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Y Saimi

Publications and source records attributed to Y Saimi.

At least 19 recordsLinked to original sources
PubMed1997

Random mutagenesis reveals a region important for gating of the yeast K+ channel Ykc1.

YKC1 (TOK1, DUK1, YORK) encodes the outwardly rectifying K+ channel of the yeast plasma membrane. Non-targeted mutations of YKC1 were isolated by their ability to completely block proliferation when expressed in yeast. All such mutations examined occurred near the cytoplasmic ends of the transmembrane segments following either of the duplicated P loops, which we termed the 'post-P loop' (PP) regions. These PP mutations specifically caused marked defects in the 'C1' states, a set of interrelated closed states that Ykc1 enters and exits at rates of tens to hundreds of milliseconds. These results indicate that the Ykc1 PP region plays a role in determining closed state conformations and that non-targeted mutagenesis and microbial selection can be a valuable tool for probing structure-function relationships of ion channels.

Amino Acid Sequence↗
PubMed1996

Bonding to intact dentin.

It has been reported that the presence of a smear layer on dentinal substrates can compromise bonding. Typically, smear layers are removed by acidic agents that selectively extract calcium salts from dentin surfaces to leave a collagen-rich substrate. Acid-conditioned dentin (i.e., demineralized) is then primed and an adhesive agent applied. In the present study, we removed smear layers by "polishing" dentin specimens with a hydroxyapatite paste and ultrasonication. Bonding procedures were carried out by means of an aqueous solution of 20% 2-methacryloyloxyethyl phenyl phosphoric acid (phenyl-P) and 30% 2-hydroxyethyl methacrylate, referred to as 2OP-30H, a "self-etching primer". The 20P-30H solution was applied to "intact" dentin (i.e., non-demineralized) for either 30 or 60 s. Control samples received no application (O s) of the self-etching primer. Mean tensile bond strengths (10 MPa) were similar in both the 30-second- and 60-second-primed groups. The widths of formed hybrid layers varied from 0.3 +/- 0.2 micron at O s application (control) to 2.1 +/- 0.3 micron for the 30-second group and 4.1 +/- 0.2 micron for the 60-second group. SEM and TEM observations revealed that the 20P-30H self-etching primer created diffusion channels into "intact" calcium-rich dentin which permitted monomer to infiltrate dentin substrates. Hybrid layers identified under microscopic examination demonstrated resistance to both HCI and NaOCI treatments, suggesting that the hybrid layer was not defective, and that bonding was stable.

Acid Etching, Dental↗
PubMed1995

YKC1 encodes the depolarization-activated K+ channel in the plasma membrane of yeast.

Our previous patch-clamp studies showed that depolarization activates a K(+)-specific current in the plasma membrane of the budding yeast, Saccharomyces cerevisiae [Gustin et al. (1986) Science 233, 1195-1197]. The Yeast Genome Sequencing Project has now uncovered on the left arm of chromosome X an open reading frame (ORF) that predicts a 77-kDa protein reminiscent of a shaker-like alpha subunit with 6 membrane spans followed by a subunit with 2 spans. We found that deleting this ORF removes the yeast K+ current. Furnishing the ORF from plasmids restores or even greatly amplifies this current. These manipulations have no effects on the 40-pS mechanosensitive conductance also native to this membrane. Thus, this ORF, named YKC1 here, likely encodes a structure for the K(+)-specific channel of the yeast plasma membrane. This and other K+ channel subunits are compared and the possible uses of this gene in research are discussed. YKC1 has recently been shown by others to induce in frog oocytes a K+ current. Its activation is coupled to EK+ and its outward rectification depends on external divalent cations. We found the YKC1 channel in its native membrane activates at low voltages largely independent of EK+ and it remains so despite removal of divalents by chelation.

Amino Acid Sequence↗
PubMed1995

Functional reconstitution of ion channels from Paramecium cortex into artificial liposomes.

Toward isolating channel proteins from Paramecium, we have explored the possibility of functionally reconstituting ion channels in an artificial system. Proteins from Paramecium cortex reconstituted with soybean azolectin retained several channels whose activities were readily registered under patch clamp. The most commonly encountered activities were three: (i) a 71-pS cation channel that opens at all voltages unless di- or trivalent cations were added to close them, (ii) a 40 pS monovalent cation channel, and (iii) a large-conductance channel that prefers anions and exhibits many subconductance states. These channels survived mild detergent treatments without observable functional alterations. The possible origin of these channels from internal membranes, the possible role of 71-pS channel in internal Ca2+ release, and the prospects of their purification are discussed.

Animals↗
PubMed1995

Paramecium Na+ channels activated by Ca(2+)-calmodulin: calmodulin is the Ca2+ sensor in the channel gating mechanism.

Paramecium Na+ channels, which were Ca(2+)-calmodulin activated, were studied in the inside-out mode of patch clamp. After excision of the membrane patch, they were active in the presence of 10(-5) to 10(-3) M Ca+ in the bath. They became much less active in the presence of 10(-6) M Ca2+, and their activity subsided completely at 10(-8) M Ca2+. A Hill plot showed a dissociation constant of 6 microM for Ca2+ binding. This dissociation constant shifted to a submicromolar range in the presence of 1 mM Mg2+. The channels also exhibited a mild voltage dependence. When exposed to 10(-8) M Ca2+ for an extended period of 2-4 min, channels were further inactivated even after bath Ca2+ was restored to 10(-4) M. Whereas neither high voltage (+100 mV) nor high Ca2+ (10(-3) M) was effective in reactivation of the inactive channels, addition of Paramecium wild-type calmodulin together with high Ca2+ to the bath restored channel activity without a requirement of additional Mg2+ and metabolites such as ATP. The channels reactivated by calmodulin had the same ion conductance, ion selectivity and Ca2+ sensitivity as those prior to inactivation. These inactivation and reactivation of the channels could be repeated, indicating that the direct calmodulin effect on the Na+ channel was reversible. Thus, calmodulin is a physiological factor critically required for Na+ channel activation, and is the Ca2+ sensor of the Na(+)-channel gating machinery.

Animals↗
PubMed1994

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.

Amino Acid Sequence↗
PubMed1994

New non-lethal calmodulin mutations in Paramecium. A structural and functional bipartition hypothesis.

The mechanisms by which calmodulin coordinates its numerous molecular targets in living cells remain largely unknown. To further understand how this pivotal Ca(2+)-binding protein functions in vivo, we isolated and studied nine new Paramecium behavioral mutants defective in calmodulin. Nucleotide sequences of mutant calmodulin genes indicated single amino-acid substitutions in mutants cam4(E104K), cam5-1 (D95G), cam6 (A102V), cam7 (H135R), cam14-1 (G59S) and cam15 (D50G). In addition, we encountered a second occurrence of three identified substitutions; they are cam1-2 (S101F), cam5-2 (D95G) and cam14-2 (G59S). Most of these mutational changes occurred in sites that have been highly conserved throughout evolution. Furthermore, most of these changes were not among the amino acids known to interact with the basic amphiphilic peptides of calmodulin targets. Consistent with our previous finding [Kink, J. A., Maley, M. E., Preston R. R., Ling, K.-Y., Wallen-Friedman, M. A., Saimi, Y. & Kung, C. (1990) Cell 62, 165-174], mutants that under-reacted to certain stimuli (allele number above 10) had substitutions in the N-terminal lobe of calmodulin, and those that over-reacted (below 10) had substitutions in the C-terminal lobe. No mutations were found in the central helix that connects the lobes. Thus, through undirected in vivo mutation analyses of Paramecium, we discovered that each of the two lobes of calmodulin has a distinct role in regulating the function of a specific ion channel and eventually the behavior of Paramecium. We, therefore, propose a hypothesis of functional bipartition of calmodulin that reflects its structural bipartition.

Alleles↗
PubMed1992

Primary mutations in calmodulin prevent activation of the Ca(++)-dependent Na+ channel in Paramecium.

Paramecium tetraurelia behavioral mutant cam12 displays a "fast-2" behavioral phenotype: it fails to respond to Na+ stimuli. Electrophysiologically, it lacks a Ca(++)-dependent Na+ current. Genetics and DNA sequencing showed the primary defect of cam12 to be in the calmodulin gene (Kink et al., 1990). To correlate calmodulin structure and function in Paramecium, we elucidated the primary structure of cam12 calmodulin. Peptide sequencing confirmed the two point mutations predicted by the DNA sequence: a glycine-to-glutamate substitution at position 40 and an aspartate-to-asparagine substitution at position 50. Our results further showed that lysine 13 and lysine 115 were methylated normally in cam12. It is likely that the electrophysiological abnormalities of cam12 are a direct reflection of the amino-acid substitutions, as opposed to improper posttranslational modification.

Amino Acid Sequence↗
PubMed1992

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.

Animals↗
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

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.

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

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↗