Biomedical subjects

M Covarrubias

Publications and source records attributed to M Covarrubias.

At least 19 recordsLinked to original sources

PubMed2001

Kv4 channels exhibit modulation of closed-state inactivation in inside-out patches.

The mechanisms of inactivation gating of the neuronal somatodendritic A-type K(+) current and the cardiac I(to) were investigated in Xenopus oocyte macropatches expressing Kv4.1 and Kv4.3 channels. Upon membrane patch excision (inside-out), Kv4.1 channels undergo time-dependent acceleration of macroscopic inactivation accompanied by a parallel partial current rundown. These changes are readily reversible by patch cramming, suggesting the influence of modulatory cytoplasmic factors. The consequences of these perturbations were investigated in detail to gain insights into the biophysical basis and mechanisms of inactivation gating. Accelerated inactivation at positive voltages (0 to +110 mV) is mainly the result of reducing the time constant of slow inactivation and the relative weight of the slow component of inactivation. Concomitantly, the time constants of closed-state inactivation at negative membrane potentials (-90 to -50 mV) are substantially decreased in inside-out patches. Deactivation is moderately accelerated, and recovery from inactivation and the peak G--V curve exhibit little or no change. In agreement with more favorable closed-state inactivation in inside-out patches, the steady-state inactivation curve exhibits a hyperpolarizing shift of approximately 10 mV. Closed-state inactivation was similarly enhanced in Kv4.3. An allosteric model that assumes significant closed-state inactivation at all relevant voltages can explain Kv4 inactivation gating and the modulatory changes.

Allosteric Regulation↗

PubMed2000

The genotoxicity of 3-nitrobenzanthrone and the nitropyrene lactones in human lymphoblasts.

Polycyclic aromatic hydrocarbons (PAH) and nitrated polycyclic aromatic compounds (nitro-PAC) have been found to be mutagenic in bacterial and human cells as well as carcinogenic in rodents. In this investigation, the genotoxic effects of 3-nitrobenzanthrone (3NB) and a mixture of nitropyrene lactones (NPLs) were determined using forward mutation assays performed in two human B-lymphoblastoid cell lines, MCL-5 and h1A1v2, which are responsive to the nitro-PAC class of compounds. Mutagenicity of the compounds was determined at the heterozygous tk locus and the hemizygous hprt locus, thus, identifying both large-scale loss of heterozygosity (LOH) events as well as intragenic mutagenic events. Genotoxicity was also determined using the CREST modified micronucleus assay, which detects chromosomal loss and breakage events. Results indicate 3NB is an effective human cell mutagen, significantly inducing mutations at the tk and hprt loci in both cell lines, and inducing micronuclei in the h1A1v2 cell line. The NPL isomers are also mutagenic, inducing mutations at the two loci as well as micronuclei in both cell lines. Because of their mutagenic potencies and their presence in ambient air, further assessments should be made of human exposures to these nitro-PAC and the potential health risks involved.

B-Lymphocytes↗

PubMed2000

General anesthetic action at an internal protein site involving the S4-S5 cytoplasmic loop of a neuronal K(+) channel.

The structural bases of general anesthetic action on a neuronal K(+) channel were investigated using the series of homologous 1-alkanols, electrophysiology, and mutational analysis. Domain swapping between dShaw2 (alkanol-sensitive) and hKv3.4 (alkanol-resistant) and site-directed mutagenesis demonstrated that a 13-amino acid cytoplasmic loop (S4-S5) determines the selective inhibition of native dShaw2 channels by 1-alkanols. The S4-S5 loop may contribute to a receptor for both 1-alkanols and the inactivation particle, because the enhanced 1-alkanol sensitivity of hKv3.4 channels hosting S4-S5 mutations correlates directly with disrupted channel inactivation. Evidence of a discrete protein site was also obtained from the analysis of the relationship between potency and alkyl chain length, which begins to level off after 1-hexanol. Rapid application to the cytoplasmic side of inside-out membrane patches shows that the interaction between dShaw2 channels and 1-alkanols equilibrates in <200 ms. By contrast, the equilibration time is >1000-fold slower when the drug is applied externally to outside-out membrane patches. The data strongly favor a mechanism of inhibition involving a discrete internal site for 1-alkanols in dShaw2 K(+) channels. A new working hypothesis proposes that 1-alkanols lock dShaw2 channels in their closed conformation by a direct interaction at a crevice formed by the S4-S5 loop.

1-Butanol↗

PubMed2000

Potent inhibition of the aortic smooth muscle maxi-K channel by clinical doses of ethanol.

We investigated the effects of clinically relevant ethanol concentrations (5-20 mM) on the single-channel kinetics of bovine aortic smooth muscle maxi-K channels reconstituted in lipid bilayers (1:1 palmitoyl-oleoyl-phosphatidylethanolamine: palmitoyl-oleoyl-phosphatidylcholine). Ethanol at 10 and 20 mM decreased the channel open probability (P(o)) by 75 +/- 20.3% mainly by increasing the mean closed time (+82 to +960%, n = 7). In some instances, ethanol also decreased the mean open time (-40.8 +/- 22. 5%). The P(o)-voltage relation in the presence of 20 mM ethanol exhibited a rightward shift in the midpoint of voltage activation (DeltaV(1/2) congruent with 17 mV), a slightly steeper relationship (change in slope factor, Deltak, congruent with -2.5 mV), and a decreased maximum P(o) (from approximately 0.82 to approximately 0. 47). Interestingly, channels inhibited by ethanol at low Ca(2+) concentrations (2.5 microM) were very resistant to ethanol in the presence of increased Ca(2+) (>/= 20 microM). Alcohol consumption in clinically relevant amounts may alter the contribution of maxi-K channels to the regulation of arterial tone.

Animals↗

PubMed1999

Injection of Xenopus oocytes with mRNA from cultured neurons induces new currents and susceptibility to the damaging action of ruthenium red.

The hexacationic dye ruthenium red produce neuronal death in primary cultures. We injected messenger RNA (mRNA) from cultured neurons into Xenopus laevis oocytes to test whether this treatment can make oocytes sensitive to the damaging action of ruthenium red. Two-microelectrode voltage clamp and resting membrane potential were used to evaluate mRNA expression and to assess the effect of the dye on oocyte survival, when added to the medium or when injected into the cells, at 20, 50, or 100 microM concentrations. Injection of mRNA from cultured cortical or cerebellar granule neurons produced both new outward currents and membrane hyperpolarization. Exposure of mRNA-injected oocytes to extracellular ruthenium red for 24 h induced a remarkable depolarization, but no significant damage was observed. Injection of the dye into buffer-injected oocytes did not cause any change in membrane potential or cell survival, whereas in mRNA-injected oocytes an important depolarization was observed at 24 h after ruthenium red introduction, and 29% of the cells showed serious damage. The results suggest that oocytes become sensitive to intracellular ruthenium red toxicity because they express neuronal-specific proteins involved in cell death.

Animals↗

PubMed1999

Control of K+ channel gating by protein phosphorylation: structural switches of the inactivation gate.

Fast N-type inactivation of voltage-dependent potassium (Kv) channels controls membrane excitability and signal propagation in central neurons and occurs by a 'ball-and-chain'-type mechanism. In this mechanism an N-terminal protein domain (inactivation gate) occludes the pore from the cytoplasmic side. In Kv3.4 channels, inactivation is not fixed but is dynamically regulated by protein phosphorylation. Phosphorylation of several identified serine residues on the inactivation gate leads to reduction or removal of fast inactivation. Here, we investigate the structure-function basis of this phospho-regulation with nuclear magnetic resonance (NMR) spectroscopy and patch-clamp recordings using synthetic inactivation domains (ID). The dephosphorylated ID exhibited compact structure and displayed high-affinity binding to its receptor. Phosphorylation of serine residues in the N- or C-terminal half of the ID resulted in a loss of overall structural stability. However, depending on the residue(s) phosphorylated, distinct structural elements remained stable. These structural changes correlate with the distinct changes in binding and unbinding kinetics underlying the reduced inactivation potency of phosphorylated IDs.

Animals↗

PubMed1999

Inactivation gating of Kv4 potassium channels: molecular interactions involving the inner vestibule of the pore.

Kv4 channels represent the main class of brain A-type K+ channels that operate in the subthreshold range of membrane potentials (Serodio, P., E. Vega-Saenz de Miera, and B. Rudy. 1996. J. Neurophysiol. 75:2174- 2179), and their function depends critically on inactivation gating. A previous study suggested that the cytoplasmic NH2- and COOH-terminal domains of Kv4.1 channels act in concert to determine the fast phase of the complex time course of macroscopic inactivation (Jerng, H.H., and M. Covarrubias. 1997. Biophys. J. 72:163-174). To investigate the structural basis of slow inactivation gating of these channels, we examined internal residues that may affect the mutually exclusive relationship between inactivation and closed-state blockade by 4-aminopyridine (4-AP) (Campbell, D.L., Y. Qu, R.L. Rasmussen, and H.C. Strauss. 1993. J. Gen. Physiol. 101:603-626; Shieh, C.-C., and G.E. Kirsch. 1994. Biophys. J. 67:2316-2325). A double mutation V[404,406]I in the distal section of the S6 region of the protein drastically slowed channel inactivation and deactivation, and significantly reduced the blockade by 4-AP. In addition, recovery from inactivation was slightly faster, but the pore properties were not significantly affected. Consistent with a more stable open state and disrupted closed state inactivation, V[404,406]I also caused hyperpolarizing and depolarizing shifts of the peak conductance-voltage curve ( approximately 5 mV) and the prepulse inactivation curve (>10 mV), respectively. By contrast, the analogous mutations (V[556,558]I) in a K+ channel that undergoes N- and C-type inactivation (Kv1.4) did not affect macroscopic inactivation but dramatically slowed deactivation and recovery from inactivation, and eliminated open-channel blockade by 4-AP. Mutation of a Kv4-specific residue in the S4-S5 loop (C322S) of Kv4.1 also altered gating and 4-AP sensitivity in a manner that closely resembles the effects of V[404, 406]I. However, this mutant did not exhibit disrupted closed state inactivation. A kinetic model that assumes coupling between channel closing and inactivation at depolarized membrane potentials accounts for the results. We propose that components of the pore's internal vestibule control both closing and inactivation in Kv4 K+ channels.

4-Aminopyridine↗

PubMed1998

Interactions between multiple phosphorylation sites in the inactivation particle of a K+ channel. Insights into the molecular mechanism of protein kinase C action.

Protein kinase C inhibits inactivation gating of Kv3.4 K+ channels, and at least two NH2-terminal serines (S15 and S21) appeared involved in this interaction (. Neuron. 13:1403-1412). Here we have investigated the molecular mechanism of this regulatory process. Site-directed mutagenesis (serine --> alanine) revealed two additional sites at S8 and S9. The mutation S9A inhibited the action of PKC by approximately 85%, whereas S8A, S15A, and S21A exhibited smaller reductions (41, 35, and 50%, respectively). In spite of the relatively large effects of individual S --> A mutations, simultaneous mutation of the four sites was necessary to completely abolish inhibition of inactivation by PKC. Accordingly, a peptide corresponding to the inactivation domain of Kv3.4 was phosphorylated by specific PKC isoforms, but the mutant peptide (S[8,9,15,21]A) was not. Substitutions of negatively charged aspartate (D) for serine at positions 8, 9, 15, and 21 closely mimicked the effect of phosphorylation on channel inactivation. S --> D mutations slowed the rate of inactivation and accelerated the rate of recovery from inactivation. Thus, the negative charge of the phosphoserines is an important incentive to inhibit inactivation. Consistent with this interpretation, the effects of S8D and S8E (E = Glu) were very similar, yet S8N (N = Asn) had little effect on the onset of inactivation but accelerated the recovery from inactivation. Interestingly, the effects of single S --> D mutations were unequal and the effects of combined mutations were greater than expected assuming a simple additive effect of the free energies that the single mutations contribute to impair inactivation. These observations demonstrate that the inactivation particle of Kv3.4 does not behave as a point charge and suggest that the NH2-terminal phosphoserines interact in a cooperative manner to disrupt inactivation. Inspection of the tertiary structure of the inactivation domain of Kv3.4 revealed the topography of the phosphorylation sites and possible interactions that can explain the action of PKC on inactivation gating.

Amino Acid Sequence↗

PubMed1998

Receptor-coupled regulation of K+ channel N-type inactivation.

Phosphorylation of the inactivation gate of a K+ channel (Kv3.4) by protein kinase C (PKC) slows rapid N-type inactivation. To demonstrate that such an effect could occur under more physiological conditions, Kv3.4 and a metabotropic serotonin (5-HT) receptor were coexpressed in Xenopus oocytes. Application of 5-HT 10 microM to these oocytes produced two main effects: 1) Enhanced activity of endogenous Ca(++)-dependent Cl- channels; and 2) Kv3.4 currents exhibited significantly slower inactivation than the control currents (time constants at +50 mV: 7.1 +/- 0.6 ms and 14.7 +/- 3 ms, before and after 5-HT, respectively). These results are consistent with the presence of receptor-coupled activation of phospholipase C. 5-HT had little or no effect on Kv3.4 current kinetics when four N-terminal serines were mutated to alanine. Peak currents exhibited, however, a slow run-down. This study demonstrates that physiological activation of PKC may regulate K+ channel inactivation by a direct action.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed1997

K+ channel inactivation mediated by the concerted action of the cytoplasmic N- and C-terminal domains.

We have examined the molecular mechanism of rapid inactivation gating in a mouse Shal K+ channel (mKv4.1). The results showed that inactivation of these channels follows a complex time course that is well approximated by the sum of three exponential terms. Truncation of an amphipathic region at the N-terminus (residues 2-71) abolished the rapid phase of inactivation (r = 16 ms) and altered voltage-dependent gating. Surprisingly, these effects could be mimicked by deletions affecting the hydrophilic C-terminus. The sum of two exponential terms was sufficient to describe the inactivation of deletion mutants. In fact, the time constants corresponded closely to those of the intermediate and slow phases of inactivation observed with wild-type channels. Further analysis revealed that several basic amino acids at the N-terminus do not influence inactivation, but a positively charged domain at the C-terminus (amino acids 420-550) is necessary to support rapid inactivation. Thus, the amphipathic N-terminus and the hydrophilic C-terminus of mKv4.1 are essential determinants of inactivation gating and may interact with each other to maintain the N-terminal inactivation gate near the inner mouth of the channel. Furthermore, this inactivation gate may not behave like a simple open-channel blocker because channel blockade by internal tetraethylammonium was not associated with slower current decay and an elevated external K+ concentration retarded recovery from inactivation.

Animals↗

PubMed1995

Permeation properties and differential expression across the auditory receptor epithelium of an inward rectifier K+ channel cloned from the chick inner ear.

The auditory receptor epithelium is an excellent model system for studying the differential expression of ion channel genes. An inward rectifier potassium current is among those which have been measured in only subsets of chick cochlear hair cells. We have cloned and characterized an inward rectifier potassium channel (cIRK1) from the chick cochlear sensory epithelium. cIRK1 functional properties are similar to those of the native channel, and the transcript encoding cIRK1 is limited to the low frequency half of the epithelium. This localization is in agreement with the distribution of the native hair cell current, suggesting that the differential current expression is transcriptionally regulated. The primary structure of cIRK1 is highly homologous to the mouse inward rectifier IRK1. However, we found that cIRK1 exhibited reduced single-channel conductance (17 picosiemens) and lower sensitivity to Ba2+ block (K1/2 = 12 microM). We identified Gln-125 near the putative pore region as being responsible for these differences. Site-directed mutagenesis was used to change Gln-125 to Glu (the residue in IRK1), resulting in a channel with a single-channel conductance of 28 picosiemens and a Ba2+ block of K1/2 = 2 microM. We propose that Gln-125 may form part of the external vestibule of the pore.

Amino Acid Sequence↗

PubMed1995

Alcohols inhibit a cloned potassium channel at a discrete saturable site. Insights into the molecular basis of general anesthesia.

The molecular basis of general anesthetic action on membrane proteins that control ion transport is not yet understood. In a previous report (Covarrubias, M., and Rubin, E. (1993) Proc. Natl. Acad. Sci. 90, 6957-6960), we found that low concentrations of ethanol (17-170mM) selectively inhibited a noninactivating cloned K+ channel encoded by Drosophila Shaw2. Here, we have conducted equilibrium dos-inhibition experiments, single channel recording, and mutagenesis in vitro to study the mechanism underlying the inhibition of Shaw2K+ channels by a homologous series of n-alkanols (ethanol to 1-hexanol). The results showed that: (i) these alcohols inhibited Shaw2 whole-cell currents, the equilibrium dose-inhibition relations were hyperbolic, and competition experiments revealed the presence of a discrete site of action, possibly a hydrophobic pocket; (ii) this pocket may be part of the protein because n-alkanol sensitivity can be transferred to novel hybrid K+ channels composed of Shaw2 subunits and homologous ethanol-insensitive subunits: (iii) moreover, a hydrophobic point mutation within a cytoplasmic loop of an ethanol-insensitive K+ channel (human Kv3.4) was sufficient to allow significant inhibition by n-alkanols, with a dose-inhibition relation that closely resembled that of wildtype Shaw2 channels; and (iv) 1-butanol selectively inhibited long duration single channel openings in a manner consistent with a direct effect on channel gating. These results strongly suggest that a discrete site within the ion channel protein is the primary locus of alcohol and general anesthetic action.

Alcohols↗

PubMed1994

Elimination of rapid potassium channel inactivation by phosphorylation of the inactivation gate.

The effect of protein kinase C (PKC) on rapid N-type inactivation of K+ channels has not been reported previously. We found that PKC specifically eliminates rapid inactivation of a cloned human A-type K+ channel (hKv3.4), converting this channel from a rapidly inactivating A type to a noninactivating delayed rectifier type. Biochemical analysis showed that the N-terminal domain of hKv3.4 is phosphorylated in vitro by PKC, and mutagenesis experiments revealed that two serines within the inactivation gate at the N-terminus are sites of direct PKC action. Moreover, mutating one of these serines to aspartic acid mimics the action of PKC. Serine phosphorylation may thus prevent rapid inactivation by shielding basic residues known to be critical to the function of the inactivation gate. The regulatory mechanism reported here may have substantial effects on signal coding in the nervous system.

Amino Acid Sequence↗

PubMed1993

Ethanol selectively blocks a noninactivating K+ current expressed in Xenopus oocytes.

There is presently a debate regarding the relative merits of lipid-based and protein-based theories of anesthesia and the action of ethanol in the central nervous system. Voltage-sensitive K+ channels play a key role as regulators of neuronal electrical activity and are potential targets of ethanol and other anesthetic agents. We investigated the action of low concentrations of ethanol on four structurally homologous cloned K+ channels expressed in Xenopus oocytes. We report that only the Drosophila Shaw2 channel, which does not inactivate upon prolonged depolarization, is rapidly and reversibly blocked by ethanol in a concentration-dependent manner (17-170 mM). The concentration dependence of the blockade can be explained by assuming a bimolecular interaction between ethanol and the channel. We also found that Shaw2 K+ channels were selectively blocked by halothane (1 mM). Our results support the "protein hypothesis" of ethanol and anesthetic action. These findings open ways to elucidate directly the molecular mechanism of interaction between general anesthetics and a voltage-sensitive K+ channel.

Anesthesia↗

PubMed1993

Low molecular weight poly(A)+ mRNA species encode factors that modulate gating of a non-Shaker A-type K+ channel.

Voltage-dependent K+ channels control repolarization of action potentials and help establish firing patterns in nerve cells. To determine the nature and role of molecular components that modulate K+ channel function in vivo, we coinjected Xenopus oocytes with cRNA encoding a cloned subthreshold A-type K+ channel (mShal1, also referred to as mKv4.1) and a low molecular weight (LMW) fraction (2-4 kb) of poly(A)+ mRNA (both from rodent brain). Coinjected oocytes exhibited a significant (fourfold) increase in the surface expression of mShal1 K+ channels with no change in the open-channel conductance. Coexpression also modified the gating kinetics of mShal1 current in several respects. Macroscopic inactivation of whole oocyte currents was fitted with the sum of two exponential components. Both fast and slow time constants of inactivation were accelerated at all membrane potentials in coinjected oocytes (tau f = 47.2 ms vs 56.5 ms at 0 mV and tau s = 157 ms vs 225 ms at 0 mV), and the corresponding ratios of amplitude terms were shifted toward domination by the fast component (Af/As = 2.71 vs 1.17 at 0 mV). Macroscopic activation was characterized in terms of the time-to-peak current, and it was found to be more rapid at all membrane potentials in coinjected oocytes (9.9 ms vs 13.5 ms at 0 mV). Coexpression also leads to more rapid recovery from inactivation (approximately 2.4-fold faster at -100 mV). The coexpressed K+ currents in oocytes resemble currents expressed in mouse fibroblasts (NIH3T3) transfected only with mShal1 cDNA. These results indicate that mammalian regulatory subunits or enzymes encoded by LMW mRNA species, which are apparently missing or expressed at low levels in Xenopus oocytes, may modulate gating in some native subthreshold A-type K+ channels.

3T3 Cells↗

PubMed1992

Antigen binding thermodynamics and antiproliferative effects of chimeric and humanized anti-p185HER2 antibody Fab fragments.

The murine monoclonal antibody 4D5 (anti-p185HER2) inhibits the proliferation of human tumor cells overexpressing p185HER2 in vitro and has been "humanized" [Carter, P., Presta, L., Gorman, C. M., Ridgway, J. B. B., Henner, D., Wong, W.-L. T., Rowland, A. M., Kotts, C., Carver, M. E., & Shepard, H. M. (1992) Proc. Natl. Acad. Sci. U.S.A. (in press)] for use in human cancer therapy. We have determined the antigen binding thermodynamics and the antiproliferative activities of chimeric 4D5 Fab (ch4D5 Fab) fragment and a series of eight humanized Fab (hu4D5 Fab) fragments differing by amino acid substitutions in the framework regions of the variable domains. Fab fragments were expressed by secretion from Escherichia coli and purified from fermentation supernatants by using affinity chromatography on immobilized streptococcal protein G or staphylococcal protein A for ch4D5 and hu4D5, respectively. Circular dichroism spectroscopy indicates correct folding of the E. coli produced Fab, and scanning calorimetry shows a greater stability for hu4D5 (Tm = 82 degrees C) as compared with ch4D5 Fab (Tm = 72 degrees C). KD values for binding to the extracellular domain (ECD) of p185HER2 were determined by using a radioimmunoassay; the delta H and delta Cp for binding were determined by using isothermal titration calorimetry. ch4D5 Fab and one of the humanized variants (hu4D5-8 Fab) bind p185HER2-ECD with comparable affinity (delta G degrees = -13.6 kcal mol-1).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1992

An essential 'set' of K+ channels conserved in flies, mice and humans.

The molecular genetic approach to studying K+ channels has revealed that at least four subfamilies of voltage-gated K+ channels originally discovered in Drosophila are conserved in mice and humans. This conservation of the K+ channel subfamilies Shaker, Shal, Shab, and Shaw suggests that not only the broad outlines of membrane electrical properties but also many molecular details as well evolved in the parent species ancestral to both invertebrate and vertebrate life. Shaker, Shal, Shab, and Shaw K+ channels have similar structures, but appear to be independent channel systems: when co-expressed in Xenopus oocytes, all four function independently. These four K+ channel subfamilies may be part of an essential 'set' of excitable channels required by most nervous systems. The task now remaining is to understand the functions of each member of the set.

Animals↗

PubMed1992

High level Escherichia coli expression and production of a bivalent humanized antibody fragment.

Many clinical uses of antibodies will require large quantities of fragments which are bivalent and humanized. We therefore attempted to generate humanized F(ab')2 fragments by secretion from E. coli. Titers of 1-2 g l-1 of soluble and functional Fab' fragments have been routinely achieved as judged by antigen-binding ELISA. Surprisingly, this high expression level of Fab' in the periplasmic space of E. coli does not drive dimerization. However, we have developed a protocol to directly and efficiently recover Fab' with the single hinge cysteine in the free thiol state, allowing F(ab')2 formation by chemically-directed coupling in vitro. The E. coli derived humanized F(ab')2 fragment is indistinguishable from F(ab')2 derived from limited proteolysis of intact antibody in its binding affinity for the antigen, p185HER2, and anti-proliferative activity against the human breast tumor cell line, SK-BR-3, which over-expresses p185HER2. This system makes E. coli expression of bivalent antibody fragments for human therapy (or other uses) practical.

Amino Acid Sequence↗