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  • 1
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 112 (1989), S. 91-96 
    ISSN: 1432-1424
    Keywords: Paramecium ; patch clamp ; K channel ; Ca2+ dependence ; proteolysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The effects of proteolysis on a hyperpolarization- and Ca2+-dependent K channel from the surface membrane ofParamecium tetraurelia were examined in the inside-out excised patch mode. Treatment with trypsin, pronase or thermolysin removed the Ca2+-dependence of the channel activation, yielding an increase in channel activity greater than 2.5-fold at all Ca2+ concentrations between 10−4 and 10−8 m. Thermolysin addition-ally removed the voltage dependence of channel opening and gave the most activation among the three proteases tested. Proteolysis did not affect the single-channel conductance. In an analogy to the mechanism of activation of many Ca2+-dependent enzymes it is suggested that thisParamecium channel has a cytoplasmic inhibitory domain which can be removed by proteolysis, and that the physiological activation by Ca2+ is due to a temporary removal of this inhibition. Moreover, these findings indicate structural differences between depolarization-, Ca2+-dependent K channels (BK channels) and the hyperpolarization-, Ca2+-dependent K channels inParamecium.
    Type of Medium: Electronic Resource
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  • 2
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 112 (1989), S. 79-89 
    ISSN: 1432-1424
    Keywords: Paramecium ; patch clamp ; K channels ; Ca i 2+ -dependence ; hyperpolarization activated ; run-down
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary We have studied a class of Ca i 2+ -dependent K channels in inside-out excised membrane patches fromParamecium under patch clamp. Single channels had a conductance of 72 ±9.0 pS in a solution containing 100mM K+. The channels were selective for K+ over Rb+ with the permeability ratio of 1∶ 0.56. and over Na+, Cs+ or NH 4 + with a ratio 1∶〈0.1. The channel activity was dependent on Ca i 2+ , which was applied to the cytoplasmic side; the Ca i 2+ concentration for the half maximal activation was 2 μm. The Hill coefficient for the Ca i 2+ dependence of the channel activity was 2.58, indicating that more than two Ca i 2+ bindings are necessary for full activation. Unlike most Ca i 2+ -dependent K channels in other organisms, the channels inParamecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV pere-fold change in channel activity. While a high Ca i 2+ concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca i 2+ concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba i 2+ and Ca i 2+ were relatively ineffective in blockage. It was concluded that these Ca i 2+ -dependent K channels inParamecium are different from the previously described Ca i 2+ -dependent K channels, and are perhaps of a novel class.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 115 (1990), S. 41-50 
    ISSN: 1432-1424
    Keywords: inward rectification ; voltage-dependent K+ current ; Ca2+-dependent K+ current ; Paramecium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Hyperpolarization of voltage-clampedParamecium tetraurelia in K+ solutions elicits a complex of Ca2+ and K+ currents. The tail current that accompanies a return to holding potential (−40 mV) contains two K+ components. The tail current elicited by a step to −110 mV of ≥50-msec duration contains fast-decaying (τ≈3.5 msec) and slow-decaying (τ≈20 msec) components. The reversal potential of both components shifts by 55–57 mV/10-fold change in external [K+], suggesting that they represent pure K+ currents. The dependence of the relative amplitudes of the two tail currents on duration of hyperpolarization suggests that the slow K+ current activates slowly and is sustained, whereas the fast current activates rapidly during hyperpolarization and then rapidly inactivates. Iontophoretic injection of a Ca2+ chelator, EGTA, specifically reduces slow tail-current amplitude without affecting the fast tail component. Both K+ currents are inhibited by extracellular TEA+ in a concentration-dependent, noncooperative manner, whereas the fast K+ current alone is inhibited by 0.7mm quinidine.
    Type of Medium: Electronic Resource
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  • 4
    ISSN: 1432-1424
    Keywords: calmodulin ; mutation ; ion currents ; Paramecium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Two behavioral mutants ofParamecium tetraurelia, pantophobiacs A1 and A2, have single amino acid defects in the structure of calmodulin. The mutants exhibit several major ion current defects under voltage clamp: (i) the Ca2+-dependent K+ current activated upon depolarization ofParamecium is greatly reduced or missing in both mutants, (ii) both mutants lack a Ca2+-dependent K+ current activated upon hyperpolarization, and (iii) the Ca2+-dependent Na+ current is significantly smaller in pantophobiac A1 compared with the wild type, whereas this current is slightly increased in pantophobiac A2. Other, minor defects include a reduction in peak amplitude of the depolarization-activated Ca2+ current in pantophobiac A2, increased rates of voltage-dependent inactivation of this Ca2+ current in both pantophobiac A1 and pantophobiac A2, and an increase in the time required for the hyperpolarization-activated Ca2+ current to recover from inactivation in the pantophobiacs. The diversity of the pantophobiac mutations' effects on ion current function may indicate specific associations of calmodulin with a variety of Ca2+-related ion channel species inParamecium.
    Type of Medium: Electronic Resource
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  • 5
    ISSN: 1432-1424
    Keywords: calmodulin ; Ca2+-dependent K+ channels ; ion channel regulation ; mutations ; Paramecium
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Paramecium tetraurelia possesses two Ca2+-dependent K+ currents, activated upon depolarizationI K(Ca,d), or upon hyperpolarizationI K(Ca,h). The two currents are mediated by pharmacologically distinct ion channel populations. Three mutations ofP. tetraurelia affect these current.s Pantophobiac A mutations (pntA) cause calmodulin sequence defects, resulting in the loss of both Ca2+-dependent K+ currents. A second mutation, TEA-insensitive A (teaA), greatly enhancesI K(Ca,d) but has no affect onI K(Ca,h). A third mutation,restless (rst), also increasesI K(Ca,d) slightly, but its principle effect is in causing an early activation ofI K(Ca,h). Interactions between the products of these three genes were investigated by constructing three double mutants. BothteaA andrst restoreI K(Ca,d) andI K(Ca,h) in pantophobiac A1, but the phenotypes ofteaA andrst are not corrected by a second mutation. These observations may indicate a role for the gene products ofteaA andrst in regulating the activity ofI K(Ca,d) andI K(Ca,h), respectively.
    Type of Medium: Electronic Resource
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  • 6
    Electronic Resource
    Electronic Resource
    Springer
    The journal of membrane biology 92 (1986), S. 227-236 
    ISSN: 1432-1424
    Keywords: Paramecium ; mutants ; Ca-dependent current ; Na current
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The membrane ofParamecium generates a Ca-dependent Na current upon depolarization. There is, however, also a Na current upon hyperpolarization in this membrane. The second Na current was analyzed under voltage clamp and found to have properties identical to those of the first. Both currents could be carried by Na and Li ions and not by K, Cs or choline ion. They were eliminated by either EGTA injection into the cell or Ca removal from the bath. Both currents were eliminated by a single-gene mutation,fast-2, that had no effect on Ca currents. These findings strongly suggest that these two currents are through the same Ca-dependent Na conductance. A hyperpolarization-induced Ca current was also identified, which served to activate the second Na current. These observations support a model that theParamecium membrane has two Ca channels with different voltage dependencies and only one Na channel, which is elicited by a rise of the itternal free Ca2+ concentration. The function of the Ca-dependent Na conductance is discussed.
    Type of Medium: Electronic Resource
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  • 7
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    The @journal of eukaryotic microbiology 46 (1999), S. 0 
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: Methods for mass transformation of Paramecium tetraurelia were established using plasmids bearing neomycin-resistance or calmodulin gene fragments. Phenotypic and molecular analyses showed that, although variable, up to 5% transformation can be achieved by electroporation. Concentrations of divalent cations Ca2- and Mg2+ in the electroporation medium were crucial for efficient transformation. Strong neomycin-resistance transformation using bioballistic particle bombardment with gold particles was observed. For both methods, hybridization to transformant DNA revealed plasmid signals consistent with macronuclear transformation and correlated with transformed phenotypes. Complementation of a known calmodulin gene mutation was also achieved by mass transformation. Possible sources of variation and the general utility of these methods are discussed.
    Type of Medium: Electronic Resource
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  • 8
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    The @journal of eukaryotic microbiology 45 (1998), S. 0 
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We examined both the somatic (macro-) and the germinal (micronuclear) DNAs that encode two K+-channel isoforms. PAK1 and PAK11, in Paramecium tetraurelia. The coding regions of these two isoforms are 88% identical in nucleotides and 95% identical in amino acids. Their introns are also highly conserved. Even some of the internal eliminated sequences in PAK1 and PAK11 are clearly related. PAK1 has five IESs; PAK11 has four. The first (5′-most) IESs of the two genes are located at the same site in the coding sequence but differ in size. The 2nd IES in PAK1 (206-bp), the largest among the nine IESs, has no PAK11 counterpart. The 3rd, 4th and 5th IESs in PAK1 have a counterpart in PAK11 that is similar in size and in sequence, and identical in its position in the coding sequence. In addition, the first IES of PAK11 bears some resemblance to the 4th one of PAK1. The similarities and differences between the two sets of IESs are discussed with respect to the origin and divergence of the two K+-channel isoforms.
    Type of Medium: Electronic Resource
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  • 9
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    The @journal of eukaryotic microbiology 42 (1995), S. 0 
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Type of Medium: Electronic Resource
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  • 10
    Electronic Resource
    Electronic Resource
    Oxford, UK : Blackwell Publishing Ltd
    The @journal of eukaryotic microbiology 42 (1995), S. 0 
    ISSN: 1550-7408
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology
    Notes: We have generated a transformation marker for Paramecium using a Paramecium expression vector (pPXV) and the open reading frame (ORF) of the bacterial antibiotic resistance gene aminoglycoside 3'-phosphotransferase-II (APH-3'-II or neor) from the transposon Tn5. The expression vector contained a small multiple cloning site between the 5' and 3' non-coding regions of the calmodulin gene, and Tetrahymena telomere sequences for the stability of the plasmid in Paramecium. After the neor ORF was inserted, the plasmid was referred to as pPXV-NEO. Delivery of approximately 10–20 picoliters of linearized PXV-NEO at 〉 2000 copies/pl into the macronucleus effected 100% transformation. Southern and Northern blot hybridization showed the presence of neor-specific DNA and RNA, respectively, in all of the transformed clones but not in the untransformed clones. The degree of resistance to G-418, and the concentrations of neor-specific DNA and neor-specific RNA in the clones were proportional to the concentration of the vector injected. We have demonstrated that when the linearized plasmid was injected into the macronucleus, the prokaryotic sequence conferred an antibiotic resistance to Paramecium despite codon-usage differences.
    Type of Medium: Electronic Resource
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