Functional alpha 2-macroglobulin half-molecules induced by cadmium

Human alpha 2-macroglobulin can be reversibly dissociated by Cd2+ at low ionic strength in half-molecules which retain their ability to bind tightly plasmin and chymotrypsin. The steady state kinetic parameters of these proteinases towards chromogenic substrates when bound to half-molecules are not greatly different from those determined for these enzymes linked to whole alpha 2M molecules. Cd2+ can also induce the dissociation of plasmin- and chymotrypsin - alpha 2M complexes into proteinase-alpha 2M half-molecule conjugates. These results, taken with the fact that monomeric units of alpha 2M cannot bind these proteinases, strongly suggest that each active site of alpha 2M consists in a specific arrangement of two monomeric units linked by disulfide bridges.     

Ca2+-activated K+ conductance causes membrane hyperpolarizations in a monkey kidney cell line (JTC-12)

Summary We have previously reported hyperpolarizing membrane potential changes in a monkey kidney cell line (JTC-12) which has characteristics resembling proximal tubular cells. These hyperpolarizations could be observed spontaneously or evoked by mechanically touching adjacent cells. In this report, we have shown further evidence that these hyperpolarizations are elicited by an increase in membrane conductance to K⁺ which is caused by an increase in cytosolic Ca²⁺ concentration. In addition, we have found another type of hyperpolarization which is evoked by applying flow of extracellular fluid to the cell. Intracellular injection of Ca²⁺ and Sr²⁺ evoked hyperpolarizations, while intracellular injection of Mn²⁺ and Ba²⁺ did not. Intracellular injection of EGTA suppressed both spontaneous and mechanically evoked hyperpolarizations. In Ca²⁺-free medium, both spontaneous and flow-evoked hyperpolarizations were not observed, while mechanical stimuli consistently evoked hyperpolarization. In Na⁺-free medium, the incidence of cells showing the spontaneous or flow-evoked hyperpolarization increased, and the amplitude and the duration of the mechanically evoked hyperpolarization became greater. Quinidine inhibited all types of hyperpolarization. These data suggest that hyperpolarizations in JTC-12 cells are due to an increase in Ca²⁺-activated K⁺ conductance.     

Impaired Ca2+ homeostasis is associated with atrial fibrillation in the alpha1D L-type Ca2+ channel KO mouse

The novel alpha1D Ca2+ channel together with alpha1C Ca2+ channel contribute to the L-type Ca2+ current (I(Ca-L)) in the mouse supraventricular tissue. However, its functional role in the heart is just emerging. We used the alpha1D gene knockout (KO) mouse to investigate the electrophysiological features, the relative contribution of the alpha1D Ca2+ channel to the global I(Ca-L), the intracellular Ca2+ transient, the Ca2+ handling by the sarcoplasmic reticulum (SR), and the inducibility of atrial fibrillation (AF). In vivo and ex vivo ECG recordings from alpha1D KO mice demonstrated significant sinus bradycardia, atrioventricular block, and vulnerability to AF. The wild-type mice showed no ECG abnormalities and no AF. Patch-clamp recordings from isolated alpha1D KO atrial myocytes revealed a significant reduction of I(Ca-L) (24.5%; P < 0.05). However, there were no changes in other currents such as I(Na), I(Ca-T), I(K), I(f), and I(to) and no changes in alpha1C mRNA levels of alpha1D KO atria. Fura 2-loaded atrial myocytes showed reduced intracellular Ca2+ transient (approximately 40%; P < 0.05) and rapid caffeine application caused a 17% reduction of the SR Ca2+ content (P < 0.05) and a 28% reduction (P < 0.05) of fractional SR Ca2+ release in alpha1D KO atria. In conclusion, genetic deletion of alpha1D Ca2+ channel in mice results in atrial electrocardiographic abnormalities and AF vulnerability. The electrical abnormalities in the alpha1D KO mice were associated with a decrease in the total I(Ca-L) density, a reduction in intracellular Ca2+ transient, and impaired intracellular Ca2+ handling. These findings provide new insights into the mechanism leading to atrial electrical dysfunction in the alpha1D KO mice.     

Serum and alpha 2-macroglobulin induce transient hyperpolarizations in the membrane potential of an osteoblastlike clone

Using microelectrode techniques, we have observed that the application of serum or alpha 2-macroglobulin (alpha 2M) induces transient hyperpolarizations in the membrane potential of a rat osteosarcoma clone (ROS 17/2). Hyperpolarizations arose from activation of Ca2+-dependent K+ channels by transient increases in the concentration of intracellular free Ca2+. Hyperpolarizing spikes were observed for several h following the addition of fetal bovine serum (FBS) to cell cultures. Application of small volumes of FBS or alpha 2M rapidly induced synchronized bursts of hyperpolarizing spikes. No response was elicited by serum-free medium, latex beads, or bovine serum albumin (BSA). Immunofluorescence labeling patterns were consistent with the receptor-mediated endocytosis of alpha 2M but not BSA. The ligand specificity and kinetics of these hyperpolarizations suggest that they are associated with a receptor-mediated event, possibly an early stage of receptor-mediated endocytosis.     

Image processing of electron micrographs of human alpha 2-macroglobulin half-molecules induced by Cd2+

Human alpha 2-macroglobulin is a tetrameric plasma inhibitor of proteinases. Its dissociation by Cd2+ gives functional dimers. Electron microscopy of negatively stained dimers shows their round-ended cylindrical shape with furrows delimiting 3 main stain-excluding domains. Image processing of electron micrographs shows the existence of 2 main orientations of the dimers on the carbon support film. The dimer is composed of 2 curved monomers linked in a central domain, and related by a 90 degree rotation. Taking into account the known primary structure of alpha 2-macroglobulin and the linkage of the 2 constitutive monomers by 2 disulfide bonds, the molecular organization of the dimer is discussed, extended to the tetrameric molecule and compared to the published models of human alpha 2-macroglobulin.     

Voltage- and Ca2+-activated potassium channels in Ca2+ store control Ca2+ release

Ca2+ release from Ca2+ stores is a 'quantal' process; it terminates after a rapid release of stored Ca2+. To explain the quantal nature, it has been supposed that a decrease in luminal Ca2+ acts as a 'brake' on store release. However, the mechanism for the attenuation of Ca2+ efflux remains unknown. We show that Ca2+ release is controlled by voltage- and Ca2+-activated potassium channels in the Ca2+ store. The potassium channel was identified as the big or maxi-K (BK)-type, and was activated by positive shifts in luminal potential and luminal Ca2+ increases, as revealed by patch-clamp recordings from an exposed nuclear envelope. The blockage or closure of the store BK channel due to Ca2+ efflux developed lumen-negative potentials, as revealed with an organelle-specific voltage-sensitive dye [DiOC5(3); 3,3'-dipentyloxacarbocyanine iodide], and suppressed Ca2+ release. The store BK channels are reactivated by Ca2+ uptake by Ca2+ pumps regeneratively with K+ entry to allow repetitive Ca2+ release. Indeed, the luminal potential oscillated bistably by approximately 45 mV in amplitude. Our study suggests that Ca2+ efflux-induced store BK channel closures attenuate Ca2+ release with decreases in counter-influx of K+.     

A Ca2+-activated ATPase specifically released by Ca2+ shock from Paramecium tetraurelia

Deciliation of Paramecium tetraurelia by a Ca2+ shock procedure releases a discrete set of proteins which represent about 1% of the total cell protein. Marker enzymes for cytoplasm (hexokinase), endoplasmic reticulum (glucose-6-phosphatase), peroxisomes (catalase), and lysosomes (acid phosphatase) were not released by this treatment. Among the proteins selectively released is a Ca2+-dependent ATPase. This enzyme has a broad substrate specificity which includes GTP, ATP, and UTP, and it can be activated by Ca2+, Sr2+, or Ba2+, but not by Mg2+ or by monovalent cations. The crude enzyme has a specific activity of 2-3 mumol/min per mg; the optimal pH for activity is 7.5. ATPase, GTPase, and UTPase all reside in the same protein, which is inhibited by ruthenium red, is irreversibly denatured at 50 degrees C, and which has a sedimentation coefficient of 8-10 S. This enzyme is compared with other surface-derived ATPases of ciliated protozoans, and its possible roles are discussed.     

Subconductance behavior in a maxi Ca2(+)-activated K+ channel induced by dendrotoxin-I

Toxin I (DTX-I), a 60-residue peptide belonging to the dendrotoxin family of Mamba snake neurotoxins, is a potent inhibitor of various types of voltage-gated K+ currents. To investigate the sensitivity of another major class of K+ channels to DTX-I, the effect of this toxin was studied on single Ca2(+)-activated K+ channels from rat skeletal muscle incorporated into planar bilayers. Internal (intracellular) DTX-I was found to induce reversibly a long-lived (tau = 40 s), inwardly rectifying subconductance state with 66% of the normal open-state current at +20 mV. Analysis of the kinetics of substate formation and the current-voltage behavior of the substate suggest that binding of DTX-I modifies conduction of K+ ions through the pore without affecting the Ca2+ dependence or voltage dependence of gating. These results identify a unique internal binding site for DTX-I (Kd = 90 nM in 50 mM KCl) on a ubiquitous class of high-conductance, Ca2(+)-activated K+ channels.     

Role of Ca2+ and Ca2+-activated protease in myoblast fusion

In this report, we have examined the effects of a calcium chelator, EGTA, and a calcium ionophore, A23187, on fusion of a cloned muscle cell line, L6. Our results confirm that EGTA essentially blocks all myoblast fusion because the lateral alignment of presumptive myoblasts cannot occur in the absence of extracellular calcium. A23187, however, promotes the precocious fusion of myoblasts, apparently by facilitating Ca2+ transport into myoblasts. We have also demonstrated that a Ca2+-activated protease, CAF (mM), appears to relocate in response to the Ca2+ flux, changing from a random, dispersed distribution in proliferative myoblasts to a predominantly peripheral distribution in prefusion myoblasts. Coincident with the mM CAF relocation is an altered distribution of a surface glycoprotein, fibronectin. Extracellular fibronectin is seen in abundance in proliferating myoblasts, but is essentially absent from the surface of fusing myoblasts. We suggest that mM CAF when activated by Ca2+ influx may act to promote the release of fibronectin from the myoblast cell surface, thus providing a mechanism by which the membrane of the fusing myoblast may be rearranged to accommodate fusion.     

Neurotransmitter modulation of small-conductance Ca2+-activated K+ channels by regulation of Ca2+ gating

Small-conductance Ca2+-activated K+ (SK) channels are widely expressed in neuronal tissues where they underlie post-spike hyperpolarizations, regulate spike-frequency adaptation, and shape synaptic responses. SK channels constitutively interact with calmodulin (CaM), which serves as Ca2+ sensor, and with protein kinase CK2 and protein phosphatase 2A, which modulate their Ca2+ gating. By recording coupled activities of Ca2+ and SK2 channels, we showed that SK2 channels can be inhibited by neurotransmitters independently of changes in the activity of the priming Ca2+ channels. This inhibition involvesSK2-associated CK2 and results from a 3-fold reduction in the Ca2+ sensitivity of channel gating. CK2phosphorylated SK2-bound CaM but not KCNQ2-bound CaM, thereby selectively regulating SK2 channels. We extended these observations to sensory neurons by showing that noradrenaline inhibits SK current and increases neuronal excitability in aCK2-dependent fashion. Hence, neurotransmitter-initiated signaling cascades can dynamically regulate Ca2+ sensitivity of SK channels and directly influence somatic excitability.     

The Ca2+-activated cation channel TRPM4 is regulated by phosphatidylinositol 4,5-biphosphate

Transient receptor potential (TRP) channel, melastatin subfamily (TRPM)4 is a Ca2+-activated monovalent cation channel that depolarizes the plasma membrane and thereby modulates Ca2+ influx through Ca2+-permeable pathways. A typical feature of TRPM4 is its rapid desensitization to intracellular Ca2+ ([Ca2+]i). Here we show that phosphatidylinositol 4,5-biphosphate (PIP2) counteracts desensitization to [Ca2+]i in inside-out patches and rundown of TRPM4 currents in whole-cell patch-clamp experiments. PIP2 shifted the voltage dependence of TRPM4 activation towards negative potentials and increased the channel's Ca2+ sensitivity 100-fold. Conversely, activation of the phospholipase C (PLC)-coupled M1 muscarinic receptor or pharmacological depletion of cellular PIP2 potently inhibited currents through TRPM4. Neutralization of basic residues in a C-terminal pleckstrin homology (PH) domain accelerated TRPM4 current desensitization and strongly attenuated the effect of PIP2, whereas mutations to the C-terminal TRP box and TRP domain had no effect on the PIP2 sensitivity. Our data demonstrate that PIP2 is a strong positive modulator of TRPM4, and implicate the C-terminal PH domain in PIP2 action. PLC-mediated PIP2 breakdown may constitute a physiologically important brake on TRPM4 activity.     

Muscle aging is associated with compromised Ca2+ spark signaling and segregated intracellular Ca2+ release

Reduced homeostatic capacity for intracellular Ca2+ ([Ca2+]i) movement may underlie the progression of sarcopenia and contractile dysfunction during muscle aging. We report two alterations to Ca2+ homeostasis in skeletal muscle that are associated with aging. Ca2+ sparks, which are the elemental units of Ca2+ release from sarcoplasmic reticulum, are silent under resting conditions in young muscle, yet activate in a dynamic manner upon deformation of membrane structures. The dynamic nature of Ca2+ sparks appears to be lost in aged skeletal muscle. Using repetitive voltage stimulation on isolated muscle preparations, we identify a segregated [Ca2+]i reserve that uncouples from the normal excitation-contraction process in aged skeletal muscle. Similar phenotypes are observed in adolescent muscle null for a synaptophysin-family protein named mitsugumin-29 (MG29) that is involved in maintenance of muscle membrane ultrastructure and Ca2+ signaling. This finding, coupled with decreased expression of MG29 in aged skeletal muscle, suggests that MG29 expression is important in maintaining skeletal muscle Ca2+ homeostasis during aging.     

Large conductance Ca2+-activated K+ (BK) channel: activation by Ca2+ and voltage

Large conductance Ca2+-activated K+ (BK) channels belong to the S4 superfamily of K+ channels that include voltage-dependent K+ (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH2-terminus. The BK channel is activated by internal Ca2+, and using chimeric channels and mutagenesis, three distinct Ca2+-dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca2+-binding domains activate the BK channel when cytoplasmic Ca2+ reaches micromolar concentrations, and a low Ca2+ affinity mechanism may be involved in the physiological regulation by Mg2+. The presence in the BK channel of multiple Ca2+-binding sites explains the huge Ca2+ concentration range (0.1 microM-100 microM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca2+. Therefore, Ca2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.     

Intracellular Ca2+ activity during slow synaptic hyperpolarizations in Helix pomatia

In the "bursting" cell RPal of Helix pomatia, intracellular Ca2+ activity increased during the slow synaptic hyperpolarization termed LLH. The Ca2+ increase had a double exponential time course and the kinetics provide evidence for an intracellularly located Ca2+ source. The Ca2+ wave could be mimicked by dopamine and inhibited by ergometrine. dB-cAMP also enhanced the Ca2+ activity. Intracellular K+ was measured during LLH. An appendix by J. D. C. Labmert demonstrates that a dominant mechanism underlying LLH is the increase of K+ permeability.     

Ca2+ signalling and Ca2+-activated ion channels in exocrine acinar cells

The development of the calcium signalling field, from its early beginnings some 40 years ago to the present, is described. Calcium signalling in exocrine gland acinar cells and the effects of neurotransmitter- or hormone-elicited rises in the cytosolic calcium ion concentration on ion channel gating are reviewed. The highly polarized arrangement of the organelle systems in living acinar cells is described as well as its importance for the physiologically relevant local and polarized calcium signalling events.     

Functional modification of a Ca2+-activated K+ channel by trimethyloxonium

Single Ca2+-activated K+ channels from rat skeletal muscle plasma membranes were studied in neutral phospholipid bilayers. Channels were chemically modified by briefly exposing the external side to the carboxyl group modifying reagent trimethyloxonium (TMO). TMO modification, in a "multi-hit" fashion, reduces the single-channel conductance without affecting ion selectivity. Modification also shifts the voltage activation curve toward more depolarized voltages and reduces the affinity of the channel blocker charybdotoxin (CTX). CTX, bound to the channel during the TMO exposure, prevents the TMO-induced reduction of the single-channel conductance. These data suggest that the high-conductance Ca2+-activated K+ channel has carboxyl groups on its external surface. These groups influence ion conduction, gating, and the binding of CTX.