A Ca-Induced Na-Current in Paramecium

Under a voltage clamp, step depolarization and repolarization can induce a sustained inward current and a tail inward current in Paramecium tetraurelia bathed in a solution containing 8 mM-Na+. These currents are best seen in the ‘paranoiac’ mutant. The I-V plot of the sustained inward current can have a region of negative resistance around −20 mV. This current is absent when Na+ is excluded from the bath solution, and it increases as the Na+ concentration increases from 2 to 8 mM. Injection of Na+ into the cell suppresses this inward current. This current develops very slowly, reaching its maximum seconds after the step depolarization and decays with a time constant of hundreds of milliseconds after the repolarization. This slow current is dependent on Ca2+. It can be suppressed by reduction or deletion of external Ca2+ or by iontophoretic injection of EGTA. ‘Pawn’ mutants with defective Caconductance also lack this current. We conclude that Paramecium has a Ca-induced conductance through which the Na-current flows. Although more prominent in the ‘paranoiac’ mutant, this Ca-induced Na-current is also seen in the wild type. This conductance may function in generating plateau depolarizations lasting seconds or even minutes and the corresponding prolonged backward swimming away from sources of irritation and stress.

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The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽

10.1093/genetics/155.3.1105 ◽

2000 ◽

Vol 155 (3) ◽

pp. 1105-1117 ◽

Cited By ~ 4

Author(s):

W John Haynes ◽

Kit-Yin Ling ◽

Robin R Preston ◽

Yoshiro Saimi ◽

Ching Kung

Keyword(s):

Genomic Library ◽

Open Reading Frame ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Rt Pcr ◽

Reading Frame ◽

Close Proximity ◽

In The Wild ◽

Dna Fragment ◽

Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

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Voltage Sensitive Ca-Channels and the Transient Inward Current in Paramecium Tetraurelia

Journal of Experimental Biology ◽

10.1242/jeb.78.1.149 ◽

1979 ◽

Vol 78 (1) ◽

pp. 149-161 ◽

Cited By ~ 1

Author(s):

YOUKO SATOW ◽

CHING KUNG

Keyword(s):

Voltage Clamp ◽

Resting Potential ◽

Ca Channel ◽

Paramecium Tetraurelia ◽

Ca Channels ◽

Action Current ◽

Inward Current ◽

Transient Inward Current ◽

Inorganic Cation ◽

Inward Currents

Transient inward currents across the membrane of P. tetraurelia are recorded upon step depolarizations with a voltage clamp in solutions where Ca2+ is the only added inorganic cation. It is shown that the current is normally carried by Ca2+ through the Ca-channels which activate and inactivate in time. The transient inward current is dependent on both the size of the depolarizing step and the holding level before the step. Maximum inward current (Imax) occurs when the membrane is first held at the resting level (- 30 mV), then stepped to 0 mV in a solution containing 0.91 mM-Ca2+. The Imax is smaller when the membrane is first held at depolarized level. This is due to the depolarization-sensitive inactivation of the Ca-channels. The Imax is also smaller when the membrane is first held at a hyperpolarized level. This may be explained by the activation of hyperpolarization-sensitive K-channels known to exist in the Paramecium membrane. I max increases with concentration of Ca2+ up to 0.9 mM. Further increase in the Ca2+ concentration does not affect Imax. This apparent saturation at 0.9 mM-Ca2+ may reflect a rate-limiting step of Ca2+ permeation. The increase in Ca2+ concentration shifts the V-Ipeak curve in the direction of less sensitivity. This result is best explained as the effect of bound Ca2+ on the surface potential of the Paramecium membrane. These results provide the first detailed description of the properties of the action current through the Ca-channel in Paramecium. They also define the conditions under which future voltage-clamp studies of wild-type and mutant membranes of P. tetraurelia should be performed, i.e. to maximize the resolution of the Ca-channel activity, the membrane should be held at or near the resting potential and there should be over 0.9 mM-Ca2+ in the test solutions. The behaviour of the Paramecium Ca-channel and small Imax in the presence of K+ are discussed.

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Mutation of a single residue in the ba3 oxidase specifically impairs protonation of the pump site

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1422434112 ◽

2015 ◽

Vol 112 (11) ◽

pp. 3397-3402 ◽

Cited By ~ 16

Author(s):

Christoph von Ballmoos ◽

Nathalie Gonska ◽

Peter Lachmann ◽

Robert B. Gennis ◽

Pia Ädelroth ◽

...

Keyword(s):

Electron Transfer ◽

Time Constant ◽

Thermus Thermophilus ◽

Proton Translocation ◽

Proton Pumping ◽

Wild Type ◽

Structural Variant ◽

In The Wild ◽

Membrane Bound ◽

Proton Uptake

The ba3-type cytochrome c oxidase from Thermus thermophilus is a membrane-bound protein complex that couples electron transfer to O2 to proton translocation across the membrane. To elucidate the mechanism of the redox-driven proton pumping, we investigated the kinetics of electron and proton transfer in a structural variant of the ba3 oxidase where a putative “pump site” was modified by replacement of Asp372 by Ile. In this structural variant, proton pumping was uncoupled from internal electron transfer and O2 reduction. The results from our studies show that proton uptake to the pump site (time constant ∼65 μs in the wild-type cytochrome c oxidase) was impaired in the Asp372Ile variant. Furthermore, a reaction step that in the wild-type cytochrome c oxidase is linked to simultaneous proton uptake and release with a time constant of ∼1.2 ms was slowed to ∼8.4 ms, and in Asp372Ile was only associated with proton uptake to the catalytic site. These data identify reaction steps that are associated with protonation and deprotonation of the pump site, and point to the area around Asp372 as the location of this site in the ba3 cytochrome c oxidase.

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Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia

Journal of Experimental Biology ◽

10.1242/jeb.203.6.1059 ◽

2000 ◽

Vol 203 (6) ◽

pp. 1059-1070 ◽

Cited By ~ 2

Author(s):

U. Nagel ◽

H. Machemer

Keyword(s):

Swimming Speed ◽

Sedimentation Rates ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Centre Of Gravity ◽

In The Wild ◽

Type Population ◽

G Value ◽

Mutant Cells

Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity.

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Mechanism of block by ZD 7288 of the hyperpolarization-activated inward rectifying current in guinea pig substantia nigra neurons in vitro

Journal of Neurophysiology ◽

10.1152/jn.1995.74.6.2366 ◽

1995 ◽

Vol 74 (6) ◽

pp. 2366-2378 ◽

Cited By ~ 195

Author(s):

N. C. Harris ◽

A. Constanti

Keyword(s):

Substantia Nigra ◽

Guinea Pig ◽

Voltage Clamp ◽

Time Constant ◽

Slow Inward Current ◽

Current Clamp ◽

Inward Current ◽

Electrotonic Potential ◽

Zd 7288

1. The effects of the novel bradycardic agent 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288) (Zeneca) were investigated on the hyperpolarization-activated cationic current (Ih) in guinea pig substantia nigra pars compacta neurons in vitro, using a single-microelectrode current-clamp/voltage-clamp technique. 2. Under current-clamp conditions, injection of large negative current pulses (0.1-0.5 nA, 400 ms) evoked a slow depolarizing "sag" in the electrotonic potential due to activation of the slow inward (anomalous) rectifier. In voltage-clamp recordings, hyperpolarizing voltage steps from a holding potential of -60 mV (close to resting potential) elicited slow inward current relaxations with kinetic properties similar to those seen for other neuronal Ihs. 3. ZD 7288 (10-100 microM) produced a consistent abolition of the electrotonic potential sag with no effect on membrane potential or spike properties. Under voltage clamp, Ih amplitude was clearly reduced in a time- and concentration-dependent manner (apparent half-maximum blocking concentration = 2 microM); full block of Ih was typically achieved after 10-15 min of exposure to 50 microM ZD 7288, with no significant recovery observed after 1 h of washing. 4. A similar (although more rapid) block of Ih was seen after application of 3-5 mM Cs+ (partially reversible after 30 min of washing). 5. Partial block of Ih by 10 microM ZD 7288 was accompanied by a reduction in the maximum amplitude of the Ih activation curve, a small negative shift in its position on the voltage axis, and a linearization of the steady-state current-voltage relationship. The estimated Ih reversal potential, however, remained unaffected. 6. In 10 microM ZD 7288, the time course of Ih activation and deactivation was significantly slowed (within the range of -70 to -120 mV for the activation time constant and -70 to -90 mV for the inactivation time constant). 7. Blockade of Ih by ZD 7288 or Cs+ was independent of prior Ih activation (i.e., non-use dependent). 8. Intracellular loading with ZD 7288 also abolished the sag in the electrotonic voltage response and Ih relaxations, suggesting an intracellular site of action. By contrast, intracellular Cs+ had no effect on Ih properties. 9. Block of Ih by ZD 7288 (but not Cs+) was relieved by prolonged cell hyperpolarization, manifested as a slowly developing (half-time approximately 20 s) inward current at a holding potential of -100 mV. 10. We propose that ZD 7288, when applied externally, may behave as a "lipophilic" quaternary cation, capable of passing into the cell interior to block Ih channels in their closed state; this compound may thus prove a useful research tool, in place of Cs+, for studying the properties and significance of Ih currents in controlling neuronal function.

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Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia.

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1133 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1133-1137 ◽

Cited By ~ 22

Author(s):

Y You ◽

K Aufderheide ◽

J Morand ◽

K Rodkey ◽

J Forney

Keyword(s):

Cell Line ◽

Dna Sequences ◽

Mutant Cell ◽

Restriction Pattern ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Coding Region ◽

Cytoplasmic Factor ◽

In The Wild ◽

Dna Restriction

A previously isolated mutant cell line called d48 contains a complete copy of the A surface antigen gene in the micronuclear genome, but the gene is not incorporated into the macronucleus. Previous experiments have shown that a cytoplasmic factor made in the wild-type macronucleus can rescue the mutant. Recently, S. Koizumi and S. Kobayashi (Mol. Cell. Biol. 9:4398-4401, 1989) observed that injection of a plasmid containing the A gene into the d48 macronucleus rescued the cell line after autogamy. It is shown here that an 8.8-kb EcoRI fragment containing only a portion of the A gene coding region is sufficient for the rescue of d48. The inability of other A gene fragments to rescue the mutant shows that this effect is dependent upon specific Paramecium DNA sequences. Rescue results in restoration of the wild-type DNA restriction pattern in the macronucleus. These results are consistent with a model in which the macronuclear A locus normally makes an additional gene product that is required for correct processing of the micronuclear copy of the A gene.

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Macronuclear transformation with specific DNA fragments controls the content of the new macronuclear genome in Paramecium tetraurelia

Molecular and Cellular Biology ◽

10.1128/mcb.11.2.1133-1137.1991 ◽

1991 ◽

Vol 11 (2) ◽

pp. 1133-1137

Author(s):

Y You ◽

K Aufderheide ◽

J Morand ◽

K Rodkey ◽

J Forney

Keyword(s):

Cell Line ◽

Dna Sequences ◽

Mutant Cell ◽

Restriction Pattern ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Coding Region ◽

Cytoplasmic Factor ◽

In The Wild ◽

Dna Restriction

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Expression of Mutated Paramecium Telomerase RNAs In Vivo Leads to Templating Errors That Resemble Those Made by Retroviral Reverse Transcriptase

Molecular and Cellular Biology ◽

10.1128/mcb.19.4.2887 ◽

1999 ◽

Vol 19 (4) ◽

pp. 2887-2894 ◽

Cited By ~ 5

Author(s):

Amanda J. Ye ◽

W. John Haynes ◽

Daniel P. Romero

Keyword(s):

Reverse Transcriptase ◽

De Novo ◽

Telomerase Rna ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Telomeric Dna ◽

Telomeric Repeats ◽

Immunodeficiency Virus ◽

In The Wild

ABSTRACT Telomeric DNA consists of short, tandemly repeated sequences at the ends of chromosomes. Telomeric DNA in the ciliate Paramecium tetraurelia is synthesized by an error-prone telomerase with an RNA template specific for GGGGTT repeats. We have previously shown that misincorporation of TTP residues at the telomerase RNA templating nucleotide C52 accounts for the 30% GGGTTT repeats randomly distributed in wild-type telomeres. To more completely characterize variable repeat synthesis in P. tetraurelia, telomerase RNA genes mutated at C52 (A, U, and G) were expressed in vivo. De novo telomeric repeats from transformants indicate that the predominant TTP misincorporation error seen in the wild-type telomerase is dependent on the presence of a C residue at template position 52. Paradoxically, the effects of various other telomerase RNA template and alignment region mutations on de novo telomeres include significant changes in fidelity, as well as the synthesis of aberrant, 5-nucleotide telomeric repeats. The occurrence of deletion errors and the altered fidelity of mutatedP. tetraurelia telomerase, in conjunction with misincorporation by the wild-type enzyme, suggest that the telomerase RNA template domain may be analogous to homopolymeric mutational hot spots that lead to similar errors by the human immunodeficiency virus proofreading-deficient reverse transcriptase.

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GENETIC ANALYSES OF "PARANOIAC" MUTANTS OF PARAMECIUM TETRAURELIA

Genetics ◽

10.1093/genetics/86.1.113 ◽

1977 ◽

Vol 86 (1) ◽

pp. 113-120

Author(s):

Judith Van Houten ◽

Sheng-Yung Chang ◽

Ching Kung

Keyword(s):

Close Linkage ◽

Paramecium Tetraurelia ◽

Wild Type ◽

Allelic Variants ◽

Genetic Analyses ◽

Behavioral Phenotypes ◽

Membrane Excitation ◽

The Future ◽

In The Wild

ABSTRACT 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.

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Membrane currents of pawn mutants of the pwA group in Paramecium tetraurelia

Journal of Experimental Biology ◽

10.1242/jeb.84.1.57 ◽

1980 ◽

Vol 84 (1) ◽

pp. 57-71 ◽

Cited By ~ 1

Author(s):

Y. Satow ◽

C. Kung

Keyword(s):

Time Course ◽

Membrane Currents ◽

Voltage Sensitivity ◽

Paramecium Tetraurelia ◽

Ca Channels ◽

Wild Type ◽

Outward Currents ◽

In The Wild ◽

Inward Currents ◽

K Current

Membrane currents were recorded from the wild type and two pawn mutants of the pwA complementation group in Paramecium tetraurelia under a voltage clamp. Most currents are not changed by the mutations. Transient inward currents of a leaky mutant, pwA132, upon step depolarizations are less than those in the wild type. The inward transient is completely lacking in a non-leaky mutant, pwA500. The time course of the residual inward currents in the leaky mutant is not significantly different from that of wild type. The voltage sensitivity of the Ca channels in the leaky mutant is also similar to that of wild type. The inward currents upon membrane hyperpolarizations in the mutants show normal characteristics in the presence or absence of external K+. With sufficiently large, prolonged depolarization, outward currents progressively develop in the wild type but decay in the mutants. The simplest conclusion we can draw is that the pwA mutations reduce the number of functional Ca channels but do not change the channel characteristics. From the conductance measurements, 45% of the Ca channels remain in the leaky mutant pwA132, and none remain in the non-leaky mutant pwA500. By subtracting the outward currents of pwA500 from the slow and prolonged outward currents of the wild type, we have tentatively separated a Ca-induced K+ current from the voltage-dependent K+ current. The time courses of these two currents differ by two orders of magnitude.

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