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M Covarrubias

Publications and source records attributed to M Covarrubias.

31 records · Page 2Linked to original sources
PubMed1991

mShal, a subfamily of A-type K+ channel cloned from mammalian brain.

We have cloned and expressed a mouse brain cDNA, mShal, that encodes a transient, A-type K+ current. mShal, the vertebrate homolog of the Drosophila Shal gene, defines a distinct subfamily of voltage-gated K+ channels. The Shal deduced proteins are more highly conserved between mouse and Drosophila than other presently known K+ channels. mShal carries a "low-threshold" A-type current with a hyperpolarized steady-state inactivation midpoint. Marked similarity was observed between mShal and its Drosophila homolog, fShal, with regard to voltage sensitivity of activation, macroscopic inactivation, steady-state inactivation, and 4-aminopyridine sensitivity. Sequence conservation for Shal proteins is unusually high at the amino terminus, an area considered important for inactivation. Removal of conserved amino-terminal residues from mShal modifies macroscopic inactivation but the transient nature of the current is preserved. Underlying the very high conservation of mShal and fShal may be a role in the nervous system that is conserved in widely divergent species.

Amino Acid Sequence↗
PubMed1991

Shaker, Shal, Shab, and Shaw express independent K+ current systems.

Although many K+ channel genes encoding homologous subunits have been cloned, a central question remains: how do these subunits associate to produce the diversity of K+ currents observed in living cells? Previous work has shown that different subunits encoded by the Shaker gene subfamily are able to form heteromultimers, which add to the diversity of currents. However, the unrestrained mixing of subunits from all genes to form hybrid channels would be undesirable for some cells that clearly require functionally discrete K+ currents. We show that Drosophila Shaker, Shal, Shab, and Shaw subunits form functional homomultimers, but that a molecular barrier to heteropolymerization is present. Coexpression of all four K+ channel systems does not alter their individual properties in any way. These experiments also demonstrate that multiple, independent A-current systems together with multiple, independent delayed rectifier systems can coexist in single cells.

Animals↗
PubMed1991

A mouse brain homolog of the Drosophila Shab K+ channel with conserved delayed-rectifier properties.

We have cloned and expressed a mouse brain K+ channel that is the homolog of the Drosophila Shab K+ channel. Mouse and Drosophila Shab K+ channels (mShab and fShab, respectively) represent an instance of K+ channels and structurally related species that are both functionally and structurally conserved; most kinetic, voltage-sensitive, and pharmacological properties are similar for the 2 channels. The greatest functional difference between the currents is recovery from inactivation, which is several times slower for mShab than for fShab currents. In addition to conserved structure, the mShab polypeptide has an unusually long nonconserved region at the carboxyl end of the protein. Truncation of 293 residues from the carboxyl end produced no noticeable change in voltage-sensitive, kinetic, or pharmacological properties. Thus, the measured functional properties of mShab are determined by the remaining 564 residues, most of which are conserved. The mShab and fShab channels are naturally occurring structural variants having substitutions in conserved portions that appear relatively neutral with respect to all measured properties except for, possibly, the rate of recovery from inactivation. The mShab current closely resembles a native delayed-rectifier-type potassium current, IK, in hippocampal neurons.

Amino Acid Sequence↗
PubMed1990

K+ current diversity is produced by an extended gene family conserved in Drosophila and mouse.

The Drosophila Shaker gene on the X chromosome has three sister genes, Shal, Shab, and Shaw, which map to the second and third chromosomes. This extended gene family encodes voltage-gated potassium channels with widely varying kinetics (rate of macroscopic current activation and inactivation) and voltage sensitivity of steady-state inactivation. The differences in the currents of the various gene products are greater than the differences produced by alternative splicing of the Shaker gene. In Drosophila, the transient (A current) subtype of the potassium channel (Shaker and Shal) and the delayed-rectifier subtype (Shab and Shaw) are encoded by homologous genes, and there is more than one gene for each subtype of channel. Homologs of Shaker, Shal, Shab, and Shaw are present in mammals; each Drosophila potassium-channel gene may be represented as a multigene subfamily in mammals.

Amino Acid Sequence↗
PubMed1990

Excision of membrane patches reduces the mean open time of nicotinic acetylcholine receptors.

Excision of membrane patches from clonal BC3H1 cells to the inside-out configuration reduces the mean open time and burst duration of skeletal muscle nicotinic acetylcholine receptors. The channel conductance is not altered. Our results indicate that the rates for several steps in receptor activation are affected. The altered receptors apparently form a homogeneous population. Patterns of activity similar to that seen after excision are seen infrequently in recordings from cell-attached patches, suggesting that receptors with this type of altered function are present in intact membranes. The extent and rate of the reduction in mean open time after excision are similar at acetylcholine concentration over a 1000-fold range, indicating that the alteration does not depend on the state of ligation of the receptor. The extent and rate also are similar at room temperature and at 11 degrees C. The changes seen indicate that careful comparisons to intact preparations are necessary when studying the gating of channels in excised patches or following reconstitution into artificial membranes.

Animals↗
PubMed1989

Inhibitors of asparagine-linked oligosaccharide processing alter the kinetics of the nicotinic acetylcholine receptor.

We used selective inhibitors of the asparagine-linked oligosaccharide processing pathway to study the effect of sugar trimming on the functional properties of the nicotinic acetylcholine (ACh) receptor expressed in clonal mammalian BC3H-1 cells. Inhibitors of initial steps of the processing pathway (1-deoxynojirimycin[DNJ] and castanospermine[CS]) reduced the density of ACh receptors on the cell surface (3- to 5-fold) but their responsiveness to ACh was more reduced (5- to 10-fold). These results suggest that the function of the ACh receptor was altered. When the ACh receptors were expressed in the presence of DNJ or CS, analysis of ACh-evoked single-channel currents (-100 mV and 11 degrees C) revealed an approximate threefold reduction in the opening rate (control: 600-650 s(-1)), treated: 130-250 s(-1)) and an approximate twofold reduction in the rate of agonist dissociation (control: 900-1,000 s(-1), treated: 400-500 s(-1)). In addition, the proportion of brief duration bursts (tau = 50-100 microseconds) was increased (1.5- to 2-fold) by treatments with DNJ or CS. In contrast, an inhibitor of a late processing step (swainsonine) did not produce such alterations. The single-channel conductance was not altered by any of the three inhibitors, and the slopes of log-log dose-response curves at low concentrations and desensitization did not appear to be affected. Each inhibitor altered the electrophoretic mobility of the ACh receptor subunits. We conclude that early sugar trimming can influence the kinetics of the nicotinic ACh receptor in BC3H-1 cells.

1-Deoxynojirimycin↗
PubMed1986

Equilibrium binding of cholinergic ligands to the membrane-bound acetylcholine receptor.

We have studied the binding equilibria of the membrane-bound acetylcholine receptor from Torpedo marmorata with representative cholinergic ligands by means of two fluorescence and a rapid centrifugation assay. Based on the established mechanism of acetylcholine binding to the receptor (Fels, G., Wolff, E. K., and Maelicke, A. (1982) Eur. J. Biochem. 127, 31-38), the obtained binding and competition data were analyzed assuming two classes of interacting sites for all ligands studied. The experimental data were consistent with this assumption and, based on the obtained KD values, suggest weak positively cooperative interactions of binding sites when occupied by agonists but independent (or negatively cooperative interacting) sites when occupied by antagonists. Based on the fluorescence binding assay employed, agonists and antagonists induce different conformational states of the liganded receptor. These states seem to be similar for all antagonists tested but differ for the different agonists tested. The existence of ligand-specific conformational states suggests a close link of these states with receptor function.

4-Chloro-7-nitrobenzofurazan↗
PubMed1984

Ligand-specific state transitions of the membrane-bound acetylcholine receptor.

We have developed a simple, direct and time-resolved method to monitor ligand-induced changes in agonist affinity of the membrane-bound acetylcholine receptor. The assay is based on the quenching of fluorescence of NBD-5-acylcholine observed upon binding of this cholinergic agonist to the receptor. Under conditions of partial saturation with the fluorescent agonist, agonists and local anesthetics but not antagonists can induce an increase in affinity of the receptor for NBD-5-acylcholine. The effect is not observed with receptor fully saturated with the fluorescent agonist. The half-life of the observed change in affinity is independent of the nature of the agonist or local anesthetic applied (t1/2 approximately 60 s at 22 degrees C). We conclude that the same state transition of the receptor can be induced by two groups of cholinergic ligands that are assumed to be non-competitive with each other and to have distinctly different modes of action. The time course of the transition is reminiscent of the slow process of desensitization observed in vivo.

4-Chloro-7-nitrobenzofurazan↗
PubMed1982

A procedure for detecting changes in the internal Ca2+ concentration in isolated nerve endings using the metallochromic dye arsenazo III.

A method for detecting changes in the internal concentration of Ca2+ in synaptosomes from mouse brain is described. Synaptosomes were fused with phosphatidylcholine-phosphatidylserine unilamellar liposomes previously loaded with the metalochromic, Ca2+ -sensitive dye arsenazo III, in order to introduce the dye into the synaptosomes. The fusion was promoted by La3+. Changes in the differential absorption between 660 and 690 nm, which indicate changes in Ca2+ concentration, were followed in a double beam spectrophotometer. It was found that both in the dye-loaded liposomes and in the fused synaptosomes, the addition of the Ca2+ ionophore A23187 to the medium containing Ca2+ produced a notable change in the differential absorbance 660-690 nm. When the depolarizing alkaloid veratridine was added, there was no response in the liposomes, whereas a change in the differential absorbance 660-690 nm was detected in the fused synaptosomes containing arsenazo but only when Na+ was present in the medium. These fused synaptosomes were able to release labeled gamma-aminobutyric acid as a response to veratridine, in a Na+ -dependent manner, similarly to control non-fused synaptosomes. These results demonstrate the feasibility of fusion to introduce Ca2+ -sensitive dyes into isolated nerve endings from the mammalian brain and therefore to detect changes in their internal Ca2+ concentration.

Animals↗