⁺ selectivity of Ca²⁺ release-activated Ca²⁺ (CRAC) channels arises from tight Ca²⁺ binding to a high affinity site within the pore, thereby blocking monovalent ion flux. Here, we examined the contribution of high affinity Ca²⁺ binding for Ca²⁺ selectivity in recombinant Orai3 channels, which function as highly Ca²⁺-selective channels when gated by the endoplasmic reticulum Ca²⁺ sensor STIM1 or as poorly Ca²⁺-selective channels when activated by the small molecule 2-aminoethoxydiphenyl borate (2-APB). Extracellular Ca²⁺ blocked Na⁺ currents in both gating modes with a similar inhibition constant (K_i; ∼25 µM). Thus, equilibrium binding as set by the K_i of Ca²⁺ blockade cannot explain the differing Ca²⁺ selectivity of the two gating modes. Unlike STIM1-gated channels, Ca²⁺ blockade in 2-APB–gated channels depended on the extracellular Na⁺ concentration and exhibited an anomalously steep voltage dependence, consistent with enhanced Na⁺ pore occupancy. Moreover, the second-order rate constants of Ca²⁺ blockade were eightfold faster in 2-APB–gated channels than in STIM1-gated channels. A four-barrier, three–binding site Eyring model indicated that lowering the entry and exit energy barriers for Ca²⁺ and Na⁺ to simulate the faster rate constants of 2-APB–gated channels qualitatively reproduces their low Ca²⁺ selectivity, suggesting that ion entry and exit rates strongly affect Ca²⁺ selectivity. Noise analysis indicated that the unitary Na⁺ conductance of 2-APB–gated channels is fourfold larger than that of STIM1-gated channels, but both modes of gating show a high open probability (P_o; ∼0.7). The increase in current noise during channel activation was consistent with stepwise recruitment of closed channels to a high P_o state in both cases, suggesting that the underlying gating mechanisms are operationally similar in the two gating modes. These results suggest that both high affinity Ca²⁺ binding and kinetic factors contribute to high Ca²⁺ selectivity in CRAC channels.     

Ca2+-induced Ca2+ Release in Chinese Hamster Ovary (CHO) Cells Co-expressing Dihydropyridine and Ryanodine Receptors

Combined patch-clamp and Fura-2 measurements were performed on chinese hamster ovary (CHO) cells co-expressing two channel proteins involved in skeletal muscle excitation-contraction (E-C) coupling, the ryanodine receptor (RyR)-Ca²⁺ release channel (in the membrane of internal Ca²⁺ stores) and the dihydropyridine receptor (DHPR)-Ca²⁺ channel (in the plasma membrane). To ensure expression of functional L-type Ca²⁺ channels, we expressed α₂, β, and γ DHPR subunits and a chimeric DHPR α₁ subunit in which the putative cytoplasmic loop between repeats II and III is of skeletal origin and the remainder is cardiac. There was no clear indication of skeletal-type coupling between the DHPR and the RyR; depolarization failed to induce a Ca²⁺ transient (CaT) in the absence of extracellular Ca²⁺ ([Ca²⁺]_o). However, in the presence of [Ca²⁺]_o, depolarization evoked CaTs with a bell-shaped voltage dependence. About 30% of the cells tested exhibited two kinetic components: a fast transient increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) (the first component; reaching 95% of its peak <0.6 s after depolarization) followed by a second increase in [Ca²⁺]_i which lasted for 5–10 s (the second component). Our results suggest that the first component primarily reflected Ca²⁺ influx through Ca²⁺ channels, whereas the second component resulted from Ca²⁺ release through the RyR expressed in the membrane of internal Ca²⁺ stores. However, the onset and the rate of Ca²⁺ release appeared to be much slower than in native cardiac myocytes, despite a similar activation rate of Ca²⁺ current. These results suggest that the skeletal muscle RyR isoform supports Ca²⁺-induced Ca²⁺ release but that the distance between the DHPRs and the RyRs is, on average, much larger in the cotransfected CHO cells than in cardiac myocytes. We conclude that morphological properties of T-tubules and/or proteins other than the DHPR and the RyR are required for functional “close coupling” like that observed in skeletal or cardiac muscle. Nevertheless, some of our results imply that these two channels are potentially able to directly interact with each other.     

Menthol increases human glioblastoma intracellular Ca2+, BK channel activity and cell migration

This study examined the effect of menthol, an agonist for transient receptor potential melastatin 8 (TRPM8) ion channels, to increase intracellular Ca²⁺ concentration, [Ca²⁺]_i, in human glioblastoma cells (DBTRG cells), which resulted in activation of the large-conductance Ca²⁺-activated K⁺ membrane ion channels (BK channels). Voltage ramps applied over 300 ms from -100 to 100 mV resulted in membrane currents with marked inwardly- and outwardly-rectifying components. Paxilline (2 μM) abolished the outwardly-rectifying current. Outwardly-rectifying on-cell patch currents were increased markedly by menthol (100 μM) added to the bath. The estimated on-cell conductance of these channels was 253 pS. Kinetic analysis showed that added menthol increased channel open probability and mean open frequency after 5 min. In a similar time course menthol increased [Ca²⁺]_i, and this increase was abolished either by added paxilline, tetraethylammonium ion or by Ca²⁺-free external solution. Finally, menthol stimulated the rate of DBTRG cell migration into scratch wounds made in confluent cells, and this also was inhibited by paxilline or by tetraethylammonium ion. We conclude that menthol, a TRPM8 agonist, increases DBTRG cell [Ca²⁺]_i that in turn activates membrane BK ion channels. Inhibition of BK channels by paxilline reverses menthol-stimulated increase of [Ca²⁺]_i and of cell migration. Thus, BK channels function to maintain elevations in [Ca²⁺]_i needed to sustain increases in DBTRG cell migration.     

Ca as second messenger in PTTH-stimulated prothoracic glands of the silkworm, Bombyx mori

Measurements of Ca²⁺ influx and [Ca²⁺]_i changes in Fura-2/AM-loaded prothoracic glands (PGs) of the silkworm, Bombyx mori, were used to identify Ca²⁺ as the actual second messenger of the prothoracicotropic hormone (PTTH) of this insect. Dose-dependent increases of [Ca²⁺]_i in PG cells were recorded in the presence of recombinant PTTH (rPTTH) within 5 minutes. The rPTTH-mediated increases of [Ca²⁺]_i levels were dependent on extracellular Ca²⁺. They were not blocked by the dihydropyridine derivative, nitrendipine, an antagonist of high-voltage-activated (HVA) Ca²⁺ channels, and by bepridil, an antagonist of low-voltage-activated (LVA) Ca²⁺ channels. The trivalent cation La³⁺, a non-specific blocker of plasma membrane Ca²⁺ channels, eliminated the rPTTH-stimulated increase of [Ca²⁺]_i levels in PG cells and so did amiloride, an inhibitor of T-type Ca²⁺ channels. Incubation of PG cells with thapsigargin resulted in an increase of [Ca²⁺]_i levels, which was also dependent on extracellular Ca²⁺ and was quenched by amiloride, suggesting the existence of store-operated plasma membrane Ca²⁺ channels, which can also be inhibited by amiloride. Thapsigargin and rPTTH did not operate independently in stimulating increases of [Ca²⁺]_i levels and one agent’s mediated increase of [Ca²⁺]_i was eliminated in the presence of the other. TMB-8, an inhibitor of intracellular Ca²⁺ release from inositol 1,4,5 trisphosphate (IP₃)-sensitive Ca²⁺ stores, blocked the rPTTH-stimulated increases of [Ca²⁺]_i levels, suggesting an involvement of IP₃ in the initiation of the rPTTH signaling cascade, whereas ryanodine did not influence the rPTTH-stimulated increases of [Ca²⁺]_i levels. The combined results indicate the presence of a cross-talk mechanism between the [Ca²⁺]_i levels, filling state of IP₃-sensitive intracellular Ca²⁺ stores and the PTTH-receptor’s-mediated Ca²⁺ influx.     

Permeation of Ca2+ through K+ channels in the plasma membrane of Vicia faba guard cells

Summary The whole-cell patch-clamp method has been used to measure Ca²⁺ influx through otherwise K⁺-selective channels in the plasma membrane surrounding protoplasts from guard cells of Vicia faba. These channels are activated by membrane hyperpolarization. The resulting K⁺ influx contributes to the increase in guard cell turgor which causes stomatal opening during the regulation of leaf-air gas exchange. We find that after opening the K⁺ channels by hyperpolarization, depolarization of the membrane results in tail current at voltages where there is no electrochemical force to drive K⁺ inward through the channels. Tail current remains when the reversal potential for permeant ions other than Ca²⁺ is more negative than or equal to the K⁺ equilibrium potential (–47 mV), indicating that the current is due to Ca²⁺ influx through the K⁺ channels prior to their closure. Decreasing internal [Ca²⁺] (Ca _i ) from 200 to 2 nm or increasing the external [Ca²⁺] (Ca _o ) from 1 to 10 mm increases the amplitude of tail current and shifts the observed reversal potential to more positive values. Such increases in the electrochemical force driving Ca²⁺ influx also decrease the amplitude of time-activated current, indicating that Ca²⁺ permeation is slower than K⁺ permeation, and so causes a partial block. Increasing Ca _o also (i) causes a positive shift in the voltage dependence of current, presumably by decreasing the membrane surface potential, and (ii) results in a U-shaped current-voltage relationship with peak inward current ca. –160 mV, indicating that the Ca^2– block is voltage dependent and suggesting that the cation binding site is within the electric field of the membrane. K⁺ channels in Zea mays guard cells also appear to have a Ca _i -, and Ca _o -dependent ability to mediate Ca²⁺ influx. We suggest that the inwardly rectiying K⁺ channels are part of a regulatory mechanism for Ca _i . Changes in Ca _o and (associated) changes in Ca _i regulate a variety of intracellular processes and ion fluxes, including the K⁺ and anion fluxes associated with stomatal aperture change.This work was supported by grants to S.M.A. from NSF (DCB-8904041) and from the McKnight Foundation. K.F.-G. is a Charles Gilbert Heydon Travelling Fellow. The authors thank Dr. R. MacKinnon (Harvard Medical School) and two anonymous reviewers for helpful comments.     

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration

Membrane currents and changes in intracellular calcium ion concentration ([Ca²⁺]_i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca²⁺-indicator, fura-2, and changes in [Ca²⁺]_i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs⁺, Ni²⁺), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni²⁺-sensitive current elicited by ramp repolarization from +90 mV to –80 mV. Ramps were sufficiently rapid that little change in [Ca²⁺]_i occured during the ramp. The (constant) [Ca²⁺]_i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni²⁺-sensitive ramp current varied linearly with 1n([Ca²⁺])_i. The I-V relations at different [Ca²⁺]_i over the range –60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca²⁺]_i had changed. The relationship between [Ca²⁺]_i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca²⁺-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca²⁺ to the exchanger during the [Ca²⁺]_i-transient and the effects of membrane voltage on the exchanger.     

Distinct Contributions of Orai1 and TRPC1 to Agonist-Induced [Ca2+]i Signals Determine Specificity of Ca2+-Dependent Gene Expression

Regulation of critical cellular functions, including Ca²⁺-dependent gene expression, is determined by the temporal and spatial aspects of agonist-induced Ca²⁺ signals. Stimulation of cells with physiological concentrations of agonists trigger increases [Ca²⁺]_i due to intracellular Ca²⁺ release and Ca²⁺ influx. While Orai1-STIM1 channels account for agonist-stimulated [Ca²⁺]_i increase as well as activation of NFAT in cells such as lymphocytes, RBL and mast cells, both Orai1-STIM1 and TRPC1-STIM1 channels contribute to [Ca²⁺]_i increases in human submandibular gland (HSG) cells. However, only Orai1-mediated Ca²⁺ entry regulates the activation of NFAT in HSG cells. Since both TRPC1 and Orai1 are activated following internal Ca²⁺ store depletion in these cells, it is not clear how the cells decode individual Ca²⁺ signals generated by the two channels for the regulation of specific cellular functions. Here we have examined the contributions of Orai1 and TRPC1 to carbachol (CCh)-induced [Ca²⁺]_i signals and activation of NFAT in single cells. We report that Orai1-mediated Ca²⁺ entry generates [Ca²⁺]_i oscillations at different [CCh], ranging from very low to high. In contrast, TRPC1-mediated Ca²⁺ entry generates sustained [Ca²⁺]_i elevation at high [CCh] and contributes to frequency of [Ca²⁺]_i oscillations at lower [agonist]. More importantly, the two channels are coupled to activation of distinct Ca²⁺ dependent gene expression pathways, consistent with the different patterns of [Ca²⁺]_i signals mediated by them. Nuclear translocation of NFAT and NFAT-dependent gene expression display “all-or-none” activation that is exclusively driven by local [Ca²⁺]_i generated by Orai1, independent of global [Ca²⁺]_i changes or TRPC1-mediated Ca²⁺ entry. In contrast, Ca²⁺ entry via TRPC1 primarily regulates NFκB-mediated gene expression. Together, these findings reveal that Orai1 and TRPC1 mediate distinct local and global Ca²⁺ signals following agonist stimulation of cells, which determine the functional specificity of the channels in activating different Ca²⁺-dependent gene expression pathways.     

Ca2+ influx into identified leech neurons induced by 5‐hydroxytryptamine

The role of 5‐hydroxytryptamine (5‐HT, serotonin) in the control of leech behavior is well established and has been analyzed extensively on the cellular level; however, hitherto little is known about the effect of 5‐HT on the cytosolic free calcium concentration ([Ca²⁺]_i) in leech neurons. As [Ca²⁺]_i plays a pivotal role in numerous cellular processes, we investigated the effect of 5‐HT on [Ca²⁺]_i (measured by Fura‐2) in identified leech neurons under different experimental conditions, such as changed extracellular ion composition and blockade of excitatory synaptic transmission. In pressure (P), lateral nociceptive (N1), and Leydig neurons, 5‐HT induced a [Ca²⁺]_i increase which was predominantly due to Ca²⁺ influx since it was abolished in Ca²⁺‐free solution. The 5‐HT‐induced Ca²⁺ influx occurred only if the cells depolarized sufficiently, indicating that it was mediated by voltage‐dependent Ca²⁺ channels. In P and N1 neurons, the membrane depolarization was due to Na⁺ influx through cation channels coupled to 5‐HT receptors, whereby the dose‐dependency suggests an involvement in excitatory synaptic transmission. In Leydig neurons, 5‐HT receptor‐coupled cation channels seem to be absent. In these cells, the membrane depolarization activating the voltage‐dependent Ca²⁺ channels was evoked by 5‐HT‐triggered excitatory glutamatergic input. In Retzius, anterior pagoda (AP), annulus erector (AE), and median nociceptive (N2) neurons, 5‐HT had no effect on [Ca²⁺]_i. © 2004 Wiley Periodicals, Inc. J Neurobiol, 2005     

Regulation of Ca2+ Release by InsP3 in Single Guinea Pig Hepatocytes and Rat Purkinje Neurons

The repetitive spiking of free cytosolic [Ca²⁺] ([Ca²⁺]_i) during hormonal activation of hepatocytes depends on the activation and subsequent inactivation of InsP₃-evoked Ca²⁺ release. The kinetics of both processes were studied with flash photolytic release of InsP₃ and time resolved measurements of [Ca²⁺]_i in single cells. InsP₃ evoked Ca²⁺ flux into the cytosol was measured as d[Ca²⁺]_i/dt, and the kinetics of Ca²⁺ release compared between hepatocytes and cerebellar Purkinje neurons. In hepatocytes release occurs at InsP₃ concentrations greater than 0.1–0.2 μM. A comparison with photolytic release of metabolically stable 5-thio-InsP₃ suggests that metabolism of InsP₃ is important in determining the minimal concentration needed to produce Ca²⁺ release. A distinct latency or delay of several hundred milliseconds after release of low InsP₃ concentrations decreased to a minimum of 20–30 ms at high concentrations and is reduced to zero by prior increase of [Ca²⁺]_i, suggesting a cooperative action of Ca²⁺ in InsP₃ receptor activation. InsP₃-evoked flux and peak [Ca²⁺]_i increased with InsP₃ concentration up to 5–10 μM, with large variation from cell to cell at each InsP₃ concentration. The duration of InsP₃-evoked flux, measured as 10–90% risetime, showed a good reciprocal correlation with d[Ca²⁺]_i/dt and much less cell to cell variation than the dependence of flux on InsP₃ concentration, suggesting that the rate of termination of the Ca²⁺ flux depends on the free Ca²⁺ flux itself. Comparing this data between hepatocytes and Purkinje neurons shows a similar reciprocal correlation for both, in hepatocytes in the range of low Ca²⁺ flux, up to 50 μM · s⁻¹ and in Purkinje neurons at high flux up to 1,400 μM · s⁻¹. Experiments in which [Ca²⁺]_i was controlled at resting or elevated levels support a mechanism in which InsP₃-evoked Ca²⁺ flux is inhibited by Ca²⁺ inactivation of closed receptor/channels due to Ca²⁺ accumulation local to the release sites. Hepatocytes have a much smaller, more prolonged InsP₃-evoked Ca²⁺ flux than Purkinje neurons. Evidence suggests that these differences in kinetics can be explained by the much lower InsP₃ receptor density in hepatocytes than Purkinje neurons, rather than differences in receptor isoform, and, more generally, that high InsP₃ receptor density promotes fast rising, rapidly inactivating InsP₃-evoked [Ca²⁺]_i transients.     

Cooperative regulation of Cav1.2 channels by intracellular Mg2+, the proximal C-terminal EF-hand,and the distal C-terminal domain

L-type Ca²⁺ currents conducted by Ca_v1.2 channels initiate excitation–contraction coupling in cardiac myocytes. Intracellular Mg²⁺ (Mg_i) inhibits the ionic current of Ca_v1.2 channels. Because Mg_i is altered in ischemia and heart failure, its regulation of Ca_v1.2 channels is important in understanding cardiac pathophysiology. Here, we studied the effects of Mg_i on voltage-dependent inactivation (VDI) of Ca_v1.2 channels using Na⁺ as permeant ion to eliminate the effects of permeant divalent cations that engage the Ca²⁺-dependent inactivation process. We confirmed that increased Mg_i reduces peak ionic currents and increases VDI of Ca_v1.2 channels in ventricular myocytes and in transfected cells when measured with Na⁺ as permeant ion. The increased rate and extent of VDI caused by increased Mg_i were substantially reduced by mutations of a cation-binding residue in the proximal C-terminal EF-hand, consistent with the conclusion that both reduction of peak currents and enhancement of VDI result from the binding of Mg_i to the EF-hand (K_D ≈ 0.9 mM) near the resting level of Mg_i in ventricular myocytes. VDI was more rapid for L-type Ca²⁺ currents in ventricular myocytes than for Ca_v1.2 channels in transfected cells. Coexpression of Ca_vβ_2b subunits and formation of an autoinhibitory complex of truncated Ca_v1.2 channels with noncovalently bound distal C-terminal domain (DCT) both increased VDI in transfected cells, indicating that the subunit structure of the Ca_v1.2 channel greatly influences its VDI. The effects of noncovalently bound DCT on peak current amplitude and VDI required Mg_i binding to the proximal C-terminal EF-hand and were prevented by mutations of a key divalent cation-binding amino acid residue. Our results demonstrate cooperative regulation of peak current amplitude and VDI of Ca_v1.2 channels by Mg_i, the proximal C-terminal EF-hand, and the DCT, and suggest that conformational changes that regulate VDI are propagated from the DCT through the proximal C-terminal EF-hand to the channel-gating mechanism.     

Ionic mechanism and role of phytochrome-mediated membrane depolarisation in caulonemal side branch initial formation in the mossPhyscomitrella patens

In caulonemal filaments of the mossPhyscomitrella patens (Hedw.), red light triggers a phytochrome-mediated transient depolarisation of the plasma membrane and the formation of side branch initials. Three-electrode voltage clamp and ion flux measurements were employed to elucidate the ionic mechanism and physiological relevance of the red-light-induced changes in ion transport. Current-voltage analyses indicated that ion channels permeable to K⁺ and Ca²⁺ are activated at the peak of the depolarisation. Calcium influx evoked by red light coincided with the depolarisation in various conditions, suggesting the involvement of voltage-gated Ca²⁺ channels. Respective K⁺ fluxes showed a small initial influx followed by a dramatic transient efflux. A role of anion channels in the depolarising current is suggested by the finding that Cl^– efflux was also increased after red light irradiation. In the presence of tetraethylammonium (10 mM) or niflumic acid (1 M), which block the red-light-induced membrane depolarisation and ion fluxes, the red-light-promoted formation of side branch initials was also abolished. Lanthanum (100 M), which inhibits K⁺ fluxes and part of the initial Ca²⁺ influx activated by red light, reduced the development of side branch initials in red light by 50%. The results suggest a causal link between the red-light-induced ion fluxes and the physiological response. The sequence of events underlying the red-light-triggered membrane potential transient and the role of ion transport in stimulus-response coupling are discussed in terms of a new model for ion-channel interaction at the plasma membrane during signalling.Abbreviations [Ca²⁺]_c cytosolic free Ca²⁺ - I-V current-voltage - E equilibrium potential - Pr red-light-absorbing phytochrome form - Pr far-red-light-absorbing phytochrome form - SPQ 6-methoxy-l-(3-sulphonatopropyl)quinolinium - TEA tetraethylammonium     

Cellular calcium deficiency plays a role in neuronal death caused by proteasome inhibitors

Cytosolic Ca²⁺ concentration ([Ca²⁺]_i) is reduced in cultured neurons undergoing neuronal death caused by inhibitors of the ubiquitin proteasome system. Activation of calcium entry via voltage‐gated Ca²⁺ channels restores cytosolic Ca²⁺ levels and reduces this neuronal death ( Snider et al. 2002 ). We now show that this reduction in [Ca²⁺]_i is transient and occurs early in the cell death process, before activation of caspase 3. Agents that increase Ca²⁺ influx such as activation of voltage‐gated Ca²⁺ channels or stimulation of Ca²⁺ entry via the plasma membrane Na–Ca exchanger attenuate neuronal death only if applied early in the cell death process. Cultures treated with proteasome inhibitors had reduced current density for voltage‐gated Ca²⁺ channels and a less robust increase in [Ca²⁺]_i after depolarization. Levels of endoplasmic reticulum Ca²⁺ were reduced and capacitative Ca²⁺ entry was impaired early in the cell death process. Mitochondrial Ca²⁺ was slightly increased. Preventing the transfer of Ca²⁺ from mitochondria to cytosol increased neuronal vulnerability to this death while blockade of mitochondrial Ca²⁺ uptake via the uniporter had no effect. Programmed cell death induced by proteasome inhibition may be caused in part by an early reduction in cytosolic and endoplasmic reticulum Ca^2+, possibly mediated by dysfunction of voltage‐gated Ca²⁺ channels. These findings may have implications for the treatment of disorders associated with protein misfolding in which proteasome impairment and programmed cell death may occur.     

High-conductance K+ channel in pancreatic islet cells can be activated and inactivated by internal calcium

Summary The Ca²⁺-activated K⁺ channel in rat pancreatic islet cells has been studied using patch-clamp single-channel current recording in excised inside-out and outside-out membrane patches. In membrane patches exposed to quasi-physiological cation gradients (Na⁺ outside, K⁺ inside) large outward current steps were observed when the membrane was depolarized. The single-channel current voltage (I/V) relationship showed outward rectification and the null potential was more negative than –40 mV. In symmetrical K⁺-rich solutions the single-channelI/V relationship was linear, the null potential was 0 mV and the singlechannel conductance was about 250 pS. Membrane depolarization evoked channel opening also when the inside of the membrane was exposed to a Ca²⁺-free solution containing 2mm EGTA, but large positive membrane potentials (70 to 80 mV) were required in order to obtain open-state probabilities (P) above 0.1. Raising the free Ca²⁺ concentration in contact with the membrane inside ([Ca²⁺]_i) to 1.5×10^–7 m had little effect on the relationship between membrane potential andP. When [Ca²⁺]_i was increased to 3×10^–7 m and 6×10^–7 m smaller potential changes were required to open the channels. Increasing [Ca²⁺]_i further to 8×10^–7 m again activated the channels, but the relationship between membrane potential andP was complex. Changing the membrane potential from –50 mV to +20 mV increasedP from near 0 to 0.6 but further polarization to +50 mV decreasedP to about 0.2. The pattern of voltage activation and inactivation was even more pronounced at [Ca²⁺]_i=1 and 2 m. In this situation a membrane potential change from –70 to +20 mV increasedP from near 0 to about 0.7 but further polarization to +80 mV reducedP to less than 0.1. The high-conductance K⁺ channel in rat pancreatic islet cells is remarkably sensitive to changes in [Ca²⁺]_i within the range 0.1 to 1 m which suggests a physiological role for this channel in regulating the membrane potential and Ca²⁺ influx through voltage-activated Ca²⁺ channels.     

Dissection of Calcium Signaling Events in Exocrine Secretion

The secretion of fluid and electrolytes by salivary gland acinar cells requires the coordinated regulation of multiple ion channel and transporter proteins, signaling components, and water transport. Importantly, neurotransmitter stimulated increase in the cytosolic free [Ca²⁺] ([Ca²⁺]_i) is critical for the regulation of salivary gland secretion as it regulates several major ion fluxes that together establish the sustained osmotic gradient to drive fluid secretion. The mechanisms that act to modulate these increases in [Ca²⁺]_i are therefore central to the process of salivary fluid secretion. Such modulation involves membrane receptors for neurotransmitters, as well as mechanisms that mediate intracellular Ca²⁺ release, and Ca²⁺ entry, as well as those that maintain cellular Ca²⁺ homeostasis. Together, these mechanisms determine the spatial and temporal aspects of the [Ca²⁺]_i signals that regulate fluid secretion. Molecular cloning of these transporters and channels as well as development of mice lacking these proteins has established the physiological significance of key components that are involved in regulating [Ca²⁺]_i in salivary glands. This review will discuss these important studies and the findings which have led to resolution of the Ca²⁺ signaling mechanisms that determine salivary gland fluid secretion.     

On the Adjacency Matrix of RyR2 Cluster Structures

In the heart, electrical stimulation of cardiac myocytes increases the open probability of sarcolemmal voltage-sensitive Ca²⁺ channels and flux of Ca²⁺ into the cells. This increases Ca²⁺ binding to ligand-gated channels known as ryanodine receptors (RyR2). Their openings cause cell-wide release of Ca²⁺, which in turn causes muscle contraction and the generation of the mechanical force required to pump blood. In resting myocytes, RyR2s can also open spontaneously giving rise to spatially-confined Ca²⁺ release events known as “sparks.” RyR2s are organized in a lattice to form clusters in the junctional sarcoplasmic reticulum membrane. Our recent work has shown that the spatial arrangement of RyR2s within clusters strongly influences the frequency of Ca²⁺ sparks. We showed that the probability of a Ca²⁺ spark occurring when a single RyR2 in the cluster opens spontaneously can be predicted from the precise spatial arrangements of the RyR2s. Thus, “function” follows from “structure.” This probability is related to the maximum eigenvalue (λ ₁) of the adjacency matrix of the RyR2 cluster lattice. In this work, we develop a theoretical framework for understanding this relationship. We present a stochastic contact network model of the Ca²⁺ spark initiation process. We show that λ ₁ determines a stability threshold for the formation of Ca²⁺ sparks in terms of the RyR2 gating transition rates. We recapitulate these results by applying the model to realistic RyR2 cluster structures informed by super-resolution stimulated emission depletion (STED) microscopy. Eigendecomposition of the linearized mean-field contact network model reveals functional subdomains within RyR2 clusters with distinct sensitivities to Ca²⁺. This work provides novel perspectives on the cardiac Ca²⁺ release process and a general method for inferring the functional properties of transmembrane receptor clusters from their structure.