Endothelial Ca2+ waves preferentially originate at specific loci in caveolin-rich cell edges

Stimulation of endothelial cells (ECs) with ATP evoked an increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i). In a single bovine aortic EC, the [Ca²⁺]_i rise started at a specific peripheral locus and propagated throughout the entire cell as a Ca²⁺ wave. The initiation locus was constant upon repeated stimulation with ATP or other agonists (bradykinin and thrombin). The Ca²⁺ wave was unaffected by the removal of extracellular Ca²⁺, demonstrating its dependence on intracellular Ca²⁺ release. Microinjection of heparin into the cell inhibited the ATP-induced Ca²⁺ responses, indicating that the Ca²⁺ wave is at least partly mediated by the inositol 1,4,5-trisphosphate receptor. Immunofluorescence staining revealed that caveolin, a marker protein for caveolae, is distributed heterogeneously in the cell and that Ca²⁺ waves preferentially originate at caveolin-rich cell edges. In contrast to caveolin, internalized transferrin and subunits of the clathrin-associated adaptor complexes such as adaptor protein-1 and -2 were diffusely distributed. Disruption of microtubules by Colcemid led to redistribution of caveolin away from the edges into the perinuclear center of the cell, and the ATP-induced [Ca²⁺]_i increase was initiated on the rim of the centralized caveolin. Thus, caveolae may be involved in the initiation of ATP-induced Ca²⁺ waves in ECs.     

Inhibition of inositol 1,4,5-trisphosphate-induced Ca2+ release by cAMP-dependent protein kinase in a living cell

Interaction of intracellular free calcium ([Ca²⁺]_i) and cAMP signaling mechanisms was examined in intact single megakaryocytes by using a combination of single-cell fluorescence microscopy to measure [Ca²⁺]_i and flash photolysis of caged Ca²⁺, inositol 1,4,5-trisphosphate (IP₃), or cAMP to elevate rapidly the concentration of these compounds inside the cell. Photolysis of caged IP₃ stimulated Ca²⁺ release from an IP₃-sensitive store. The cAMP-elevating agent carbacyclin inhibited this IP₃-induced rise in [Ca²⁺]_i but did not affect the rate of Ca²⁺ removal from the cytoplasm after photolysis of caged Ca²⁺. Photolysis of caged cAMP during ADP-induced [Ca²⁺]_i oscillations caused the [Ca²⁺]_i oscillation to transiently cease without affecting the rate of Ca²⁺ uptake and/or extrusion. We conclude that the principal mechanism of cAMP-dependent inhibition of Ca²⁺ mobilization in megakaryocytes appears to be by inhibition of IP₃-induced Ca²⁺ release and not by stimulation of Ca²⁺ removal from the cytoplasm. Two inhibitors of cAMP-dependent protein kinase, a specific peptide inhibitor of the catalytic subunit of cAMP protein kinase and KT5720, blocked the inhibitory effect of carbacyclin, indicating that the inhibition of IP₃-induced Ca²⁺-release by carbacyclin is mediated by cAMP-dependent protein kinase.     

Novel paracrine signaling mechanism in the ocular ciliary epithelium

The ciliary body contains an epithelial bilayer consisting of an outer pigmented cell layer (PE) and an inner nonpigmented cell layer (NPE) responsible for aqueous humor secretion. Secretion may be mediated in part by cytosolic Ca²⁺ concentration ([Ca²⁺]_i), but whether or how the two layers could coordinate their Ca²⁺ signals to regulate secretion is unclear. To investigate interactions between PE and NPE, we examined [Ca²⁺]_i signaling in isolated intact ciliary epithelial bilayers using confocal microscopy. Phenylephrine selectively increased [Ca²⁺]_i in PE and acetylcholine increased [Ca²⁺]_i in NPE, but epinephrine increased [Ca²⁺]_i in both layers. This increase spread from PE to NPE, and [Ca²⁺]_i signaling across the bilayer remained coordinated during [Ca²⁺]_i oscillations. All epinephrine-induced [Ca²⁺]_i signaling was blocked by the α₁-adrenergic antagonist prazosin, whereas signaling in the NPE but not PE was blocked by the β-adrenergic antagonist propranolol, the gap junction blockers octanol and 18α-glycyrrhetinic acid, or the A kinase inhibitor R_p diastereomer of adenosine 3′,5′-cyclic monophosphothioate. The β-adrenergic agonist isoproterenol failed to increase Ca²⁺ by itself, but isoproterenol plus phenylephrine-induced [Ca²⁺]_i signals across the bilayer similar to those induced by epinephrine. Finally, isoproterenol increased cell-to-cell spread of lucifer yellow via gap junctions, whereas cell-to-cell spread of [Ca²⁺]_i signals could be induced by photorelease of caged inositol 1,4,5-trisphosphate. Thus, calcium signals are coordinated in the epithelial bilayer so that adrenergic stimulation can increase [Ca²⁺]_i in NPE, but only if NPE are primed by activation of endogenous adenylyl cyclase, whereupon they receive stimulation from adjacent PE via gap junctions. This novel interplay between endocrine and paracrine pathways may coordinate [Ca²⁺]_i signaling across the ciliary epithelial bilayer.     

UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor

The presence of the P2Y₂ (P_2U-purinergic) receptor on the apical surface of airway tissue raises the possibility that aerosolized UTP might be used therapeutically to induce Cl⁻ secretion in individuals with cystic fibrosis. However, the duration of the effects of UTP may be limited by enzymatic degradation. We therefore have analyzed the metabolism of UTP and its metabolite UDP on polarized human nasal epithelium (HNE), and have compared the pharmacological activities of these two uridine nucleotides. HPLC analysis of medium bathing the mucosal surface of HNE cells revealed the presence of an ecto-nucleotidase(s) that hydrolyzed [³H]UTP and [³H]UDP with t_½ values (at 1 μM nucleotide) of 14 and 27 min, respectively. An ecto-nucleoside diphosphokinase activity also was observed, which promoted conversion of [³H]UDP into [³H]UTP in the presence of ATP. The effects of UDP on [³H]inositol phosphate accumulation, intracellular calcium levels ([Ca²⁺]_i), and Cl⁻ secretory rates (I_Cl⁻) were quantitated in HNE cells in the presence of hexokinase and glucose to ensure that no UTP (or ATP) contaminated UDP solutions during the assays. Although UDP does not activate the human P2Y₂ receptor, mucosal addition of UDP promoted [³H]inositol phosphate accumulation with an EC₅₀ of 190 nM. Mucosal addition of UTP stimulated [³H]inositol phosphate accumulation with an EC₅₀ of 280 nM. The maximal effects of mucosal UDP on [³H]inositol phosphate, [Ca²⁺]_i, and I_Cl⁻ responses were approximately one-half of those observed with mucosal UTP. Serosal application of UTP promoted a 50% greater [³H]inositol phosphate and calcium response than did mucosal application of UTP. In contrast, UDP had no effect when added to the serosal medium. Repetitive mucosal applications of UDP to HNE cells resulted in a progressive loss, i.e., desensitization, of the [Ca²⁺]_i and I_Cl⁻ response to UDP, whereas the corresponding responses to UTP remained unchanged. Our results provide evidence for the existence of a UDP receptor on HNE cells that is pharmacologically distinct from the P2Y₂ receptor. The relative stability of UDP on the airway surface and the apparent predominant mucosal expression of this putative UDP receptor make it a potential target for cystic fibrosis treatment.     

Calcium influx factor is synthesized by yeast and mammalian cells depleted of organellar calcium stores

Depletion of endoplasmic reticulum Ca²⁺ stores leads to the entry of extracellular Ca²⁺ into the cytoplasm, a process termed capacitative or store-operated Ca²⁺ entry. Partially purified extracts were prepared from the human Jurkat T lymphocyte cell line and yeast in which Ca²⁺ stores were depleted by chemical and genetic means, respectively. After microinjection into Xenopus laevis oocytes, the extracts elicited a wave of increased cytoplasmic free Ca²⁺ ([Ca²⁺]_i) that spread from the point of injection across the oocyte. Extracts from cells with replete organellar Ca²⁺ stores were inactive. The increases depended on extracellular Ca²⁺, were unaffected by the inositol 1,4,5-trisphosphate (IP₃) inhibitor heparin or an anti-IP₃ receptor antibody and were unchanged when the endoplasmic reticulum was segregated to the hemisphere opposite the injection site by centrifugation. Confocal microscopy revealed that [Ca²⁺]_i increases were most pronounced at the periphery of the oocyte. The patterns of [Ca²⁺]_i increases were replicated by computer simulations based on a diffusible messenger of about 700 Da that directly activates Ca²⁺ influx. In addition, I_CRAC, a Ca²⁺ release-activated Ca²⁺ current monitored in Jurkat cells by whole-cell patch clamp recordings, was more rapidly activated when active extracts were included in the patch pipette than by the inclusion of a Ca²⁺ chelator or IP₃. These data support the existence in yeast and mammalian cells depleted of Ca²⁺ stores of a functionally conserved diffusible calcium influx factor that directly activates Ca²⁺ influx.     

Inositol 1,4,5-tris-phosphate activation of inositol tris-phosphate receptor Ca2+ channel by ligand tuning of Ca2+ inhibition

Inositol 1,4,5-tris-phosphate (IP₃) binding to its receptors (IP₃R) in the endoplasmic reticulum (ER) activates Ca²⁺ release from the ER lumen to the cytoplasm, generating complex cytoplasmic Ca²⁺ concentration signals including temporal oscillations and propagating waves. IP₃-mediated Ca²⁺ release is also controlled by cytoplasmic Ca²⁺ concentration with both positive and negative feedback. Single-channel properties of the IP₃R in its native ER membrane were investigated by patch clamp electrophysiology of isolated Xenopus oocyte nuclei to determine the dependencies of IP₃R on cytoplasmic Ca²⁺ and IP₃ concentrations under rigorously defined conditions. Instead of the expected narrow bell-shaped cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_i) response centered at ≈300 nM–1 μM, the open probability remained elevated (≈0.8) in the presence of saturating levels (10 μM) of IP₃, even as [Ca²⁺]_i was raised to high concentrations, displaying two distinct types of functional Ca²⁺ binding sites: activating sites with half-maximal activating [Ca²⁺]_i (K_act) of 210 nM and Hill coefficient (H_act) ≈2; and inhibitory sites with half-maximal inhibitory [Ca²⁺]_i (K_inh) of 54 μM and Hill coefficient (H_inh) ≈4. Lowering IP₃ concentration was without effect on Ca²⁺ activation parameters or H_inh, but decreased K_inh with a functional half-maximal activating IP₃ concentration (K_IP3) of 50 nM and Hill coefficient (H_IP3) of 4 for IP₃. These results demonstrate that Ca²⁺ is a true receptor agonist, whereas the sole function of IP₃ is to relieve Ca²⁺ inhibition of IP₃R. Allosteric tuning of Ca²⁺ inhibition by IP₃ enables the individual IP₃R Ca²⁺ channel to respond in a graded fashion, which has implications for localized and global cytoplasmic Ca²⁺ concentration signaling and quantal Ca²⁺ release.     

Bradykinin inhibits M current via phospholipase C and Ca2+ release from IP3-sensitive Ca2+ stores in rat sympathetic neurons

A variety of intracellular signaling pathways can modulate the properties of voltage-gated ion channels. Some of them are well characterized. However, the diffusible second messenger mediating suppression of M current via G protein-coupled receptors has not been identified. In superior cervical ganglion neurons, we find that the signaling pathways underlying M current inhibition by B₂ bradykinin and M₁ muscarinic receptors respond very differently to inhibitors. The bradykinin pathway was suppressed by the phospholipase C inhibitor U-73122, by blocking the IP₃ receptor with pentosan polysulfate or heparin, and by buffering intracellular calcium, and it was occluded by allowing IP₃ to diffuse into the cytoplasm via a patch pipette. By contrast, the muscarinic pathway was not disrupted by any of these treatments. The addition of bradykinin was accompanied by a [Ca²⁺]_i rise with a similar onset and time to peak as the inhibition of M current. The M current inhibition and the rise of [Ca²⁺]_i were blocked by depletion of Ca²⁺ internal stores by thapsigargin. We conclude that bradykinin receptors inhibit M current of sympathetic neurons by activating phospholipase C and releasing Ca²⁺ from IP₃-sensitive Ca²⁺ stores, whereas muscarinic receptors do not use the phospholipase C pathway to inhibit M current channels.     

Phosphorylation of photolyzed rhodopsin is calcium-insensitive in retina permeabilized by α-toxin

Light triggers the phototransduction cascade by activating the visual pigment rhodopsin (Rho → Rho*). Phosphorylation of Rho* by rhodopsin kinase (RK) is necessary for the fast recovery of sensitivity after intense illumination. Ca²⁺ ions, acting through Ca²⁺-binding proteins, have been implicated in the desensitization of phototransduction. One such protein, recoverin, has been proposed to regulate RK activity contributing to adaptation to background illumination in retinal photoreceptor cells. In this report, we describe an in vitro assay system using isolated retinas that is well suited for a variety of biochemical assays, including assessing Ca²⁺ effects on Rho* phosphorylation. Pieces of bovine retina with intact rod outer segments were treated with pore-forming staphylococcal α-toxin, including an α-toxin mutant that forms pores whose permeability is modulated by Zn²⁺. The pores formed through the plasma membranes of rod cells permit the diffusion of small molecules <2 kDa but prevent the loss of proteins, including recoverin (25 kDa). The selective permeability of these pores was confirmed by using the small intracellular tracer N-(2-aminoethyl) biotinamide hydrochloride. Application of [γ-³²P]ATP to α-toxin-treated, isolated retina allowed us to monitor and quantify phosphorylation of Rho*. Under various experimental conditions, including low and high [Ca²⁺]_free, the same level of Rho* phosphorylation was measured. No differences were observed between low and high [Ca²⁺]_free conditions, even when rods were loaded with ATP and the pores were closed by Zn²⁺. These results suggest that under physiological conditions, Rho* phosphorylation is insensitive to regulation by Ca²⁺ and Ca²⁺-binding proteins, including recoverin.     

Neuregulins stimulate the functional expression of Ca2+-activated K+ channels in developing chicken parasympathetic neurons

The developmental expression of macroscopic Ca²⁺-activated K⁺ currents (I_K[Ca]) in chicken ciliary ganglion (CG) neurons is dependent in part on trophic factors released from preganglionic nerve terminals. Neuregulins are expressed in the preganglionic neurons that innervate the chicken CG and are therefore plausible candidates for this activity. Application of 1 nM β1-neuregulin peptide for 12 hr evokes a large (7- to 10-fold) increase in I_K[Ca] in embryonic day 9 CG neurons, even in the presence of a translational inhibitor. A similar posttranslational effect is produced by high concentrations (10 nM) of epidermal growth factor and type α transforming growth factor but not by 10 nM α2-neuregulin peptide or by neurotrophins at 40 ngml⁻¹. β1-neuregulin treatment for 12 hr also confers Ca²⁺ sensitivity onto large-conductance (285 pS) K⁺ channels observed in inside–out patches. β-Neuregulins have no effect on voltage-activated Ca²⁺ currents of CG neurons. These data support the hypothesis that β-neuregulins mediate the trophic effects of preganglionic nerve terminals on the electrophysiological differentiation of developing CG neurons.     

Abscisic acid induces oscillations in guard-cell cytosolic free calcium that involve phosphoinositide-specific phospholipase C

Oscillations in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) are an important component of Ca²⁺-based signal transduction pathways. This fact has led us to investigate whether oscillations in [Ca²⁺]_cyt are involved in the response of stomatal guard cells to the plant hormone abscisic acid (ABA). We show that ABA induces oscillations in guard-cell [Ca²⁺]_cyt. The pattern of the oscillations depended on the ABA concentration and correlated with the final stomatal aperture. We examined the mechanism by which ABA generates oscillations in guard-cell [Ca²⁺]_cyt by using 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-1H-pyrrole-2,5-dione (U-73122), an inhibitor of phosphoinositide-specific phospholipase C (PI-PLC)-dependent processes in animals. U-73122 inhibited the hydrolysis of phosphatidylinositol 4,5-bisphosphate by a recombinant PI-PLC, isolated from a guard-cell-enriched cDNA library, in a dose-dependent manner. This result confirms that U-73122 is an inhibitor of plant PI-PLC activity. U-73122 inhibited both ABA-induced oscillations in [Ca²⁺]_cyt and stomatal closure. In contrast, U-73122 did not inhibit external Ca²⁺-induced oscillations in guard-cell [Ca²⁺]_cyt and stomatal closure. Furthermore, there was no effect of the inactive analogue 1-(6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl)-2,5-pyrrolidinedione on recombinant PI-PLC activity or ABA-induced and external Ca²⁺-induced oscillations in [Ca²⁺]_cyt and stomatal closure. This lack of effect suggests that the effects of U-73122 in guard cells are the result of inhibition of PI-PLC and not a consequence of nonspecific effects. Taken together, our data suggest a role for PI-PLC in the generation of ABA-induced oscillations in [Ca²⁺]_cyt and point toward the involvement of oscillations in [Ca²⁺]_cyt in the maintenance of stomatal aperture by ABA.     

Human autoantibodies specific for the α1A calcium channel subunit reduce both P-type and Q-type calcium currents in cerebellar neurons

The pharmacological properties of voltage-dependent calcium channel (VDCC) subtypes appear mainly to be determined by the α₁ pore-forming subunit but, whether P-and Q-type VDCCs are encoded by the same α₁ gene presently is unresolved. To investigate this, we used IgG antibodies to presynaptic VDCCs at motor nerve terminals that underlie muscle weakness in the autoimmune Lambert–Eaton myasthenic syndrome (LEMS). We first studied their action on changes in intracellular free Ca²⁺ concentration [Ca²⁺]_i in human embryonic kidney (HEK293) cell lines expressing different combinations of human recombinant VDCC subunits. Incubation for 18 h with LEMS IgG (2 mg/ml) caused a significant dose-dependent reduction in the K⁺-stimulated [Ca²⁺]_i increase in the α_1A cell line but not in the α_1B, α_1C, α_1D, and α_1E cell lines, establishing the α_1A subunit as the target for these autoantibodies. Exploiting this specificity, we incubated cultured rat cerebellar neurones with LEMS IgG and observed a reduction in P-type current in Purkinje cells and both P- and Q-type currents in granule cells. These data are consistent with the hypothesis that the α_1A gene encodes for the pore-forming subunit of both P-type and Q-type VDCCs.     

Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca²⁺) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca²⁺ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca²⁺ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (I_Ba) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed I_Ba recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of I_Ba recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca²⁺ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca²⁺ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.     

Defective insulin secretion and enhanced insulin action in KATP channel-deficient mice

ATP-sensitive K⁺ (K_ATP) channels regulate many cellular functions by linking cell metabolism to membrane potential. We have generated K_ATP channel-deficient mice by genetic disruption of Kir6.2, which forms the K⁺ ion-selective pore of the channel. The homozygous mice (Kir6.2^−/−) lack K_ATP channel activity. Although the resting membrane potential and basal intracellular calcium concentrations ([Ca²⁺]_i) of pancreatic beta cells in Kir6.2^−/− are significantly higher than those in control mice (Kir6.2^+/+), neither glucose at high concentrations nor the sulfonylurea tolbutamide elicits a rise in [Ca²⁺]_i, and no significant insulin secretion in response to either glucose or tolbutamide is found in Kir6.2^−/−, as assessed by perifusion and batch incubation of pancreatic islets. Despite the defect in glucose-induced insulin secretion, Kir6.2^−/− show only mild impairment in glucose tolerance. The glucose-lowering effect of insulin, as assessed by an insulin tolerance test, is increased significantly in Kir6.2^−/−, which could protect Kir6.2^−/− from developing hyperglycemia. Our data indicate that the K_ATP channel in pancreatic beta cells is a key regulator of both glucose- and sulfonylurea-induced insulin secretion and suggest also that the K_ATP channel in skeletal muscle might be involved in insulin action.     

In situ activation of the type 2 ryanodine receptor in pancreatic beta cells requires cAMP-dependent phosphorylation

Molecular mechanisms that regulate in situ activation of ryanodine receptors (RY) in different cells are poorly understood. Here we demonstrate that caffeine (10 mM) released Ca²⁺ from the endoplasmic reticulum (ER) in the form of small spikes in only 14% of cultured fura-2 loaded beta cells from ob/ob mice. Surprisingly, when forskolin, an activator of adenylyl cyclase was present, caffeine induced larger Ca²⁺ spikes in as many as 60% of the cells. Forskolin or the phosphodiesterase-resistant PKA activator Sp-cAMPS alone did not release Ca²⁺ from ER. 4-Chloro-3-ethylphenol (4-CEP), an agent that activates RYs in other cell systems, released Ca²⁺ from ER, giving rise to a slow and small increase in [Ca²⁺]_i in beta cells. Prior exposure of cells to forskolin or caffeine (5 mM) qualitatively altered Ca²⁺ release by 4-CEP, giving rise to Ca²⁺ spikes. In glucose-stimulated beta cells forskolin induced Ca²⁺ spikes that were enhanced by 3,9-dimethylxanthine, an activator of RYs. Analysis of RNA from islets and insulin-secreting βTC-3-cells by RNase protection assay, using type-specific RY probes, revealed low-level expression of mRNA for the type 2 isoform of the receptor (RY₂). We conclude that in situ activation of RY₂ in beta cells requires cAMP-dependent phosphorylation, a process that recruits the receptor in a functionally operative form.     

Two-dimensional confocal images of organization, density, and gating of focal Ca2+ release sites in rat cardiac myocytes

In cardiac myocytes Ca²⁺ cross-signaling between Ca²⁺ channels and ryanodine receptors takes place by exchange of Ca²⁺ signals in microdomains surrounding dyadic junctions, allowing first the activation and then the inactivation of the two Ca²⁺-transporting proteins. To explore the details of Ca²⁺ signaling between the two sets of receptors we measured the two-dimensional cellular distribution of Ca²⁺ at 240 Hz by using a novel confocal imaging technique. Ca²⁺ channel-triggered Ca²⁺ transients could be resolved into dynamic “Ca²⁺ stripes” composed of hundreds of discrete focal Ca²⁺ releases, appearing as bright fluorescence spots (radius 0.5 μm) at reproducible sites, which often coincided with t-tubules as visualized with fluorescent staining of the cell membrane. Focal Ca²⁺ releases triggered stochastically by Ca²⁺ current (I_Ca) changed little in duration (7 ms) and size (100,000 Ca ions) between −40 and +60 mV, but their frequency of activation and first latency mirrored the kinetics and voltage dependence of I_Ca. The resolution of 0.95 ± 0.13 reproducible focal Ca²⁺ release sites per μm³ in highly Ca²⁺-buffered cells, where diffusion of Ca²⁺ is limited to 50 nm, suggests the presence of about one independent, functional Ca²⁺ release site per half sarcomere. The density and distribution of Ca²⁺ release sites suggest they correspond to dyadic junctions. The abrupt onset and termination of focal Ca²⁺ releases indicate that the cluster of ryanodine receptors in individual dyadic junctions may operate in a coordinated fashion.     

Depletion of Ca2+ from the sarcoplasmic reticulum of cardiac muscle prompts phosphorylation of phospholamban to stimulate store refilling

Nonmuscle cells have almost ubiquitously evolved a mechanism to detect and prevent Ca²⁺ store depletion—store operated calcium entry. No such mechanism has, as yet, been reported in cardiac myocytes. However, it is conceivable that such a mechanism may play an important role in cardiac Ca²⁺ homeostasis to ensure the availability of sufficient stored Ca²⁺ to maintain normal excitation contraction coupling. We present data that confirms the presence of a mechanism that is able to monitor the Ca²⁺ load of the SR and initiate a signaling process to accelerate Ca²⁺ uptake by the SR when store depletion is detected. Depletion of SR Ca²⁺ activates a protein kinase, the principal SR substrate of which is phospholamban. Phosphorylation of this SR protein promotes Ca²⁺ pump activity and therefore store refilling. Furthermore, a protein kinase activity associated with the SR that is inhibited by Ca²⁺ ions has been identified. We have measured lumenal [Ca²⁺] by using a fluorescent Ca²⁺ indicator and found that by initiating Ca²⁺ uptake and increasing Ca²⁺ load, we can inhibit the protein kinase activity associated with the SR. This confirms that a protein kinase, that is regulated by lumenal [Ca²⁺], has been identified and represents part of a previously unidentified signalling cascade. This local feedback mechanism would allow the myocyte to detect and prevent SR Ca²⁺ load depletion.     

A substance P (neurokinin-1) receptor mutant carboxyl-terminally truncated to resemble a naturally occurring receptor isoform displays enhanced responsiveness and resistance to desensitization

Two isoforms of the substance P (SP) receptor, differing in the length of the cytoplasmic carboxyl-terminus by ≈8 kDa, have been detected previously in rat salivary glands and other tissues. The binding and functional properties of these two isoforms have been investigated using full-length (407 amino acids) and carboxyl-terminally truncated (324 amino acids) rat SP receptors transfected stably into Chinese hamster ovary cells. Both the full-length and the truncated receptor bound radiolabeled SP with a similar K_d (≈0.1 nM). The average number of high affinity SP binding sites per cell was 1.0 × 10⁵ and 0.3 × 10⁵ for the full-length and the truncated SP receptor, respectively. In both cell lines, SP induced a rapid but transient increase in cytosolic calcium concentration ([Ca²⁺]_i), which consisted of the release of Ca²⁺ from intracellular stores and the influx of extracellular Ca²⁺. Both components are dependent on phospholipase C activation. Although the full-length and the truncated receptor utilize the same calcium pathways, they differ in their EC₅₀ values (0.28 nM for the full-length; 0.07 nM for the truncated). These differences in responsiveness may be related to the observed differences in receptor desensitization. The truncated receptor, in contrast to the full-length receptor, does not undergo rapid and long-lasting desensitization. Cells possessing the short isoform of the SP receptor would thus be expected to exhibit a prolonged responsiveness.     

Phosphorylation of a twitchin-related protein controls catch and calcium sensitivity of force production in invertebrate smooth muscle

“Catch” is a condition of prolonged, high-force maintenance at resting intracellular Ca²⁺ concentration ([Ca²⁺]) and very low energy usage, occurring in invertebrate smooth muscles, including the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Relaxation from catch is rapid on serotonergic nerve stimulation in intact muscles and application of cAMP in permeabilized muscles. This release of catch occurs by protein kinase A-mediated phosphorylation of a high (≈600 kDa) molecular mass protein, the regulator of catch. Here, we identify the catch-regulating protein as a homologue of the mini-titin, twitchin, based on (i) a partial cDNA of the purified isolated protein showing 77% amino acid sequence identity to the kinase domain of Aplysia californica twitchin; (ii) a polyclonal antibody to a synthetic peptide in this sequence reacting with the phosphorylated catch-regulating protein band from permeabilized ABRM; and (iii) the similarity of the amino acid composition and molecular weight of the protein to twitchin. In permeabilized ABRM, at all but maximum [Ca²⁺], phosphorylation of twitchin results in a decreased calcium sensitivity of force production (half-maximum at 2.5 vs. 1.3 μM calcium). At a given submaximal force, with equal numbers of force generators, twitchin phosphorylation increased unloaded shortening velocity ≈2-fold. These data suggest that aspects of the catch state exist not only at resting [Ca²⁺], but also at higher submaximal [Ca²⁺]. The mechanism that gives rise to force maintenance in catch probably operates together, to some extent, with that of cycling myosin crossbridges.     

Ca2+-independent inhibition of inositol trisphosphate receptors by calmodulin: Redistribution of calmodulin as a possible means of regulating Ca2+ mobilization

The interactions between calmodulin, inositol 1,4,5-trisphosphate (InsP₃), and pure cerebellar InsP₃ receptors were characterized by using a scintillation proximity assay. In the absence of Ca²⁺, ¹²⁵I-labeled calmodulin reversibly bound to multiple sites on InsP₃ receptors and Ca²⁺ increased the binding by 190% ± 10%; the half-maximal effect occurred when the Ca²⁺ concentration was 184 ± 14 nM. In the absence of Ca²⁺, calmodulin caused a reversible, concentration-dependent (IC₅₀ = 3.1 ± 0.2 μM) inhibition of [³H]InsP₃ binding by decreasing the affinity of the receptor for InsP₃. This effect was similar at all Ca²⁺ concentrations, indicating that the site through which calmodulin inhibits InsP₃ binding has similar affinities for calmodulin and Ca²⁺-calmodulin. Calmodulin (10 μM) inhibited the Ca²⁺ release from cerebellar microsomes evoked by submaximal, but not by maximal, concentrations of InsP₃. Tonic inhibition of InsP₃ receptors by the high concentrations of calmodulin within cerebellar Purkinje cells may account for their relative insensitivity to InsP₃ and limit spontaneous activation of InsP₃ receptors in the dendritic spines. Inhibition of InsP₃ receptors by calmodulin at all cytosolic Ca²⁺ concentrations, together with the known redistribution of neuronal calmodulin evoked by protein kinases and Ca²⁺, suggests that calmodulin may also allow both feedback control of InsP₃ receptors and integration of inputs from other signaling pathways.     

A transient outward-rectifying K+ channel current down-regulated by cytosolic Ca2+ in Arabidopsis thaliana guard cells

Sustained (noninactivating) outward-rectifying K⁺ channel currents have been identified in a variety of plant cell types and species. Here, in Arabidopsis thaliana guard cells, in addition to these sustained K⁺ currents, an inactivating outward-rectifying K⁺ current was characterized (plant A-type current: I_AP). I_AP activated rapidly with a time constant of 165 ms and inactivated slowly with a time constant of 7.2 sec at +40 mV. I_AP was enhanced by increasing the duration (from 0 to 20 sec) and degree (from +20 to −100 mV) of prepulse hyperpolarization. Ionic substitution and relaxation (tail) current recordings showed that outward I_AP was mainly carried by K⁺ ions. In contrast to the sustained outward-rectifying K⁺ currents, cytosolic alkaline pH was found to inhibit I_AP and extracellular K⁺ was required for I_AP activity. Furthermore, increasing cytosolic free Ca²⁺ in the physiological range strongly inhibited I_AP activity with a half inhibitory concentration of ≈ 94 nM. We present a detailed characterization of an inactivating K⁺ current in a higher plant cell. Regulation of I_AP by diverse factors including membrane potential, cytosolic Ca²⁺ and pH, and extracellular K⁺ and Ca²⁺ implies that the inactivating I_AP described here may have important functions during transient depolarizations found in guard cells, and in integrated signal transduction processes during stomatal movements.