Biomedical subjects

G F Tomaselli

Publications and source records attributed to G F Tomaselli.

At least 19 recordsLinked to original sources

PubMed1997

Proton inhibition of sodium channels: mechanism of gating shifts and reduced conductance.

Extracellular acidosis affects both permeation and gating of the expressed rat skeletal muscle Na+ channel (micro1). Reduction of the extracellular pH produced a progressive decrease in the maximal whole-cell conductance and a depolarizing shift in the whole-cell current-voltage relationship. A smaller depolarizing shift in the steady-state inactivation curve was observed. The pK of the reduction of maximal conductance was 6.1 over the pH range studied. An upper limit estimate of the pK of the shift of the half-activation voltage was 6.1. The relative reduction in the maximal whole-cell conductance did not change with higher [Na+]o. The conductance of single fenvalerate-modified Na+ channels was reduced by extracellular protons. Although the single-channel conductance increased with higher [Na+]o, the maximal conductances at pH 7.6, 7.0 and 6.0 did not converge at [Na+]o up to 280 mm, inconsistent with a simple electrostatic effect. A model incorporating both Na+ and H+ binding in the pore and cation binding to a Gouy-Chapman surface charge provided a robust fit to the single-channel conductance data with an estimated surface charge density of 1e-/439A2. Neither surface charge nor proton block alone suffices to explain the effects of extracellular acidosis on Na+ channel permeation; both effects play major roles in mediating the response to extracellular pH.

Animals↗

PubMed1996

Local anesthetics as effectors of allosteric gating. Lidocaine effects on inactivation-deficient rat skeletal muscle Na channels.

Time- and voltage-dependent local anesthetic effects on sodium (Na) currents are generally interpreted using modulated receptor models that require formation of drug-associated nonconducting states with high affinity for the inactivated channel. The availability of inactivation-deficient Na channels has enabled us to test this traditional view of the drug-channel interaction. Rat skeletal muscle Na channels were mutated in the III-IV linker to disable fast inactivation (F1304Q: FQ). Lidocaine accelerated the decay of whole-cell FQ currents in Xenopus oocytes, reestablishing the wild-type phenotype; peak inward current at -20 mV was blocked with an IC50 of 513 microM, while plateau current was blocked with an IC50 of only 74 microM (P < 0.005 vs. peak). In single-channel experiments, mean open time was unaltered and unitary current was only reduced at higher drug concentrations, suggesting that open-channel block does not explain the effect of lidocaine on FQ plateau current. We considered a simple model in which lidocaine reduced the free energy for inactivation, causing altered coupling between activation and inactivation. This model readily simulated macroscopic Na current kinetics over a range of lidocaine concentrations. Traditional modulated receptor models which did not modify coupling between gating processes could not reproduce the effects of lidocaine with rate constants constrained by single-channel data. Our results support a reinterpretation of local anesthetic action whereby lidocaine functions as an allosteric effector to enhance Na channel inactivation.

Allosteric Regulation↗

PubMed1996

Coupling between fast and slow inactivation revealed by analysis of a point mutation (F1304Q) in mu 1 rat skeletal muscle sodium channels.

1. We sought to elucidate the mechanism of the defective inactivation that characterizes sodium channels containing mutations in the cytoplasmic loop between the third and fourth domains (the III-IV linker). Specifically, we measured whole-cell and single-channel currents through wild-type and F1304Q mutant mu 1 rat skeletal muscle Na+ channels expressed in Xenopus laevis oocytes. 2. In wild-type channels, inactivation is complete and the faster of two decay components predominates. In F1304Q, inactivation is incomplete; the slow decay component is larger in amplitude and slower than in wild-type. The fraction of non-inactivating current is substantial (37 +/- 2% of peak current at -20 mV) in F1304Q. 3. Cell-attached patch recordings confirmed the profound kinetic differences and indicated that permeation was not altered by the F1304Q mutation. The F1304Q phenotype must be conferred entirely by changes in gating properties and is not remedied by coexpression with the beta 1-subunit. 4. Recovery from inactivation of F1304Q channels is faster than for wild-type channels and three exponentials are required to describe recovery adequately following long (5 s) depolarizations. Thus, there are three inactivated states even in 'inactivation-deficient' F1304Q channels. 5. The steady-state voltage dependence of F1304Q inactivation is right-shifted by 26 +/- 2 mV. 6. A gating model incorporating three inactivated states, all directly accessible from multiple closed states or the open state, was constrained to fit wild-type and F1304Q inactivation (h infinitive) data and repriming data simultaneously. While it was necessary to alter the rate constants entering and exiting all three inactivated states, the model accounted for the F1304Q-induced rightward shift in steady-state inactivation without imposing voltage dependence on the inactivation rate constants. 7. We conclude that the F1304Q mutation in mu 1 sodium channels modifies several inactivation processes simultaneously. The fact that a single amino acid substitution profoundly alters both fast and slow inactivation indicates that these processes share physical determinants in Na+ channels.

Animals↗

PubMed1996

External pore residue mediates slow inactivation in mu 1 rat skeletal muscle sodium channels.

1. Upon depolarization, voltage-gated sodium channels assume non-conducting inactivated states which may be characterized as "fast' or "slow' depending on the length of the repolarization period needed for recovery. Skeletal muscle Na+ channel alpha-subunits expressed in Xenopus laevis oocytes display anomalous gating behaviour, with substantial slow inactivation after brief depolarizations. We exploited this kinetic behaviour to examine the structural basis for slow inactivation. 2. While fast inactivation in Na+ channels is mediated by cytoplasmic occlusion of the pore by III-IV linker residues, the structural features of slow inactivation are unknown. Since external pore-lining residues modulate C-type inactivation in potassium channels, we performed serial cysteine mutagenesis in the permeation loop (P-loop) of the rat skeletal muscle Na+ channel (mu 1) to determine whether similarly placed residues are involved in Na+ channel slow inactivation. 3. Wild-type and mutant alpha-subunits were heterologously expressed in Xenopus oocytes, and Na+ currents were recorded using a two-electrode voltage clamp. Slow inactivation after brief depolarizations was eliminated by the W402C mutation in domain I. Cysteine substitution of the homologous tryptophan residues in domains II, III and IV did not alter slow inactivation. 4. Analogous to the W402C mutation, coexpression of the wild-type alpha-subunit with rat brain Na+ channel beta 1-subunit attenuated slow inactivation. However, the W402C mutation imposed a delay on recovery from fast inactivation, while beta 1-subunit coexpression did not. We propose that the W402C mutation and the beta 1-subunit modulate gating through distinct mechanisms. 5. Removal of fast inactivation in wild-type alpha-subunits with the III-IV linker mutation I1303Q; F1304Q; M1305Q markedly slowed the development of slow inactivation. We propose that slow inactivation in Na+ channels involves conformational changes in the external pore. Mutations that affect fast and slow inactivation appear to interact despite their remote positions in the channel.

Amino Acid Sequence↗

PubMed1996

Adjacent pore-lining residues within sodium channels identified by paired cysteine mutagenesis.

The pores of voltage-gated ion channels are lined by protein loops that determine selectivity and conductance. The relative orientations of these "P" loops remain uncertain, as do the distances between them. Using site-directed mutagenesis, we introduced pairs of cysteines into the P loops of micro1 rat skeletal muscle sodium channels and sought functional evidence of proximity between the substituted residues. Only cysteinyl residues that are in close proximity can form disulfide bonds or metal-chelating sites. The mutant Y401C (domain I) spontaneously formed a disulfide bond when paired with E758C in the P loop of domain II; the same residue, when coupled with G1530C in domain IV, created a high-affinity binding site for Cd2+ ions. The results provide the first specific constraints for intramolecular dimensions of the sodium channel pore.

Amino Acid Sequence↗

PubMed1996

Control of ion flux and selectivity by negatively charged residues in the outer mouth of rat sodium channels.

1. The sodium channel has a ring of negatively charged amino acids on its external face. This common structural feature of cation-selective channels has been proposed to optimize conduction by electrostatic attraction of permeant cations into the channel mouth. We tested this idea by mutagenesis of mu1 rat skeletal sodium channels expressed in Xenopus oocytes. 2. Replacement of the external glutamate residue in domain II by cysteine reduces sodium current by decreasing single-channel conductance. While this effect can be reversed by the negatively charged sulfhydryl modifying reagent methanethiosulphonate ethylsulphonate (MTSES), the flux saturation behaviour cannot be rationalized simply by changes in the surface charge. 3. The analogous mutations in domains I, III and IV affect not only conductance but also selectivity. These changes in selectivity are only partially reversed by exposure to MTSES. 4. Our findings necessitate revision of prevailing concepts regarding the role of superficial negatively charged residues in the process of ion permeation. These residues do not act solely by electrostatic attraction of permeant ions, but instead may help to form ion-specific binding sites within the pore.

Amino Acids↗

PubMed1996

Structure of the sodium channel pore revealed by serial cysteine mutagenesis.

The pores of voltage-gated cation channels are formed by four intramembrane segments that impart selectivity and conductance. Remarkably little is known about the higher order structure of these critical pore-lining or P segments. Serial cysteine mutagenesis reveals a pattern of side-chain accessibility that contradicts currently favored structural models based on alpha-helices or beta-strands. Like the active sites of many enzymes of known structure, the sodium channel pore consists of irregular loop regions.

Amino Acid Sequence↗

PubMed1996

Single-channel analysis of inactivation-defective rat skeletal muscle sodium channels containing the F1304Q mutation.

The intracellular linker between domains III and IV of the voltage-gated Na channel mediates fast inactivation. Targeted alteration of one or more of a triplet of hydrophobic amino acids within this linker region results in a marked slowing in the decay of ionic current. The mechanism of this defective inactivation was explored in rat skeletal muscle sodium channels (mu 1) containing the F1304Q mutation in Xenopus laevis oocytes with and without coexpression of the rat brain beta 1 subunit. Cell-attached single-channel patch-clamp recordings revealed that the mu 1-F1304Q channel reopens multiple times with open times that are prolonged compared with those of the wild-type channel. Coexpression of the beta 1 subunit stabilized a dominant nonbursting gating mode and accelerated the activation kinetics of mu 1-F1304Q but did not modify mean open time or fast-inactivation kinetics. A Markov gating model incorporating separate fast- and slow-inactivation particles reproduced the results by assuming that the F1304Q mutation specifically influences transitions to and from fast-inactivated states. These effects are independent of interactions of the mutant channel with the beta 1 subunit and do not result from a change in modal gating behavior. These results indicate that F1304Q mutant channels can still enter the inactivated state but do so reversibly and with altered kinetics.

Animals↗

PubMed1996

Depth asymmetries of the pore-lining segments of the Na+ channel revealed by cysteine mutagenesis.

We used serial cysteine mutagenesis to study the structure of the outer vestibule and selectivity region of the voltage-gated Na channel. The voltage dependence of Cd(2+) block enabled us to determine the locations within the electrical field of cysteine-substituted mutants in the P segments of all four domains. The fractional electrical distances of the substituted cysteines were compared with the differential sensitivity to modification by sulfhydryl-specific modifying reagents. These experiments indicate that the P segment of domain II is external, while the domain IV P segment is displaced internally, compared with the first and third domain P segments. Sulfhydryls with a steep voltage dependence for Cd(2+) block produced changes in monovalent cation selectivity; these included substitutions at the presumed selectivity filter, as well as residues in the domain IV P segment not previously recognized as determinants of selectivity. A new structural model is presented in which each of the P segments contribute unique loops that penetrate the membrane to varying depths to form the channel pore.

Amino Acid Sequence↗

PubMed1996

Functional consequences of lidocaine binding to slow-inactivated sodium channels.

Na channels open upon depolarization but then enter inactivated states from which they cannot readily reopen. After brief depolarizations, native channels enter a fast-inactivated state from which recovery at hyperpolarized potentials is rapid (< 20 ms). Prolonged depolarization induces a slow-inactivated state that requires much longer periods for recovery (> 1 s). The slow-inactivated state therefore assumes particular importance in pathological conditions, such as ischemia, in which tissues are depolarized for prolonged periods. While use-dependent block of Na channels by local anesthetics has been explained on the basis of delayed recovery of fast-inactivated Na channels, the potential contribution of slow-inactivated channels has been ignored. The principal (alpha) subunits from skeletal muscle or brain Na channels display anomalous gating behavior when expressed in Xenopus oocytes, with a high percentage entering slow-inactivated states after brief depolarizations. This enhanced slow inactivation is eliminated by coexpressing the alpha subunit with the subsidiary beta 1 subunit. We compared the lidocaine sensitivity of alpha subunits expressed in the presence and absence of the beta 1 subunit to determine the relative contributions of fast-inactivated and slow-inactivated channel block. Coexpression of beta 1 inhibited the use-dependent accumulation of lidocaine block during repetitive (1-Hz) depolarizations from -100 to -20 mV. Therefore, the time required for recovery from inactivated channel block was measured at -100 mV. Fast-inactivated (alpha + beta 1) channels were mostly unblocked within 1 s of repolarization; however, slow-inactivated (alpha alone) channels remained blocked for much longer repriming intervals (> 5 s). The affinity of the slow-inactivated state for lidocaine was estimated to be 15-25 microM, versus 24 microM for the fast-inactivated state. We conclude that slow-inactivated Na channels are blocked by lidocaine with an affinity comparable to that of fast-inactivated channels. A prominent functional consequence is potentiation of use-dependent block through a delay in repriming of lidocaine-bound slow-inactivated channels.

Anesthetics, Local↗

PubMed1996

Ionic mechanism of action potential prolongation in ventricular myocytes from dogs with pacing-induced heart failure.

Membrane current abnormalities have been described in human heart failure. To determine whether similar current changes are observed in a large animal model of heart failure, we studied dogs with pacing-induced cardiomyopathy. Myocytes isolated from the midmyocardium of 13 dogs with heart failure induced by 3 to 4 weeks of rapid ventricular pacing and from 16 nonpaced control dogs did not differ in cell surface area or resting membrane potential. Nevertheless, action potential duration (APD) was significantly prolonged in myocytes isolated from failing ventricles (APD at 90% repolarization, 1097 +/- 73 milliseconds [failing hearts, n = 30] versus 842 +/- 56 milliseconds [control hearts, n = 25]; P < .05), and the prominent repolarizing notch in phase 1 was dramatically attenuated. Basal L-type Ca2+ current and whole-cell Na+ current did not differ in cells from failing and from control hearts, but significant differences in K+ currents were observed. The density of the inward rectifier K+ current (IKl) was reduced in cells from failing hearts at test potentials below -90 mV (at -150 mV, -19.1 +/- 2.2 pA/pF [failing hearts, n = 18] versus -32.2 +/- 5.1 pA/pF [control hearts, n = 15]; P < .05). The small outward current component of IKl was also reduced in cells from failing hearts (at -60 mV, 1.7 +/- 0.2 pA/pF [failing hearts] versus 2.5 +/- 0.2 pA/pF [control hearts]; P < .05). The peak of the Ca(2+)-independent transient outward current (Ito) was dramatically reduced in myocytes isolated from failing hearts compared with nonfailing control hearts (at +80 mV, 7.0 +/- 0.9 pA/pF [failing hearts, n = 20] versus 20.4 +/- 3.2 pA/pF [control hearts, n = 15]; P < .001), while the steady state component was unchanged. There were no significant differences in Ito kinetics or single-channel conductance. A reduction in the number of functional Ito channels was demonstrated by nonstationary fluctuation analysis (0.4 +/- 0.03 channels per square micrometer [failing hearts, n = 5] versus 1.2 +/- 0.1 channels per square micrometer [control hearts, n = 3]; P < .001). Pharmacological reduction of Ito by 4-aminopyridine in control myocytes decreased the notch amplitude and prolonged the APD. Current clamp-release experiments in which current was injected for 8 milliseconds to reproduce the notch sufficed to shorten the APD significantly in cells from failing hearts. These data support the hypothesis that downregulation of Ito in pacing-induced heart failure is at least partially responsible for the action potential prolongation. Because the repolarization abnormalities mimic those in cells isolated from failing human ventricular myocardium, canine pacing-induced cardiomyopathy may provide insights into the development of repolarization abnormalities and the mechanisms of sudden death in patients with heart failure.

Action Potentials↗

PubMed1996

Reversal of potassium channel deficiency in cells from failing hearts by adenoviral gene transfer: a prototype for gene therapy for disorders of cardiac excitability and contractility.

Heart failure is a common, often lethal disorder in which conventional pharmacologic strategies have achieved limited success. Failing hearts exhibit a delay of electrical repolarization which predisposes to fatal arrhythmias. To explore the feasibility of gene therapy for this condition, we isolated myocytes from normal and failing dog hearts and quantified electrophysiologic and contractile parameters in primary culture. Action potentials were prolonged in failing cells as a result of diminished potassium currents. Exposure to AdShK, an adenovirus that overexpresses potassium channels, reversed the action potential prolongation of failing cells. The precise phenotype varied as a function of the density of expressed channels; modest increases in potassium current sufficed to mimic the non-diseased state most faithfully, while more robust expression of the transgene excessively abbreviated excitation and contraction. Our results demonstrate that viral gene transfer can modify the electrical properties of adult mammalian heart cells in a manner appropriate to reverse a fundamental disorder of excitability. Realistic application of this form of therapy will need to include a sensitive mechanism for control of the level and distribution of transgene expression.

Action Potentials↗

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

Functional association of the beta 1 subunit with human cardiac (hH1) and rat skeletal muscle (mu 1) sodium channel alpha subunits expressed in Xenopus oocytes.

Native cardiac and skeletal muscle Na channels are complexes of alpha and beta 1 subunits. While structural correlates for activation, inactivation, and permeation have been identified in the alpha subunit and the expression of alpha alone produces functional channels, beta 1-deficient rat skeletal muscle (mu 1) and brain Na channels expressed in Xenopus oocytes do not gate normally. In contrast, the requirement of a beta 1 subunit for normal function of Na channels cloned from rat heart or human heart (hH1) has been disputed. Coinjection of rat brain beta 1 subunit cRNA with hH1 (or mu 1) alpha subunit cRNA into oocytes increased peak Na currents recorded 2 d after injection by 240% (225%) without altering the voltage dependence of activation. In mu 1 channels, steady state inactivation was shifted to more negative potentials (by 6 mV, p < 0.01), but the shift of 2 mV was not significant for hH1 channels. Nevertheless, coexpression with beta 1 subunit speeded the decay of macroscopic current of both isoforms. Ensemble average hH1 currents from cell-attached patches revealed that coexpression of beta 1 increases the rate of inactivation (quantified by time to 75% decay of current; p < 0.01 at -30, -40, and -50 mV). Use-dependent decay of hH1 Na current during repeated pulsing to -20 mV (1 s, 0.5 Hz) after a long rest was reduced to 16 +/- 2% of the first pulse current in oocytes coexpressing alpha and beta 1 subunits compared to 35 +/- 8% use-dependent decay for oocytes expressing the alpha subunit alone. Recovery from inactivation of mu 1 and hH1 Na currents after 1-s pulses to -20 mV is multiexponential with three time constants; coexpression of beta 1 subunit decreased all three recovery time constants. We conclude that the beta 1 subunit importantly influences the function of Na channels produced by coexpression with either the hH1 or mu 1 alpha subunits.

Animals↗

PubMed1995

Cardiac sodium channels (hH1) are intrinsically more sensitive to block by lidocaine than are skeletal muscle (mu 1) channels.

When lidocaine is given systemically, cardiac Na channels are blocked preferentially over those in skeletal muscle and nerve. This apparent increased affinity is commonly assumed to arise solely from the fact that cardiac Na channels spend a large fraction of their time in the inactivated state, which exhibits a high affinity for local anesthetics. The oocyte expression system was used to compare systematically the sensitivities of skeletal (mu 1-beta 1) and cardiac (hH1-beta 1) Na channels to block by lidocaine, under conditions in which the only difference was the choice of alpha subunit. To check for differences in tonic block, Na currents were elicited after 3 min of exposure to various lidocaine concentrations at -100 mV, a potential at which both hH1-beta 1 and mu 1-beta 1 channels were fully reprimed. Surprisingly, hH1-beta 1 Na channels were threefold more sensitive to rested-state block by lidocaine (402 +/- 36 microM, n = 4-22) than were mu 1-beta 1 Na channels (1,168 +/- 34 microM, n = 7-19). In contrast, the inactivated state binding affinities determined at partially depolarized holding potentials (h infinity approximately 0.2) were similar (Kd = 16 +/- 1 microM, n = 3-9 for hH1-beta 1 and 12 +/- 2 microM, n = 4-11 for mu 1-beta 1). Lidocaine produced more use-dependent block of peak hH1-beta 1 Na current elicited by trains of short-(10 ms) or long- (1 s) duration step depolarizations (0.5 Hz, -20 mV) than of mu 1-beta 1 Na current. During exposure to lidocaine, hH1-beta 1 channels recover from inactivation at -100 mV after a prolonged delay (20 ms), while mu 1-beta 1 channels begin repriming immediately. The overall time course of recovery from inactivation in the presence of lidocaine is much slower in hH1-beta 1 than in mu 1-beta 1 channels. These unexpected findings suggest that structural differences in the alpha subunits impart intrinsically different lidocaine sensitivities to the two isoforms. The differences in steady state affinities and in repriming kinetics are both in the correct direction to help explain the increased potency of cardiac Na channel block by local anesthetics.

Animals↗

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↗