Biomedical subjects
Y Saimi
Publications and source records attributed to Y Saimi.
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.
Patch clamp studies of microbial ion channels.
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Genetic analysis of ion channels of prokaryotes and lower eukaryotes.
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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.
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.
Calmodulin mutants and Ca2(+)-dependent channels in Paramecium.
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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.
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.
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.
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.
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.
Calcium-dependent potassium channel in Paramecium studied under patch clamp.
We have studied a class of Ca2+i-dependent K channels in inside-out excised membrane patches from Paramecium under patch clamp. single channels had a conductance of 72 +/- 9.0 pS in a solution containing 100 mM 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: less than 0.1. The channel activity was dependent on Ca2+i, which was applied to the cytoplasmic side; the Ca2+i concentration for the half maximal activation was 2 microM. The Hill coefficient for the Ca2+i dependence of the channel activity was 2.58, indicating that more than two Ca2+i bindings are necessary for full activation. Unlike most Ca2+i-dependent K channels in other organisms, the channels in Paramecium were slightly more active upon hyperpolarization than upon depolarization. The voltage dependence was fitted to a Boltzmann curve with 41.2 mV per e-fold change in channel activity. While a high Ca2+i concentration activated the channels, it also irreversibly reduced the channel activity over time. The decay of channel activity occurred faster at higher Ca2+i concentrations. Quaternary ammonium ions suppressed ion passage through the channel; more highly alkylated quaternary ammonium ions were more efficient in blocking. Ba2+i and Ca2+i were relatively ineffective in blockage. it was concluded that these Ca2+i-dependent K channels in Paramecium are different from the previously described Ca2+i-dependent K channels, and are perhaps of a novel class.
Proteolytic activation of a hyperpolarization- and calcium-dependent potassium channel in Paramecium.
The effects of proteolysis on a hyperpolarization- and Ca2+-dependent K channel from the surface membrane of Paramecium 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 additionally 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 this Paramecium 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 in Paramecium.
Ion channels in Paramecium, yeast and Escherichia coli.
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Ion channels in Paramecium, yeast, and Escherichia coli.
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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.