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G F Tomaselli

Publications and source records attributed to G F Tomaselli.

14 recordsLinked to original sources
PubMed1995

A mutation in the pore of the sodium channel alters gating.

Ion permeation and channel gating are classically considered independent processes, but site-specific mutagenesis studies in K channels suggest that residues in or near the ion-selective pore of the channel can influence activation and inactivation. We describe a mutation in the pore of the skeletal muscle Na channel that alters gating. This mutation, I-W53C (residue 402 in the mu 1 sequence), decreases the sensitivity to block by tetrodotoxin and increases the sensitivity to block by externally applied Cd2+ relative to the wild-type channel, placing this residue within the pore near the external mouth. Based on contemporary models of the structure of the channel, this residue is remote from the regions of the channel known to be involved in gating, yet this mutation abbreviates the time to peak and accelerates the decay of the macroscopic Na current. At the single-channel level we observe a shortening of the latency to first opening and a reduction in the mean open time compared with the wild-type channel. The acceleration of macroscopic current kinetics in the mutant channels can be simulated by changing only the activation and deactivation rate constants while constraining the microscopic inactivation rate constants to the values used to fit the wild-type currents. We conclude that the tryptophan at position 53 in the domain IP-loop may act as a linchpin in the pore that limits the opening transition rate. This effect could reflect an interaction of I-W53 with the activation voltage sensors or a more global gating-induced change in pore structure.

Amino Acid Sequence↗
PubMed1995

Inward rectifier K+ channel from human heart and brain: cloning and stable expression in a human cell line.

We have cloned the human homologue of the inward rectifier K+ channel from both heart and brain tissue (HHBIRK1). The human clones were identical to each other in their coding regions and were highly homologous to the mouse macrophage (IRK1) channel. The inward rectifier currents from human and mouse clones were characterized using a novel strategy for stable ion channel expression in a human cell line. The permeability of the expressed inwardly rectifying channels was greater for K+ than for Rb+, whereas no current was observed when K+ was replaced by Na+. A prominent time- and voltage-dependent block was observed in the presence of Ba2+, whereas a small decay in the steady-state current was observed with millimolar concentrations of Na+. Single-channel conductances of 49.1 +/- 3.3 pS (n = 6) and 40.2 +/- 2.5 pS (n = 3) (P = 0.005) were obtained for the HHBIRK1 and IRK1 clones, respectively. These results indicate that sequence dissimilarities between human and mouse inward rectifier K+ channels may have significant functional consequences.

Adult↗
PubMed1994

Simultaneous expression of cardiac and skeletal muscle isoforms of the L-type Ca2+ channel in a rat heart muscle cell line.

1. We have investigated the identity of the L-type Ca2+ channels present in the H9c2 myoblast line derived from embryonic rat ventricle. To this end, we characterized macroscopic and unitary Ba2+ currents through Ca2+ channels, and looked for specific genetic messages encoding different L-type Ca2+ channel isoforms. 2. The macroscopic Ba2+ current (recorded in 10 mM BaCl2) revealed two components with different time courses of activation. The fast component (IBa,fast) activates with a time constant of 23 +/- 12 ms (at +10 mV), while the slow component activates with a time constant of 125 +/- 12 ms (at +10 mV). 3. Single-channel recordings revealed the presence of two independent channels with conductance values of 11 and 25 pS (in 70 mM Ba2+). These values are identical to those reported previously for skeletal muscle and cardiac Ca2+ channels, respectively. 4. The mean ensemble current from the 11 pS channel reproduced the time course of the slow component observed at the macroscopic level, while the 25 pS ensemble time course paralleled that of the fast component. 5. Reverse transcriptase polymerase chain reaction (PCR) with alpha 1-isoform-specific primers revealed the presence of two distinct transcripts in H9c2 cells. The sequences of the PCR products showed a high degree of homology with the corresponding segments of the rabbit cardiac and skeletal muscle L-type Ca2+ channel isoforms. Adult rat skeletal and cardiac muscle expressed only one type of transcript. 6. H9c2 cells appear to be unique in that they simultaneously express both skeletal muscle and cardiac isoforms of the L-type Ca2+ channel alpha 1-subunit. Thus, the H9c2 cell line may prove to be useful when studying the regulation of subtype-specific Ca2+ channel gene expression.

Animals↗
PubMed1994

Dynamic actin structures stabilized by profilin.

We describe the production and analysis of clonal cell lines in which we have overexpressed human profilin, a small ubiquitous actin monomer binding protein, to assess the role of profilin on actin function in vivo. The concentration of filamentous actin is increased in cells with higher profilin levels, and actin filament half-life measured in these cells is directly proportional to the steady-state profilin concentration. The distribution of actin filaments is altered by profilin overexpression. While parallel actin bundles crossing the cells are virtually absent in cells overexpressing profilin, the submembranous actin network of these cells is denser than in control cells. These results suggest that in vivo profilin regulates the stability, and thereby distribution, of specific dynamic actin structures.

Actins↗
PubMed1994

Multiway sequential hypothesis testing for tachyarrhythmia discrimination.

A multiway sequential hypothesis testing (M-SHT) algorithm is proposed for simultaneous discrimination of cardiac tachyarrhythmias--supraventricular tachycardia (SVT) and ventricular tachycardia (VT)--from normal sinus rhythm (NSR). The M-SHT algorithm calculates a likelihood function from atrio-ventricular delay measurements, and compares this function with thresholds derived from specified error probabilities for the arrhythmias to be discriminated. Performance of this algorithm was evaluated on dual channel endocardial electrograms recorded in the cardiac electrophysiology laboratory. Two databases were developed, one for development of the algorithm and another for evaluation. The M-SHT algorithm accurately classified 26 out of 28 NSR (2 misclassified as SVT), 31 out of 31 cases of SVT, and 41 out of 43 VT (2 misclassified as NSR). The average length of time taken for classification of the three rhythms was: 3.6 s for NSR, 5.0 s for SVT, and 1.6 s for VT. Unique features of this algorithm are that acceptable error rates for each arrhythmia are independently specified and accuracy can be traded off for a faster detection time, and vice versa.

Adult↗
PubMed1994

Sudden cardiac death in heart failure. The role of abnormal repolarization.

Congestive heart failure is a common, highly lethal cardiovascular disorder claiming over 200,000 lives a year in the United States alone. Some 50% of the deaths in heart failure patients are sudden, and most of these are probably the result of ventricular tachyarrhythmias. Methods designed to identify patients at risk have been remarkably unrewarding, as have attempts to intervene and prevent sudden death in these patients. The failure to impact favorably on the incidence of sudden death in heart failure patients stems largely from a lack of understanding of the underlying mechanisms of arrhythmogenesis. This article explores the role of abnormalities of ventricular repolarization in heart failure patients. We will examine evidence for the hypothesis that alteration of repolarizing K+ channel expression in failing myocardium predisposes to abnormalities in repolarization that are arrhythmogenic. The possible utility of novel electrophysiological and ECG measures of altered ventricular repolarization will be explored. Understanding the mechanism of sudden death in heart failure may lead to effective therapy and more accurate identification of patients at greatest risk.

Action Potentials↗
PubMed1992

Karyoplasmic interaction selection strategy: a general strategy to detect protein-protein interactions in mammalian cells.

We describe a strategy and reagents for study of protein-protein interactions in mammalian cells, termed the karyoplasmic interaction selection strategy (KISS). With this strategy, specific protein-protein interactions are identified by reconstitution of the functional activity of the yeast transcriptional activator GAL4 and the resultant transcription of a GAL4-regulated reporter gene. Reconstitution of GAL4 function results from specific interaction between two chimeric proteins: one contains the DNA-binding domain of GAL4; the other contains a transcriptional activation domain. Transcription of the reporter gene occurs if the two chimeric proteins can form a complex that reconstitutes the DNA-binding and transcriptional activation functions of GAL4. Using the KISS system, we demonstrate specific interactions for sequences from three different pairs of proteins that complex in the cytoplasm. In addition, we demonstrate that reporter genes encoding cell surface or drug-resistance markers can be specifically activated as a result of protein-protein interactions. With these selectable markers, the KISS system can be used to screen specialized cDNA libraries to identify novel protein interactions.

Amino Acid Sequence↗
PubMed1992

Molecular localization of an ion-binding site within the pore of mammalian sodium channels.

Sodium channels are the major proteins that underlie excitability in nerve, heart, and skeletal muscle. Chemical reaction rate theory was used to analyze the blockage of single wild-type and mutant sodium channels by cadmium ions. The affinity of cadmium for the native tetrodotoxin (TTX)-resistant cardiac channel was much higher than its affinity for the TTX-sensitive skeletal muscle isoform of the channel (microliters). Mutation of Tyr401 to Cys, the corresponding residue in the cardiac sequence, rendered microliters highly susceptible to cadmium blockage but resistant to TTX. The binding site was localized approximately 20% of the distance down the electrical field, thus defining the position of a critical residue within the sodium channel pore.

Amino Acid Sequence↗
PubMed1991

Mutations affecting agonist sensitivity of the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (AChR) is a pentameric transmembrane protein (alpha 2 beta gamma delta) that binds the neurotransmitter acetylcholine (ACh) and transduces this binding into the opening of a cation selective channel. The agonist, competitive antagonist, and snake toxin binding functions of the AChR are associated with the alpha subunit (Kao et al., 1984; Tzartos and Changeux, 1984; Wilson et al., 1985; Kao and Karlin, 1986; Pederson et al., 1986). We used site-directed mutagenesis and expression of AChR in Xenopus oocytes to identify amino acid residues critical for ligand binding and channel activation. Several mutations in the alpha subunit sequence were constructed based on information from sequence homology and from previous biochemical (Barkas et al., 1987; Dennis et al., 1988; Middleton and Cohen, 1990) and spectroscopic (Pearce and Hawrot, 1990; Pearce et al., 1990) studies. We have identified one mutation, Tyr190 to Phe (Y190F), that had a dramatic effect on ligand binding and channel activation. These mutant channels required more than 50-fold higher concentrations of ACh for channel activation than did wild type channels. This functional change is largely accounted for by a comparable shift in the agonist binding affinity, as assessed by the ability of ACh to compete with alpha-bungarotoxin binding. Other mutations at nearby conserved positions of the alpha subunit (H186F, P194S, Y198F) produce less dramatic changes in channel properties. Our results demonstrate that ligand binding and channel gating are separable properties of the receptor protein, and that Tyr190 appears to play a specific role in the receptor site for acetylcholine.

Amino Acid Sequence↗
PubMed1990

Human cardiac sodium channels expressed in Xenopus oocytes.

We report the expression of voltage-dependent Na+ channels in Xenopus oocytes injected with total RNA isolated from explanted human hearts. The expressed channels demonstrate characteristic voltage-dependent gating, inhibition by tetrodotoxin, and selectivity for Na+. Oocytes injected with sterile water or intentionally degraded RNA had no similar channel activity. The antiarrhythmic agent lidocaine (20 microM) inhibits current flow through the channel in a voltage-dependent fashion. Na+ channels expressed by injection of human cardiac RNA into Xenopus oocytes qualitatively resemble channels in the native tissue.

Animals↗
PubMed1989

Sodium channels from human brain RNA expressed in Xenopus oocytes. Basic electrophysiologic characteristics and their modification by diphenylhydantoin.

We describe the expression and characterization of sodium channels from human brain RNA in the Xenopus oocyte. The expressed channel, studied by whole-cell voltage clamp, reveals characteristic selectivity for sodium as the permeant ion, voltage-dependent gating, and block by nanomolar concentrations of tetrodotoxin. Such channels are not seen in control oocytes injected with solvent only. The anticonvulsant diphenylhydantoin (DPH) inhibits the expressed channel in a voltage- and use-dependent manner, much like the effect seen in primary mammalian neuronal preparations. The inhibition of the expressed human sodium channel by DPH can be described by models previously developed to explain block of Na channels by local anesthetics. The preferential block of Na channels during depolarization helps explain the selectivity of DPH for neurons involved in seizure activity.

Animals↗
PubMed1988

Morphologic differences of the endocardial electrogram in beats of sinus and ventricular origin.

The lack of accurate arrhythmia detection and identification is one of the major obstacles to improvement in the efficacy of antitachycardia devices. We evaluated a method for detection of beats of ventricular origin compared to sinus rhythm based on the morphology of the endocardial electrogram. In order to compare mechanically induced ventricular beats to normal sinus beats, endocardial electrograms from a standard pacing electrode were recorded from eight open-chested dogs. Time and frequency domain features analyzed included peak-to-peak amplitude (AMP), maximal slew rate (dV/dT), and frequency content (-3 dB downpoint). Quantitative morphologic comparison of the waveforms was performed using standard correlation and by the absolute area of difference between the waveform and a sinus beat template. The AMP and dV/dT for a group of ventricular beats did not differ significantly from beats of sinus origin. In the unipolar configuration -3 dB for ventricular beats was significantly different from sinus beats (p = .01), but overlap occurred in three of eight cases. Conversely, using either method of assessment of morphological differences, all ventricular beats could be identified without overlapping the values for normal beats. We concluded that morphologic analysis of the endocardial electrogram by such methods may be a highly accurate means of distinguishing between beats of sinus and ventricular origins. This technique may also be applicable to the problem of automatic rhythm identification by implanted devices.

Animals↗
PubMedDate not supplied

Ion channels: structure and function.

Ion channels are ubiquitous membrane proteins in mammalian cells. Their critical physiological roles include control of the electrical potential across the membrane, facilitation of neuromuscular and neuronal transmission, signal transduction, and regulation of secretion and contractility. The alliance of techniques in biochemistry, electrophysiology, pharmacology, and molecular biology has provided insights into the three-dimensional structure of channel proteins and has allowed specific aspects of functional correlates to be better understood. Promises for the future include molecular genetic approaches to the treatment of disorders such as long QT syndrome and more rational design of drugs targeted to ion channels, including antiarrhythmic agents.

Action Potentials↗