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R R Preston

Publications and source records attributed to R R Preston.

At least 37 records · Page 2Linked to original sources
PubMed1992

Calcium-dependent inactivation of the calcium current activated upon hyperpolarization of Paramecium tetraurelia.

The Ca2+ current activated upon hyperpolarization of Paramecium tetraurelia decays over a period of 150-200 ms during sustained steps under voltage clamp. At membrane potentials between -70 and approximately -100 mV, the time course of this inactivation is described by a single exponential function. Steps negative to approximately -100 mV elicit currents that decay biexponentially, however. Three lines of evidence suggest that this current's inactivation is a function of intracellular Ca2+ concentration rather than membrane potential: (a) Comparing currents with similar amplitudes but elicited at widely differing membrane potentials suggests that their time course of decay is a sole function of inward current magnitude. (b) The extent of current inactivation is correlated with the amount of Ca2+ entering the cell during hyperpolarization. (c) The onset and time course of recovery from inactivation can be hastened significantly by injecting cells with EGTA. We suggest that the decay of this current during hyperpolarization involves a Ca(2+)-dependent pathway.

Animals↗
PubMed1991

Efficient transformation of cam2, a behavioral mutant of Paramecium tetraurelia, with the calmodulin gene.

An Ile-136----Thr substitution in calmodulin reduces the Ca(2+)-dependent K+ currents of cam2, a behavioral mutant of Paramecium tetraurelia, and renders it overly susceptible to BaCl2. DNA fragments carrying the wild-type CAM gene injected into cam2 macronuclei reverted these phenotypes in the clonal descendants of the recipients. Tetrahymena telomeric sequences, added in vitro to the fragment termini before injection, enhanced the efficiency and quality of transformation. Five times 10(4) copies of such fragments consistently restored the phenotypes to near normal; even 10(3) or fewer copies could still effect weak transformation. The restored phenotypes were stable for greater than 20 fissions in many clones and were lost after autogamy. We examined the fate of the injected fragments in the transformed clones and discuss the possible application of this efficient transformation in the cloning of other genes of P. tetraurelia.

Amino Acid Sequence↗
PubMed1990

A magnesium current in Paramecium.

Recent reappraisals of the role of ionized magnesium in cell function suggest that many cells maintain intracellular free Mg2+ at low concentrations (0.1 to 0.7 mM) and that external agents can influence cell function via changes in intracellular Mg2+ concentration. Depolarization and hyperpolarization of voltage-clamped Paramecium elicited a Mg2(+)-specific current, IMg. Both Co2+ and Mn2+ were able to substitute for Mg2+ as charge carriers, but the resultant currents were reduced compared with Mg2+ currents. Intracellular free Mg2+ concentrations were estimated from the reversal potential of IMg to be about 0.39 mM. The IMg was inhibited when external Ca2+ was removed or a Ca2+ chelator was injected, suggesting that its activation was Ca2(+)-dependent.

Animals↗
PubMed1990

Mutations in paramecium calmodulin indicate functional differences between the C-terminal and N-terminal lobes in vivo.

We examined calmodulin and its gene from the wild-type and viable mutants of P. tetraurelia. The mutants, selected for their behavioral aberrations, have little or no defects in growth rates, secretion, excretion, or motility. They can be grouped according to whether they underreact or overreact behaviorally to certain stimuli, reflecting their respective loss of either a Ca2(+)-dependent Na+ current or a Ca2(+)-dependent K+ current. Sequence analyses showed that all three underreactors have amino acid substitutions in the N-terminal lobe of the calmodulin dumbbell, whereas all three overreactors have substitutions in the C-terminal lobe. No mutations fell in the central helix connecting the two lobes. These results may indicate that the sites defined by these mutations are important in membrane excitation but not in other biological functions. They also suggest that the two lobes of calmodulin may be used differentially for the activation of different Ca2(+)-dependent channels.

Amino Acid Sequence↗
PubMed1990

Genetic dissection of Ca2(+)-dependent ion channel function in Paramecium.

The ciliated protozoan, Paramecium, broadcasts the activity of its individual ion channel classes through its swimming behaviour. This fact has made it possible to isolate mutants with defective ion currents, simply by selecting individuals with abnormal swimming patterns. At least four of Paramecium's ion currents are activated by rising intracellular calcium concentration, including two K+ currents and a Na+ current. A variety of cell lines with defects in these Ca2(+)-dependent currents have been isolated: in several cases, the defects have been traced to mutations in the structural gene for calmodulin. Sequence analysis of calmodulins from these and other Ca2(+)-dependent ion-current mutants may enable a detailed mapping of putative channel interaction domains on the surface of the calmodulin molecule.

Amino Acid Sequence↗
PubMed1990

Evidence for two K+ currents activated upon hyperpolarization of Paramecium tetraurelia.

Hyperpolarization of voltage-clamped Paramecium 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 greater than or equal to 50-msec duration contains fast-decaying (tau approximately 3.5 msec) and slow-decaying (tau approximately 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 0.7 mM quinidine.

Animals↗
PubMed1990

Calmodulin defects cause the loss of Ca2(+)-dependent K+ currents in two pantophobiac mutants of Paramecium tetraurelia.

Two behavioral mutants of Paramecium 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 of Paramecium 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 in Paramecium.

Amino Acid Sequence↗
PubMed1990

Interactions between mutants with defects in two Ca2(+)-dependent K+ currents of Paramecium tetraurelia.

Paramecium tetraurelia possesses two Ca2(+)-dependent K+ currents, activated upon depolarization IK(Ca,d), or upon hyperpolarization IK(Ca,h). The two currents are mediated by pharmacologically distinct ion channel populations. Three mutations of P. tetraurelia affect these currents. 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 enhances IK(Ca,d) but has no affect on IK(Ca,h). A third mutation, restless (rst), also increases IK(Ca,d) slightly, but its principle effect is in causing an early activation of IK(Ca,h). Interactions between the products of these three genes were investigated by constructing three double mutants. Both teaA and rst restore IK(Ca,d) and IK(Ca,h) in pantophobiac A1, but the phenotypes of teaA and rst are not corrected by a second mutation. These observations may indicate a role for the gene products of teaA and rst in regulating the activity of IK(Ca,d) and IK(Ca,h), respectively.

Animals↗
PubMed1990

Transcapillary oncotic pressure in the edema of congestive heart failure.

Tissue fluid and plasma oncotic pressure levels were measured in an unselected group of 13 patients presenting with congestive heart failure. Patients had a mean serum albumin of 3.6 g/dl +/- (SD) 0.35; serum oncotic pressure was 22.23 +/- 1.8 mm Hg; tissue fluid (lower leg) oncotic pressure was 0.985 +/- 0.34 mm Hg. The oncotic pressure gradient between plasma and tissue fluid was 21.25 Hg +/- 1.7. This is about 10 mm Hg greater than delta pi in normal man. The high value in congestive heart failure is a mechanism to protect against the formation of edema. The possible mechanisms responsible for the formation of edema in congestive heart failure are discussed.

Aged↗
PubMed1988

L-glutamate-induced membrane hyperpolarization and behavioural responses in Paramecium tetraurelia.

Paramecium tetraurelia is attracted to L-glutamic acid concentrations of 10(-9) M to 10(-4) M in a behavioural assay. Electrophysiological studies show that P. tetaurelia responds to L-glutamate application with hyperpolarization. This response is transient, even in the continued presence of the stimulus. The concentration dependence of the membrane potential response is similar to that of the behavioural responses, although the threshold concentration of L-glutamate required for hyperpolarization is three orders of magnitude lower than for attraction. The membrane potential response to L-glutamate persists following artificial deciliation of P. tetraurelia. While application of L-glutamate to P. tetraurelia invariably elicits a hyperpolarization, withdrawal of the stimulus frequently results in a second transient membrane response, in the form of either a hyperpolarization or a depolarization. It is suggested that these 'off-responses' may have a significant role in maintaining a behavioural response to L-glutamate.

Animals↗
PubMed1987

Correlations between cyclic AMP binding and chemoreception in Paramecium.

Paramecium tetraurelia is attracted to cyclic AMP, which probably, as other attractants, signifies the presence of food. Attraction to cyclic AMP was specific, saturable, and, therefore, likely to be receptor-mediated. In these studies, we measured the binding of cyclic [3H]AMP to whole cells and found it to be saturable, reversible, and displaying specificity similar to that of attraction. An HPLC method of separating nucleotides was devised and used to determine that external cyclic AMP was degraded in the absence of IBMX, a phosphodiesterase inhibitor, and that cyclic AMP was taken into the cells in small amounts. Since binding and attraction were subsequently measured in the presence of IMBX, it was cyclic AMP and not a degradation product that served as the attractant stimulus for Paramecium.

1-Methyl-3-isobutylxanthine↗
PubMed1987

Chemoreception in Paramecium tetraurelia: acetate and folate-induced membrane hyperpolarization.

Acetic and folic acids hyperpolarize the membrane potential of Paramecium tetraurelia in a concentration-dependent manner. The membrane responses are accompanied by small changes in cell resistance, and are significantly reduced by increasing extracellular cation concentrations, suggesting that the attractants bring about the membrane potential change by increasing cell permeability to cations. The inability to show a reversal potential for the hyperpolarization to attractants suggests that the effects of cations on the response are non-specific, however. The possible roles of Ca++, K+, and Na+ in the attractant-induced responses were further investigated by applying acetate and folate to cells with genetic defects in specific ion conductances, by collapsing the driving forces for these ions, and by testing the effects of ion channel blockers on the responses. These studies suggest that the membrane responses to attractants are not due to the direct effects of increased or decreased membrane permeability to cations. Attempts to block the acetate and folate-induced hyperpolarization by collapsing surface potential or using a mutant with reduced surface charge were inconclusive, as were studies on the possible role of attractant transport in the membrane responses. We hypothesize that the membrane hyperpolarization may be due to either the indirect effects of increased calcium permeability, to extrusion of calcium through activation of a calcium pump, or to a proton efflux.

Acetates↗
PubMed1987

Localization of the chemoreceptive properties of the surface membrane of Paramecium tetraurelia.

The plasma membrane of Paramecium tetraurelia comprises two morphologically distinct components; a membrane that encloses the cell body and a ciliary membrane. In order to investigate the relative contributions of the two membranes to attractant-induced membrane potential changes, cells were deciliated with ethanol and their subsequent responses to attractants examined. Deciliation did not significantly affect the magnitude of the hyperpolarizations evoked by acetic or lactic acids, and had no effect on the concentration dependence of responses to folic acid. We conclude that the components necessary for detection and response to attractants are not exclusive to the ciliary membrane of P. tetraurelia. Deciliation of Paramecium concomitantly permits localized chemical stimuli to be applied directly to the cell surface in the absence of strong fluid currents that are generated by the activity of the locomotory organelles. By systematically applying K2 folate to a number of sites on the cell surface, it has been possible to demonstrate an anterior-posterior gradient of chemosensitivity on the cell body of P. tetraurelia.

Acetates↗
PubMed1987

Eukaryotic unicells: how useful in studying chemoreception?

The description of the chemoreception pathway in Paramecium is incomplete, but the technical means are available to study these pathways at the molecular level. The hallmark of ciliates is their versatility and their most important attribute is the availability of useful mutants. It is just this versatility and amenability to genetic manipulation that will move the study of Paramecium chemoreception forward and provide useful information for chemoreceptor cell function in general.

Animals↗
PubMed1986

Rectilinear particle arrays in freeze-fracture replicas of the surface membrane of Paramecium tetraurelia.

Freeze-fracture replicas of the plasma membrane of unfixed, uncryoprotected Paramecium tetraurelia bear large rectilinear arrays of 11 nm particles arranged in 7-11 parallel rows. The arrays are of sufficient size to leave impressions in replicas of the underlying outer alveolar membrane, and are apparent as parallel ridges in replicas of the surface coat of deep-etched cells. By noting the location of arrays in replicas of identified portions of the cortex of P. tetraurelia, it has been possible to map the distribution of arrays over the cell surface. The arrays are found primarily over the anterior surfaces of the cell, covering an area that extends from the preoral suture over the left adoral field and a large portion of the anterior dorsal surface. Freeze-fracture analyses of cells taken from a number of different stages of a culture cycle suggest that the particle arrays are not replicated as an integral part of the cortex during cell division, but are assembled and oriented in the membrane as the cells mature. The appearance of small intramembranous particle complexes in the plasma membrane of cells in logarithmic growth phase supports this hypothesis, possibly representing an assembly stage in the formation of the larger particle arrays. The facts that the particle arrays are apparent in replicas of the surface coat of cells, are found primarily at the anterior of the cell body, and have a highly specific orientation with respect to the cell surface, strongly suggest that they function as chemoreceptors in P. tetraurelia.

Animals↗