Intra- and interspecific complementation of membrane-inexcitable mutants of Paramecium.

Membrane excitation was the basis for backward swimming of Paramecium facing stimulus. According to standard genetic tests, inexcitable mutants fell into three complementation groups for both Paramecium tetraurelia (pwA, pwB, and pwC) and Paramecium caudatum (cnrA, cnrB, and cnrC). Cytoplasm from a wild type transferred to a mutant through microinjection restored the excitability. Transfusions between genetically defined complementation groups of the same species effected curing, whereas transfusions between different mutants (alleles) of the same group or between sister cells of the same mutant clone did not. Cytoplasmic transfers of all combinations among the six groups of mutants of the two species showed that any cytoplasm, except those from the same group, was able to cure. Since the pawns and the caudatum nonreversals complement one another through transfusion, they appeared to belong to six different complementation groups. The extent of curing, the amount of transfer needed to cure, and the time course of curing were characteristic of the group that received the transfusion. Variations in these parameters further suggested that the six groups represented six different genes. Because the donor cytoplasms from either species were equally effective quantitatively in curing a given mutant, the curing factors were not species specific. These factors are discussed.

Microinjection of cytoplasm as a test of complementation in Paramecium.

Mutants in Paramecium tetraurelia, unable to generate action potentials, have been isolated as cells which show no backward swimming in response to ionic stimulation. These "pawn" mutants belong to at least three complementation groups designated pwA, pwB, and pwC. We have found that microinjection of cytoplasm from a wild-type donor into a pawn recipient of any of the three complementation groups restores the ability of the pawn to generate action potentials and hence swim backward. In addition, the cytoplasm from a pawn cannot restore a recipient of the same complementation group, but that from a pawn of a different group can. Electrophysiological analysis had demonstrated that the restoration of backward swimming is not due to a simple addition of ions but represents a profound change in the excitable membrane of the recipient pawn cells. Using known pawn mutants and those which had previously been unclassified, we have been able to establish a perfect concordance of genetic complementation and complementation by cytoplasmic transfer through microinjection. This method has been used to classify pawn mutants that are sterile or hard-to-mate and to examine the ability of cytoplasms from different species of ciliated protozoa to restore the ability to swim backward in the pawn mutants of P. tetraurelia. A cell homogenate has also been fractionated by centrifugation to further purify the active components. These results demonstrate that transfer of cytoplasm between cells by microinjection can be a valid and systematic method to classify mutants. This test is simpler to perform than the genetic complementation test and can be used under favorable conditions in mutants that are sterile and in cells of different species.

Antibodies to the ciliary membrane of Paramecium tetraurelia alter membrane excitability.

Immobilization of Paramecium followed the binding of antibodies to the major proteins of the ciliary membrane (the immobilization antigens, i-antigens, approximately 250,000 mol wt). Immunoelectron microscopy showed this binding to be serotype-specific and to occur over the entire cell surface. Antibody binding also reduced the current through the Ca-channel of the excitable ciliary membrane as monitored using a voltage-clamp. The residual Ca-current appeared normal in its voltage sensitivity and kinetics. As a secondary consequence of antibody binding, the Ca-induced K-current was also reduced. The resting membrane characteristics and other activatable currents, however, were not significantly altered by the antibody treatment. Since monovalent fragments of the antibodies also reduced the current but did not immobilize the cell, the electrophysiological effects were not the secondary consequences of immobilization. Antibodies against the second most abundant family of proteins (42,000-45,000 mol wt) had similar electrophysiological effects as revealed by experiments in which the Paramecia and the serum were heterologous with respect to the i-antigen but homologous with respect to the 42,000-45,000-mol-wt proteins. Protease treatment, shown to remove the surface antigen, also caused a reduction of the Ca-inward current. The loss of the inward Ca-current does not seem to be due to a drop in the driving force for Ca++ entry since increasing the external Ca++ or reducing the internal Ca++ (through EGTA injection) did not restore the current. Here we discuss the possibilities that (a) the major proteins define the functional environment of the Ca-channel and that (b) the Ca-channel is more susceptible to certain general changes in the membrane.

Calmodulin activation of calcium-dependent sodium channels in excised membrane patches of Paramecium.

Calmodulin is a calcium-binding protein that participates in the transduction of calcium signals. The electric phenotypes of calmodulin mutants of Paramecium have suggested that the protein may regulate some calcium-dependent ion channels. Calcium-dependent sodium single channels in excised patches of the plasma membrane from Paramecium were identified, and their activity was shown to decrease after brief exposure to submicromolar concentrations of calcium. Channel activity was restored to these inactivated patches by adding calmodulin that was isolated from Paramecium to the cytoplasmic surface. This restoration of channel activity did not require adenosine triphosphate and therefore, probably resulted from direct binding of calmodulin, either to the sodium channel itself or to a channel regulator that was associated with the patch membrane.

Are ions involved in the gating of calcium channels?

The rates of activation and deactivation of the currents carried by calcium, strontium, or barium ions through the voltage-sensitive calcium channel of Paramecium are different. The differences cannot be attributed to complications due to internal ion concentration, calcium channel inactivation, potassium current activation, surface charge effects, or incomplete space clamping. The findings indicate participation of the divalent cations in the voltage-driven calcium channel gating process.

Ion channels in yeast.

Voltage-dependent ion channels have been found in the plasma membrane of the yeast Saccharomyces cerevisiae. Ion channel activities were recorded from spheroplasts or patches of plasma membrane with the patch-clamp technique. The most prominent activities came from a set of potassium channels with the properties of activation by positive but not negative voltages, high selectivity for potassium over sodium ion, unit conductance of 20 picosiemens, inhibition by tetraethylammonium or barium ions, and bursting kinetics.

Evaluation of a new fiber-reinforced resin composite.

Efficacy of the usage of an experimental fiber-reinforced composite (FRC) on mechanical properties of an indirect composite was investigated by means of three-point bending and Charpy impact tests. Bond strength between the FRC and the indirect composite was also evaluated by tensile testing. The FRC consisted of a matrix resin with 25% silanized milled glass fiber (11-microm diameter, 150-microm length) and 5% colloidal silica. The values of strain of proportional limit, total strain, and fracture energy of the FRC during the bending test (1.2%, 10.4%, and 41.6 x 10(-3) J) were significantly higher than those of the indirect composite (0.1%, 2.5%, and 11.9 x 10(-3) J). The impact strengths of the 1-mm specimens with FRC ranged from 15.2 to 15.9 kJ/m(2), and were significantly higher than that of the control (3.1 kJ/m(2)). The 2-mm specimens showed significant difference from the control when the FRC thickness was equal or greater than 0.5 mm. The bond strength after the thermocycling was 15.2 MPa, and all of the specimens exhibited cohesive fracture inside the indirect composite. Based upon the results, it was concluded that the FRC tested in this study improved toughness and impact resistance of the indirect composite. The interfacial bonding between the FRC and the indirect composite was strong enough to prevent delamination.

Studies on cephalopod rhodopsin conformational changes in chromophore and protein during the photoregeneration process.

The ultraviolet absorbance of squid and octopus rhodopsin changes reversibly at 234 nm and near 280 nm in the interconversion of rhodopsin and metarhodopsin. The absorbance change near 280 nm is ascribed to both protein and chromophore parts. Rhodopsin is photoregenerated from metarhodopsin via an intermediate, P380, on irradiation with yellow light (lamda >520 nm). The ultraviolet absorbance decreases in the change from rhodopsin to metarhodopsin and recovers in two steps; mostly in the process from metarhodopsin to P380 and to a lesser extent in the process from P380 to rhodopsin. P380 has a circular dichroism (CD) band at 380 nm and its magnitude is the same order as that of rhodopsin. Thus it is considered that the molecular structure of P380 is close to that of rhodopsin and that the chromophore is fixed to opsin as in rhodopsin. In the change from metarhodopsin to P380, the chromophore is isomerized from the all-trans to the 11-cis form, and the conformation of opsin changes to fit 11-cis retinal. In the change from P380 to rhodopsin, a small change in the conformation of the protein part and the protonation of the Schiff base, the primary retinal-opsin link, occur.

Calcium current activated upon hyperpolarization of Paramecium tetraurelia.

Hyperpolarization of Paramecium tetraurelia under conditions where K+ currents are suppressed elicits an inward current that activates rapidly toward a peak at 25-80 ms and decays thereafter. This peak current (Ihyp) is not affected by removing Cl ions from the microelectrodes used to clamp membrane potential, or by changing extracellular Cl- concentration, but is lost upon removing extracellular Ca2+. Ihyp is also lost upon replacing extracellular Ca2+ with equimolar concentrations of Ba2+, Co2+, Mg2+, Mn2+, or Sr2+, suggesting that the permeability mechanism that mediates Ihyp is highly selective for Ca2+. Divalent cations also inhibit Ihyp when introduced extracellularly, in a concentration- and voltage-dependent manner. Ba2+ inhibits Ihyp with an apparent dissociation constant of 81 microM at -110 mV, and with an effective valence of 0.42. Ihyp is also inhibited reversibly by amiloride, with a dissociation constant of 0.4 mM. Ihyp is not affected significantly by changes in extracellular Na+, K+, or H+ concentration, or by EGTA injection. Also, it is unaffected by manipulations or mutations that suppress the depolarization-activated Ca2+ current or the various Ca(2+)-dependent currents of Paramecium. We suggest that Ihyp is mediated by a novel, hyperpolarization-activated calcium conductance that is distinct from the one activated by depolarization.

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.

A derivative of amiloride blocks both the light-regulated and cyclic GMP-regulated conductances in rod photoreceptors.

Vertebrate rod photoreceptors in the dark maintain an inward current across the outer segment membrane. The photoresponse results from a light-induced suppression of this dark current. The light-regulated current is not sensitive to either tetrodotoxin or amiloride, potent blockers of Na+ channels. Here, we report that a derivative of amiloride, 3',4'-dichlorobenzamil (DCPA), completely suppresses the dark current and light response recorded from rod photoreceptors. DCPA also blocks a cyclic GMP-activated current in excised patches of rod plasma membrane and a cGMP-induced Ca++ flux from rod disk membranes. These results are consistent with the notion that the Ca++ flux mechanism in the disk membrane and the light-regulated conductance in the plasma membrane are identical. DCPA also inhibits the Na/Ca exchange mechanism in intact rods, but at a 5-10-fold-higher concentration than is required to block the cGMP-activated flux and current. The blocking action of DCPA in 10 nM Ca++ is different from that in 1 mM Ca++, which suggests either that the conductance state of the light-regulated channel may be modified in high and low concentrations of Ca++, or that there may be two ionic channels in the rod outer segment membrane.

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 Ba2+ 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 analysis of mutants with a reduced Ca2+-dependent K+ current in Paramecium tetraurelia.

Two mutants of Paramecium tetraurelia with greatly reduced Ca2+-dependent K+ currents have been isolated and genetically analyzed. These mutants, designated pantophobiac, give much stronger behavioral responses to all stimuli than do wild-type cells. Under voltage clamp, the Ca2+-dependent K+ current is almost completely eliminated in these mutants, whereas the Ca2+ current is normal. The two mutants, pntA and pntB, are recessive and unlinked to each other. pntA is not allelic to several other ion-channel mutants of P. tetraurelia. The microinjection of a high-speed supernatant fraction of wild-type cytoplasm into either pantophobiac mutant caused a temporary restoration to the wild-type phenotype.

K(+)-channel transgenes reduce K(+) currents in Paramecium, probably by a post-translational mechanism.

PAK11 is 1 of more than 15 members in a gene family that encodes K(+)-channel pore-forming subunits in Paramecium tetraurelia. Microinjection of PAK11 DNA into macronuclei of wild-type cells results in clonal transformants that exhibit hyperexcitable swimming behaviors reminiscent of certain loss-of-K(+)-current mutants. PAK2, a distant homolog of PAK11, does not have the same effect. But PAK1, a close homolog of PAK11, induces the same hyperexcitability. Cutting the PAK11 open reading frame (ORF) with restriction enzymes before injection removes this effect entirely. Microinjection of PAK11 ORF flanked by the calmodulin 5' and 3' UTRs also induces the same hyperexcitable phenotype. Direct examination of transformed cells under voltage clamp reveals that two different Ca(2+)-activated K(+)-specific currents are reduced in amplitude. This reduction does not correlate with a deficit of PAK11 message, since RNA is clearly produced from the injected transgenes. Insertion of a single nucleotide at the start of the PAK11 ORF does not affect the RNA level but completely abolishes the phenotypic transformation. Thus, the reduction of K(+) currents by the expression of the K(+)-channel transgenes reported here is likely to be the consequence of a post-translational event. The complexity of behavioral changes, possible mechanisms, and implications in Paramecium biology are discussed.

The cloning by complementation of the pawn-A gene in Paramecium.

The genetic dissection of a simple avoidance reaction behavior in Paramecium tetraurelia has shown that ion channels are a critical molecular element in signal transduction. Pawn mutants, for example, were originally selected for their inability to swim backward, a trait that has since been shown to result from the loss of a voltage-dependent calcium current. The several genes defined by this phenotype were anticipated to be difficult to clone since the 800-ploid somatic macronucleus of P. tetraurelia is a formidable obstacle to cloning by complementation. Nonetheless, when the macronucleus of a pawn mutant (pwA/pwA) was injected with total wild-type DNA or a fractional library of DNA, its clonal descendants all responded to stimuli like the wild type. By sorting a fractional library, we cloned and sequenced a 2.3-kb fragment that restores the Ca2+ current and excitability missing in pawn-A. Data from RNase protection assays, followed by the sequencing of mutant alleles and cDNA clones, established an open reading frame. The conceptually translated product suggests a novel protein that may be glycophosphatidylinositol anchored. We also discuss the general usefulness of this method in cloning other unknown DNA sequences from Paramecium that are functionally responsible for various mutant phenotypes.

The cloning and molecular analysis of pawn-B in Paramecium tetraurelia.

Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca(2+) 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 Ca(2+) 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.

Effects of filler composition on flexibility of microfilled resin composite.

The effects of the filler composition on physical and mechanical properties of microfilled composites was investigated by measuring water absorption, solubility, compressive, flexural, and impact strength. A series of experimental composites, consisting of UDMA/TEGDMA comonomer matrix and prepolymerized fillers, was fabricated. The prepolymerized fillers were composed of hydrophobic colloidal silica and two monomers in varying ratios, trimethylolpropanetrimethacrylate (TMPT), and polyesterdiacrylate (PEDA). TMPT/PEDA ratios were 100:0, 64:36, 46:54, 18:82, and 0:100%. There were no significant differences in water sorption and solubility, regardless of the amount of PEDA monomer. Young's modulus and modulus of resilience increased with decreasing PEDA ratio. Fracture energy exhibited drastic changes (30.1 x 10(-5) J to 93.4 x 10(-5) J). The highest value of flexural strength (96.0 +/- 3.5 MPa) was obtained when the TMPT-PEDA filler was 46:54. The impact strengths of composites fabricated with TMPT-PEDA filler of 46:54 (11.2 +/- 1.4 kJ/m(2)), 18:82 (10.6 +/- 3.2 kJ/m(2)), and 0:100 (13.1 +/- 3.8 kJ/m(2)) were significantly higher than those with 100:0 (6.0 +/- 1.8 kJ/m(2)) or 64:36 (7.1 +/- 2.4 kJ/m(2)). Based upon the results, it was concluded that the mechanical properties of microfilled composites were improved by the modification of prepolymerized filler composition.

In vivo Paramecium mutants show that calmodulin orchestrates membrane responses to stimuli.

Paramecium generates a Ca2+ action potential and can be considered a one-cell animal. Rises in internal [Ca2+] open membrane channels that specifically pass K+, or Na+. Mutational and patch-clamp studies showed that these channels, like enzymes, are activated by Ca(2+)-calmodulin. Viable CaM mutants of Paramecium have altered transmembrane currents and easily recognizable eccentricities in their swimming behavior, i.e. in their responses to ionic, chemical, heat, or touch stimuli. Their CaMs have amino-acid substitutions in either C- or N-terminal lobes but not the central helix. Surprisingly, these mutations naturally fall into two classes: C-lobe mutants (S101F, I136T, M145V) have little or no Ca(2+)-dependent K+ currents and thus over-react to stimuli. N-lobe mutants (E54K, G40E+D50N, V35I+D50N) have little or no Ca(2+)-dependent Na+ current and thus under-react to certain stimuli. Each mutation also has pleiotropic effects on other ion currents. These results suggest a bipartite separation of CaM functions, a separation consistent with the recent studies of Ca(2+)-ATPase by Kosk-Kosicka et al. [41, 55]. It appears that a major function of Ca(2+)-calmodulin in vivo is to orchestrate enzymes and channels, at or near the plasma membrane. The orchestrated actions of these effectors are not for vegetative growth at steady state but for transient responses to stimuli epitomized by those of electrically excitable cells.

A new multigene family encoding calcium-dependent calmodulin-binding membrane proteins of Paramecium tetraurelia.

Ca2+/calmodulin (CaM) regulates various physiological processes in a wide variety of organisms, metazoa and protists alike. To better understand Ca2+/CaM-dependent processes, particularly those with membrane-associated components, we studied Ca2+/CaM-binding membrane proteins in Paramecium tetraurelia, a unicellular model system. A CaM-binding protein, PCM1 (Paramecium CaM-binding membrane-bound protein), from a detergent-solubilized ciliary membrane fraction was identified and purified through Ca2+-dependent CaM-affinity chromatography. PCM1 has an apparent molecular mass of approx. 65kDa. It binds radiolabeled CaM in blot overlay assays and binds to CaM-affinity columns, both only in the presence of 10 microM or higher Ca2+. Three peptide sequences from PCM1 were obtained, and polymerase chain reaction (PCR) and Southern hybridization experiments were designed accordingly, leading to a partial cDNA clone for PCM1 and the discovery of three homologs: PCM2, PCM3 and PCM4. Amino acid sequences predicted by the full-length coding sequence for PCM3 and partial genes for PCM1, PCM2 and PCM4 are very similar (approx. 85% amino-acid identities). Their sequences indicate that they are hitherto novel proteins with beta/gamma-crystallin domains, cysteine-rich regions and potential CaM-binding domains. These protein motifs are suggested to mediate protein-protein interaction important for Ca2+/CaM signal transduction event(s) through the PCM family of proteins.

Ion channel regulation by calmodulin binding.

While many ion channels are modulated by phosphorylation, there is growing evidence that they can also be regulated by Ca(2+)-calmodulin, apparently through direct binding. In some cases, this binding activates channels; in others, it modulates channel activities. These phenomena have been documented in Paramecium, in Drosophila, in vertebrate photoreceptors and olfactory receptors, as well as in ryanodine receptor Ca(2+)-release channels. Furthermore, studies on calmodulin mutants in Paramecium have shown a clear bipartite distribution of two groups of mutations in the calmodulin gene that lead to opposite behavioral and electrophysiological phenotypes. These results indicate that the N-lobe of calmodulin specifically interacts with one class of ion-channel proteins and the C-lobe with another.