Mutants with Altered Ca2+-Channel Properties in PARAMECIUM TETRAURELIA: Isolation, Characterization and Genetic Analysis

Dancers are a group of mutants in Paramecium tetraurelia whose Ca²⁺ current inactivates poorly and are likely to be defective in the structure of their Ca²⁺ channels. These mutants show prolonged backward swimming in response to K⁺ and Ba ²⁺ in the medium and were selected by this property in a galvanotactic trough. The dancer mutants are semidominant, and all isolated mutants belong to one complementation group; they are not allelic to any of the previously isolated behavioral mutants of P. tetraurelia. The phenotypic change from the homozygous parent to heterozygous F₁ generation takes three to five fissions. There is no evidence of a cytoplasmic factor capable of converting the dancer to the wild-type phenotype, as has been demonstrated in the mutants pawn and cnr. We suggest that the dancer locus is a structural gene for the Ca²⁺ channel.     

Genetic Analyses of "Paranoiac" Mutants of PARAMECIUM TETRAURELIA

Six mutants of Paramecium tetraurelia with curious "Paranoiac" phenotypes have been isolated and examined. Instead of the normal transient avoiding reactions in Na⁺ solutions, these mutants show "violent avoidances"—backing continuously for 10 to over 60 sec. This behavior corresponds to prolonged membrane excitation.—Genetic analyses establish five genic loci at which mutations give the "Paranoiac" phenotype. Close linkage between two of these genes occurs. Allelic variants are found for two of the genes. In one case, the two alleles determine very different behavioral phenotypes ("Paranoiac" and "fast-2"). These results show that the mechanism(s) which shuts off excitation in the wild-type membrane is (are) complex, but in the future may be fruitfully pursued in mutants which are defective.     

Genetic Analysis of Mating-Type Differentiation in PARAMECIUM TETRAURELIA

Whereas each of the two complementary mating types, O and E, of Paramecium tetraulrelia normally shows cytoplasmic inheritance, an abnormal heredity of mating type was observed in the progeny of crosses between two stocks of different geographical origin of Paramecium tetraurelia (stock 51 and stock 32). The modified pattern of mating-type inheritance was shown to result from the interaction of the two wild-type alleles at the locus mtD (mtD51 and mtD32), leading to a new differentiated state O*, different from the normal O and E states observed in both stock 51 and stock 32 cells. The genetic analysis of O* clones showed that the O* phenotype involves both a new heritable cytoplasmic state and possibly a nuclear change which can be transmitted through conjugation and segregates in a Mendelian fashion. All the data can be interpreted if the assumption is made that mating-type determination is achieved only by the commitment or noncommitment to the expression of mating-type E , and that this commitment may simply reflect the activation or nonactivation of the locus mtD, under the influence of one or two "cytoplasmic factors" including the product of the gene mtD itself.     

K+-Dependent Selectivity and External Ca2+ Block of Shab K+ Channels

Potassium channels allow the selective flux of K⁺ excluding the smaller, and more abundant in the extracellular solution, Na⁺ ions. Here we show that Shab is a typical K⁺ channel that excludes Na⁺ under bi-ionic, Na_o/K_i or Na_o/Rb_i, conditions. However, when internal K⁺ is replaced by Cs⁺ (Na_o/Cs_i), stable inward Na⁺ and outward Cs⁺ currents are observed. These currents show that Shab selectivity is not accounted for by protein structural elements alone, as implicit in the snug-fit model of selectivity. Additionally, here we report the block of Shab channels by external Ca²⁺ ions, and compare the effect that internal K⁺ replacement exerts on both Ca²⁺ and TEA block. Our observations indicate that Ca²⁺ blocks the channels at a site located near the external TEA binding site, and that this pore region changes conformation under conditions that allow Na⁺ permeation. In contrast, the latter ion conditions do not significantly affect the binding of quinidine to the pore central cavity. Based on our observations and the structural information derived from the NaK bacterial channel, we hypothesize that Ca²⁺ is probably coordinated by main chain carbonyls of the pore´s first K⁺-binding site.     

Passive Ca2+ Transport and Ca2+-Dependent K+ Transport in Plasmodium falciparum-Infected Red Cells

Previous reports have indicated that Plasmodium falciparum-infected red cells (pRBC) have an increased Ca²⁺ permeability. The magnitude of the increase is greater than that normally required to activate the Ca²⁺-dependent K⁺ channel (K _Ca channel) of the red cell membrane. However, there is evidence that this channel remains inactive in pRBC. To clarify this discrepancy, we have reassessed both the functional status of the K _Ca channel and the Ca²⁺ permeability properties of pRBC. For pRBC suspended in media containing Ca²⁺, K _Ca channel activation was elicited by treatment with the Ca²⁺ ionophore A23187. In the absence of ionophore the channel remained inactive. In contrast to previous claims, the unidirectional influx of Ca²⁺ into pRBC in which the Ca²⁺ pump was inhibited by vanadate was found to be within the normal range (30–55 μmol (10¹³ cells · hr)⁻¹), provided the cells were suspended in glucose-containing media. However, for pRBC in glucose-free media the Ca²⁺ influx increased to over 1 mmol (10¹³ cells · hr)⁻¹, almost an order of magnitude higher than that seen in uninfected erythrocytes under equivalent conditions. The pathway responsible for the enhanced influx of Ca²⁺ into glucose-deprived pRBC was expressed at approximately 30 hr post-invasion, and was inhibited by Ni²⁺. Possible roles for this pathway in pRBC are considered. Received: 12 May 1999/Revised: 8 July 1999     

Genetic Analyses of Heat-Sensitve Pawn Mutants of PARAMECIUM AURELIA

Heat-sensitive Pawn (ts Pawns) of Paramecium aurelia behaved normally when grown at 23° but failed to avoid strong stimuli at 35°. Four of the five ts Pawn lines tested were found to be allelic at a locus known also to carry temperature-independent Pawn mutations. The fifth ts Pawn line complemented all the conditional and unconditional Pawn mutants of the two known loci. This result, together with the patterns of F₂ segregation from various crosses, suggested the existence of a third Pawn locus. An additive effect of the unlinked ts Pawn genes was observed. These findings and the significance of ts Pawns as experimental material in behavioral and physiological research are discussed.     

RyR2 Modulates a Ca2+-Activated K+ Current in Mouse Cardiac Myocytes

In cardiomyocytes, Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCCs) binds to and activates RyR2 channels, resulting in subsequent Ca²⁺ release from the sarcoplasmic reticulum (SR) and cardiac contraction. Previous research has documented the molecular coupling of small-conductance Ca²⁺-activated K⁺ channels (SK channels) to VDCCs in mouse cardiac muscle. Little is known regarding the role of RyRs-sensitive Ca²⁺ release in the SK channels in cardiac muscle. In this study, using whole-cell patch clamp techniques, we observed that a Ca²⁺-activated K+ current (I_K,Ca) recorded from isolated adult C57B/L mouse atrial myocytes was significantly decreased by ryanodine, an inhibitor of ryanodine receptor type 2 (RyR2), or by the co-application of ryanodine and thapsigargin, an inhibitor of the sarcoplasmic reticulum calcium ATPase (SERCA) (p<0.05, p<0.01, respectively). The activation of RyR2 by caffeine increased the I_K,Ca in the cardiac cells (p<0.05, p<0.01, respectively). We further analyzed the effect of RyR2 knockdown on I_K,Ca and Ca²⁺ in isolated adult mouse cardiomyocytes using a whole-cell patch clamp technique and confocal imaging. RyR2 knockdown in mouse atrial cells transduced with lentivirus-mediated small hairpin interference RNA (shRNA) exhibited a significant decrease in I_K,Ca (p<0.05) and [Ca²⁺]i fluorescence intensity (p<0.01). An immunoprecipitated complex of SK2 and RyR2 was identified in native cardiac tissue by co-immunoprecipitation assays. Our findings indicate that RyR2-mediated Ca²⁺ release is responsible for the activation and modulation of SK channels in cardiac myocytes.     

Genetic Analysis of Mating Type Differentiation in PARAMECIUM TETRAURELIA. II. Role of the Micronuclei in Mating-Type Determination

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.     

A Sequential Vesicle Pool Model with a Single Release Sensor and a Ca2+-Dependent Priming Catalyst Effectively Explains Ca2+-Dependent Properties of Neurosecretion

Neurotransmitter release depends on the fusion of secretory vesicles with the plasma membrane and the release of their contents. The final fusion step displays higher-order Ca²⁺ dependence, but also upstream steps depend on Ca²⁺. After deletion of the Ca²⁺ sensor for fast release – synaptotagmin-1 – slower Ca²⁺-dependent release components persist. These findings have provoked working models involving parallel releasable vesicle pools (Parallel Pool Models, PPM) driven by alternative Ca²⁺ sensors for release, but no slow release sensor acting on a parallel vesicle pool has been identified. We here propose a Sequential Pool Model (SPM), assuming a novel Ca²⁺-dependent action: a Ca²⁺-dependent catalyst that accelerates both forward and reverse priming reactions. While both models account for fast fusion from the Readily-Releasable Pool (RRP) under control of synaptotagmin-1, the origins of slow release differ. In the SPM the slow release component is attributed to the Ca²⁺-dependent refilling of the RRP from a Non-Releasable upstream Pool (NRP), whereas the PPM attributes slow release to a separate slowly-releasable vesicle pool. Using numerical integration we compared model predictions to data from mouse chromaffin cells. Like the PPM, the SPM explains biphasic release, Ca²⁺-dependence and pool sizes in mouse chromaffin cells. In addition, the SPM accounts for the rapid recovery of the fast component after strong stimulation, where the PPM fails. The SPM also predicts the simultaneous changes in release rate and amplitude seen when mutating the SNARE-complex. Finally, it can account for the loss of fast- and the persistence of slow release in the synaptotagmin-1 knockout by assuming that the RRP is depleted, leading to slow and Ca²⁺-dependent fusion from the NRP. We conclude that the elusive ‘alternative Ca²⁺ sensor’ for slow release might be the upstream priming catalyst, and that a sequential model effectively explains Ca²⁺-dependent properties of secretion without assuming parallel pools or sensors.     

Age-Correlated Changes in Expression of Micronuclear Damage and Repair in PARAMECIUM TETRAURELIA

In Paramecium, age is defined as the number of mitotic divisions which have elapsed since the previous cross-fertilization (conjugation) or self-fertilization (autogamy). As the mitotic interval between fertilizations increases, the percentage of nonviable progeny clones increases. In the current study, resolution of conflicting previous reports on the pattern of increase of death and reduced viability in progeny from aging parent cells is found. Some exautogamous clones exhibit a high mortality at young clonal ages, others show no mortality throughout their life span, but most (73%) show an abrupt increase in the percent death and reduced viability in progeny from cells 50–80 fissions old.

Ultraviolet-irradiation-induced micronuclear mutations, repairable by photoreactivation, increased with increased clonal age when monitored by percent death and reduced viability of exautogamous progeny of irradiated cells. Loss of dark repair is considered a contributor to the increased expression of micronuclear mutations with increased clonal age.

     

Coupling of K+-gating and permeation with Ca2+ block in the Ca2+-Activated K+ channel inChara australis

Summary The patch-clamp technique is used here to investigate the kinetics of Ca²⁺ block in single high-conductance Ca²⁺-activated K⁺ channels. These channels are detected in the membrane surounding cytoplasmic drops fromChara australis, a membrane which originates from the tonoplast of the parent cell. The amplitudes and durations of single channel events are measured over a wide range of membrane potential (–300 to 200 mV). Ca²⁺ on either side of the channel reduces its K⁺ conductance and alters its ion-gating characteristics in a voltage-dependent manner. This Ca²⁺-induced attenuation of conductance is analyzed using the theory of diffusion-limited ion flow through pores. Interaction of external Ca²⁺ with the channel's ion-gating mechanism is examined in terms of a kinetic model for ion-gating that includes two voltage-dependent gating mechanisms. The kinetics of channel block by external Ca²⁺ indicates that (i) external Ca²⁺ binds at two sites, a superficial site and a deep site, located at 8 and 40% along the trans-pore potential difference, (ii) the external vestibule cannot be occupied by more than one Ca²⁺ or K⁺, and (iii) the kinetics of Ca²⁺ binding at the deep site is coupled with that of a voltage-dependent gate on the external side of the channel. Kinetics of channel block by internal Ca²⁺ indicates that more than one Ca²⁺ is involved.     

Reduced transmitter release conferred by mutations in theslowpoke-encoded Ca2+-activated K+ channel gene ofDrosophila

Potassium channels control the repolarization of nerve terminals and thus play important roles in the control of synaptic transmission. Here we describe the effects of mutations in theslowpoke gene, which is the structural gene for a calcium activated potassium channel, on transmitter release at the neuromuscular junction inDrosophila melanogaster. Surprisingly, we find that theslowpoke mutant exhibits reduced transmitter release compared to normal. Similarly, theslowpoke mutation significantly suppresses the increased transmitter release conferred either by a mutation inShaker or by application of 4-aminopyridine, which blocks theShaker-encoded potassium channel at theDrosophila nerve terminal. Furthermore, theslowpoke mutation suppresses the striking increase in transmitter release that occurs following application of 4-aminopyridine to theether a go-go mutant. This suppression is most likely the result of a reduction of Ca²⁺ influx into the nerve terminal in theslowpoke mutant. We hypothesize that the effects of theslowpoke mutation are indirect, perhaps resulting from increased Ca²⁺ channel inactivation, decreased Na⁺ or Ca²⁺ channel localization or gene expression, or by increases in the expression or activity of potassium channels distinct fromslowpoke.     

Ceramide Induces Release of Pro-Apoptotic Proteins from Mitochondria by Either a Ca2+-Dependent or a Ca2+-Independent Mechanism

Several observations have been reported in the last years indicating that ceramide may activate the mitochondrial route of apoptosis. We show here that on addition of either C2- or C16-ceramide to mitochondria isolated from rat heart and suspended in a saline medium, release of cytochrome c and apoptosis-inducing factor (AIF) from the intermembrane space takes place. The release process is Ca2+ -independent and is not inhibited by Cyclosporin A (CsA). For the protein release process to occur, the presence of an oxidizable substrate is required. When mitochondria are suspended in sucrose instead of potassium medium, only short chain C2-ceramide causes cytochrome c release through a Ca2+ -dependent and CsA sensitive mitochondrial permeability transition (MPT) mechanism. The latter effect appears to be related to the membrane potential dissipating ability exhibited by short chain C2-ceramide.     

Characterization of a Maize Ca2+-Dependent Protein Kinase Phosphorylating Phosphoenolpyruvate Carboxylase

Phosphoenolpyruvate carboxylase (PEPC) [EC 4.1.1.31 [EC] ] of plantsundergoes regulatory phosphorylation in response to light ornutritional conditions. However, the nature of protein kinase(s)for this phosphorylation has not yet been fully elucidated.We separated a Ca²⁺-requiring protein kinase from Ca²⁺-independentone, both of which can phosphorylate maize leaf PEPC and characterizedthe former kinase after partial purification. Several linesof evidence indicated that the kinase is one of the characteristicCa²⁺-dependent but calmodulin-independent protein kinase (CDPK).Although the M_r, of native CDPK was estimated to be about 100kDa by gel permeation chromatography, in situ phosphorylationassay of CDPK in a SDS-polyacrylamide gel revealed that thesubunit has an M_r of about 50 kDa suggesting dimer formationor association with other protein(s). Several kinetic parameterswere also obtained using PEPC as a substrate. Although the CDPKshowed an ability of regulatory phosphorylation (Ser-15 in maizePEPC), no significant desensitization to feedback inhibitor,malate, could be observed presumably due to low extent of phosphorylation.The kinase was not specific to PEPC but phosphorylated a varietyof synthetic peptides. The possible physiological role of thiskinase was discussed. ¹Present address: NEOS Central Research Laboratory, 1-1 Ohike-machi,Kosei-cho, Shiga, 520-3213 Japan. ²Present address: Chugai Pharmaceutical Co., Ltd., 1-135 Komakado,Gotemba, 412-0038 Japan. ⁴N.O. and N.Y. contributed equally to this work.     

Extracellular pH Modulates the Voltage-dependent Ca2+ Current and Low Threshold K+ Current in Hair Cells

Extracellular protons have been shown to modulate voltage-activated ionic channels. It has been proposed that synaptic modulation by exocytosed vesicular protons would be a characteristic feature of ribbon-type synapses. Type-I hair cells have a calyceal afferent junction with a diffusionally restricted synaptic cleft. These led us to study the action of extracellular pH changes on the voltage-activated Ca²⁺ and K⁺ currents evaluated using a whole-cell patch clamp in isolated cells. The amplitude of the Ca²⁺ and the K⁺ current were reduced by extracellular acidification, but without significant changes with extracellular alkalization. A shift in the voltage dependence to a more positive membrane potential was achieved at pH < 6.8. Our results shows that the presynaptic K⁺ and Ca²⁺ currents are modulated by protons, indicating that protons released along with an afferent neurotransmitter would participate as a feedback mechanism in type-I hair cells. Special issue article in honor of Dr. Ricardo Tapia.     

Characterization of Ca2+-Dependent Protein-Protein Interactions within the Ca2+ Release Units of Cardiac Sarcoplasmic Reticulum

In the heart, excitation-contraction (E-C) coupling is mediated by Ca²⁺ release from sarcoplasmic reticulum (SR) through the interactions of proteins forming the Ca²⁺ release unit (CRU). Among them, calsequestrin (CSQ) and histidine-rich Ca²⁺ binding protein (HRC) are known to bind the charged luminal region of triadin (TRN) and thus directly or indirectly regulate ryanodine receptor 2 (RyR2) activity. However, the mechanisms of CSQ and HRC mediated regulation of RyR2 activity through TRN have remained unclear. We first examined the minimal KEKE motif of TRN involved in the interactions with CSQ2, HRC and RyR2 using TRN deletion mutants and in vitro binding assays. The results showed that CSQ2, HRC and RyR2 share the same KEKE motif region on the distal part of TRN (aa 202–231). Second, in vitro binding assays were conducted to examine the Ca²⁺ dependence of protein-protein interactions (PPI). The results showed that TRN-HRC interaction had a bell-shaped Ca²⁺ dependence, which peaked at pCa4, whereas TRN-CSQ2 or TRN-RyR2 interaction did not show such Ca²⁺ dependence pattern. Third, competitive binding was conducted to examine whether CSQ2, HRC, or RyR2 affects the TRN-HRC or TRN-CSQ2 binding at pCa4. Among them, only CSQ2 or RyR2 competitively inhibited TRN-HRC binding, suggesting that HRC can confer functional refractoriness to CRU, which could be beneficial for reloading of Ca²⁺ into SR at intermediate Ca²⁺ concentrations.     

Ca2+-dependent K+ transport in lymphocytes

The treatment of rat thymocytes with A23187 + Ca2+, ascorbate-phenazine methosulphate or propranolol induced quinine-sensitive fluxes of K+ (Rb+) suggesting the presence in the cell membrane of Ca2+-dependent K+ channels. Concanavalin A induced K+ channel activation only at very high doses (13 micrograms/ml). Neither quinine nor the increase of the K+ concentration in the medium to 30 mM prevented the stimulation of amino acid transport induced by concanavalin A, suggesting that the Ca2+-dependent K+ channel is not involved in the early phenomena of lymphocyte activation.