Biomedical subjects

H A Fozzard

Publications and source records attributed to H A Fozzard.

At least 19 recordsLinked to original sources

PubMed1995

University of Wisconsin solution preserves myocardial calcium current response to isoproterenol in isolated canine ventricular myocytes.

BACKGROUND: University of Wisconsin (UW) solution has been shown to be an effective solution for cold storage of various organs. This study was designed to evaluate the subcellular protective mechanism of UW solution during cardiac myocyte storage using patch-clamp techniques for the first time as a tool for the detection of myocyte viability. METHODS AND RESULTS: The protective effects of UW solution on the preservation of dihydropyridine-sensitive Ca2+ channel current response to catecholamine were evaluated in canine cardiac ventricular cells by measurement of single channel open probability. Single ventricular myocytes were isolated and stored in UW solution, in Stanford (SF) solution, or in St Thomas' (ST) solution at 4 degrees C for 2, 6, 12, and 24 hours, and after each storage period, recordings were made of cell-attached single Ca2+ channel currents. When 0.1 mumol/L isoproterenol was applied, percent mean open probability of the Ca2+ channel tested in freshly isolated cells was 167 +/- 4% (n = 24) of controls (100%). The response was decrescent with increased duration of the hypothermic storage and was only 130 +/- 12% (n = 4) after 24 hours of storage in SF solution and 135 +/- 9% (n = 7) in ST solution. However, it was significantly highly preserved as much as 165 +/- 9% (n = 6) in UW solution. Ca2+ channel kinetics and channel conductance were not changed after up to 24 hours of hypothermic storage. CONCLUSIONS: Hypothermic storage of canine cardiac myocytes in UW solution preserved beta-adrenergic response, which suggests that UW solution during cold storage preserved high-energy phosphates in myocytes that are responsible for Ca2+ channel phosphorylations.

Adenosine↗

PubMed1995

A structural motif for the voltage-gated potassium channel pore.

Mutation studies have identified a region of the S5-S6 loop of voltage-gated K+ channels (P region) responsible for teraethylammonium (TEA) block and permeation/selectivity properties. We previously modeled a similar region of the Na+ channel as four beta-hairpins with the C strands from each of the domains forming the external vestibule and with charged residues at the beta-turns forming the selectivity filter. However, the K+ channel P region amino acid composition is much more hydrophobic in this area. Here we propose a structural motif for the K+ channel pore based on the following postulates (Kv2.1 numbering). (i) The external TEA binding site is formed by four Tyr-380 residues; P loop residues participating in the internal TEA binding site are four Met-371 and Thr-372 residues. (ii) P regions form extended hairpins with beta-turns in sequence ITMT. (iii) only C ends of hairpins form the inner walls of the pore. (iv) They are extended nonregular strands with backbone carbonyl oxygens of segment VGYGD facing the pore with the conformation BRLRL. (v) Juxtaposition of P loops of the four subunits forms the pore. Fitting the external and internal TEA sites to TEA molecules predicts an hourglass-like pore with the narrowest point (GYG) as wide as 5.5 A, suggesting that selectivity may be achieved by interactions of carbonyls with partially hydrated K+. Other potential cation binding sites also exist in the pore.

Amino Acid Sequence↗

PubMed1995

A direct effect of forskolin on sodium channel bursting.

A long-lasting component of current through voltage-dependent Na channels is believed to contribute to the plateau phase of the cardiac action potential. Here we report that in cardiac ventricular myocytes forskolin increases the contribution of a very slow component of decay (tau = 36 +/- 16 ms, n = 13) in ensemble currents in response to step depolarizations to 0 mV. Long-lasting bursts of openings (mean duration of 27 +/- 14 ms, n = 10) accounted for this behavior. The slow time constant of decay was not altered by forskolin (5-50 microM). Rather, an increase in the probability of bursting behavior produced a forskolin concentration-dependent increase in the amplitude of this very slow component. This action of forskolin was not the result of stimulation of adenylyl cyclase because it was not affected when cAMP-dependent phosphorylation was inhibited by the protein kinase inhibitor H-89, and it could not be mimicked by addition of isoproterenol, membrane-permeant cAMP [8-(4-chlorophenylthio)-cAMP], or the phosphatase inhibitor okadaic acid. In addition, bursting was not augmented by guanosine 5'-O-(3-thiotriphosphate) (GTP [gamma S]) either applied to the bath or directly to the intracellular face of the channel in inside-out macropatches. Furthermore, 1,9-dideoxy-forskolin, which does not stimulate adenylyl cyclase and 6-(3-dimethylaminopropionyl)-forskolin, a hydrophilic derivative of forskolin, also augmented late channel activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

A mutant of TTX-resistant cardiac sodium channels with TTX-sensitive properties.

The cardiac sodium channel alpha subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (microliter) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in microliter) confers three properties of TTX-sensitive channels: (i) greater sensitivity to TTX (730-fold); (ii) lower sensitivity to cadmium (28-fold); and (iii) altered additional block by toxin upon repetitive stimulation. Thus, the primary determinant of high-affinity TTX-STX binding is a critical aromatic residue at position 374, and the interaction may take place possibly through an ionized hydrogen bond. This finding requires revision of the sodium channel pore structure that has been previously suggested by homology with the potassium channel.

Amino Acid Sequence↗

PubMed1992

Mechanisms of pharmacologic intervention at the level of the calcium channel.

Calcium channels are large, complex membrane proteins that mediate transmembrane calcium currents. At least 2 kinds of calcium channels are found in heart muscle--the transient type and the long-lasting type. Calcium currents are modulated by diverse endogenous and exogenous factors including hormones, catecholamines, and calcium antagonists. Calcium antagonists act preferentially on vascular smooth muscle and have relatively less effect on the calcium channels of heart muscle. Compared with heart muscle, vascular smooth muscle is relatively depolarized, suggesting that vascular smooth muscle cells have predominantly the long-lasting type of calcium currents. The differential binding to different types of calcium channels underlies the clinical efficacy of the calcium antagonists. A drug such as bepridil, which acts preferentially on the coronary vasculature rather than on the peripheral vasculature, dilates the coronary vessels without depressing cardiac contraction, a putative clinical advantage.

Animals↗

PubMed1992

Afterdepolarizations and triggered activity.

One of the possible cellular mechanisms for certain types of ventricular arrhythmias is afterdepolarizations. There are two types of afterdepolarization. The delayed afterdepolarization (DAD) arises from the resting potential after full repolarization of an action potential and it may reach threshold for activation. It is favored by cellular Ca overload, and rapid preceding activation rates. The inward current generating the DAD is caused by one of two mechanisms: a Ca-dependent opening of non-specific cation channels, or Ca activation of a rheogenic Na/Ca exchange. The early afterdepolarization (EAD) arises on the shoulder of a preceding action potential plateau and it is favored by slow preceding activation rate and prolonged action potentials. Ca channels are usually responsible for the inward current for EAD's, and cellular Ca overload is not related. These afterdepolarizations have characteristics that suggest their etiological role in certain arrhythmias found in heart failure.

Animals↗

PubMed1992

The cloned cardiac Na channel alpha-subunit expressed in Xenopus oocytes show gating and blocking properties of native channels.

The neonatal rat cardiac Na channel alpha-subunit directed currents in oocytes show characteristic cardiac relative resistance to tetrodotoxin (TTX) block. TTX-sensitive currents obtained by expression in Xenopus oocytes of the alpha-subunits of the rat brain (BrnIIa) and adult skeletal muscle (microI) Na channels show abnormally slow decay kinetics. In order to determine if currents directed by the cardiac alpha-subunit (RHI) exhibit kinetics in oocytes like native currents, we compared RHI-directed currents in oocytes to Na currents in freshly isolated neonatal rat myocytes. The decay rate of RHI currents approached that of neonatal myocytes and was faster than BrnIIa and microI currents in oocytes. The voltage dependence of availability and activation was the same as that in the rat myocytes except for a 12-19 mV shift in the depolarizing direction. The RHI Na currents were sensitive to Cd2+ block, and they showed use dependence of TTX and lidocaine block similar to native currents. The current expressed in oocytes following injection of the cRNA encoding for the alpha-subunit of the cardiac Na channel possesses most of the characteristic kinetic and pharmacological properties of the native cardiac Na current.

Animals↗

PubMed1992

The ECG and the single channel.

Our ability to understand and use the ECG has increased with the availability of tools to study the heart's electrical system. Great advances were achieved with direct electrical recordings from intact animal and human hearts. The microelectrode opened the way to recording of the cellular action potentials and their underlying currents. We now have two new and powerful methods to study cardiac electrophysiology--the patch clamp and molecular biology. We have begun to characterize the behavior of the elementary unit of membrane current, the single channel. The single channel can best be visualized as existing in a finite set of states related sequentially as in a Markov chain. The primary structures of several channels have now been determined by cloning, and the structural determinants of channel function can be explored. We have entered the molecular age of electrophysiology and can expect a greater understanding of the basic physiological and pathophysiological processes underlying the ECG. We can also expect powerful drugs to be designed based on the structures of their channel targets. The ECG remains a rich area for study and an ever better clinical tool.

Animals↗

PubMed1992

Phosphorylation restores activity of L-type calcium channels after rundown in inside-out patches from rabbit cardiac cells.

1. Rundown of L-type calcium channels was studied in inside-out patches made from single isolated rabbit ventricular myocytes, using barium as the charge carrier. 2. In the cell-attached patches single-channel activity was stable for more than 15 min after the patch pipette sealed. beta-Receptor stimulation by isoprenaline caused a characteristic increase in opening probability and the appearance of prolonged openings. When the patch was excised to the inside-out configuration and exposed to a simple ionic solution, channel activity disappeared within 1-2 min and never reappeared spontaneously. 3. After rundown of L-type channel activity in the excised patch, exposure of the inside face of the patch to MgATP and the catalytic subunit of the cyclic AMP-dependent protein kinase (PKAc) resulted in recovery of Ca2+ channel activity. Under these conditions channel activity could be even greater than under control cell-attached conditions, resembling channel activity after exposure to isoprenaline. This recovery of activity persisted many minutes, usually until the patch was lost. Addition of MgATP alone caused a small transient increase in channel activity in some patches. 4. Recovery of activity by MgATP and PKAc could be prevented by prior exposure of the excised patch to protein kinase inhibitor (PKI), or it could be abruptly terminated by exposure to PKI after recovery of activity. Addition to the pipette solution of okadaic acid, a protein phosphatase inhibitor, greatly slowed rundown. These findings support the proposal that dephosphorylation is an important component of rundown, and that phosphorylation is needed for channel opening activity. 5. Single-channel conductance was not altered by patch excision, but it was reduced after exposure of the excised patch to MgATP and PKAc. Mg2+ was responsible for this effect, probably by direct channel block from the inside, and Mg2+ also caused a negative shift in the channel activation, as expected from shielding of inside fixed negative charges.

Adenosine Triphosphate↗

PubMed1992

Dose-dependent modulation of the cardiac sodium channel by sea anemone toxin ATXII.

The effects of sea anemone toxin ATXII on single sodium channels were studied in cell-attached patches on rabbit ventricular myocytes at 20-22 degrees C. Exposure of patches to 1,000 nM ATXII induced long-lasting bursts of openings, which were more dramatically different from control at -20 mV than at -50 mV. Mean open duration, which had a biphasic dependence on voltage in control patches, was monotonically dependent on voltage in toxin-exposed patches, being 3.5 times longer than control at -20 mV and 4.5 times longer at -10 mV. Multiple mean open durations were detected at depolarized potentials. To test whether the multiple mean open durations resulted from a mixture of modified and unmodified openings, histograms of late openings (when unmodified channels would be inactivated) were constructed. Because in most cases these fit a single exponential with a mean open duration like that of modified channels, we conclude that voltage-dependent toxin unbinding produced a mixed population of unmodified and modified openings. Consistent with this hypothesis, lower concentrations of toxin most often produced open-duration histograms best fit with two exponentials. Ensembles revealed complex decay kinetics, which could be interpreted within the context of the toxin-induced increase in mean open duration and burst duration and the summation of modified and unmodified events. Analysis of the numbers of early versus late events at -20 mV for patches exposed to 20 nM, 100 nM, and 1,000 nM ATXII predicted the ED50 for ATXII block to be 285 nM at this potential. Using a five-state Markovian model, the action of ATXII could be explained as a reduction of the open-to-inactivated rate constant without effect on inactivation from closed states or other rate transitions.

Animals↗

PubMed1991

Excitation-contraction coupling in the heart.

There has been dramatic progress in our understanding of normal cardiac excitation-contraction coupling and in control of contraction strength, as the result of the new patch pipette method of voltage clamping of single cells and the new methods for monitoring Cai. Several abnormalities have been shown to exist in hypertrophied muscle; the action potential is changed and the contraction is slower. A kinetic change appears to exist in the L-type Ca current, associated with a slower decay of Cai. The next few years should bring a much improved understanding of the molecular and cellular basis for the changes of hypertrophy.

Animals↗

PubMed1991

External site for local anesthetic block of cardiac Na+ channels.

We report patch clamp studies of single Na+ channels from cardiac ventricular and Purkinje cells that support the hypothesis that local anesthetics can act from the outside of the membrane, and that demonstrate some aspects of their mechanism of action. Inclusion of lidocaine (0.1 mM) or QX-314 (0.5 mM), a membrane-impermeant, quaternary ammonium derivative of lidocaine, in the pipette solution for on-cell single channel recording demonstrated four important findings. (1) The open probability of the channel is reduced by drug in a use-dependent way. (2) Late openings are preferentially reduced. (3) Mean open time is shortened. (4) Hyperpolarization enhances recovery of the drug-bound channels. These findings are consistent with a hyperpolarizing shift of the transition rates for drug-bound channel. Further, we postulate that there is a drug-bound channel conformation which conducts current. At least some of the properties of local anesthetic interaction with the cardiac Na+ channels may be the result of kinetic effects mediated by binding to an external site.

Anesthetics, Local↗

PubMed1990

Functional expression of the rat heart I Na+ channel isoform. Demonstration of properties characteristic of native cardiac Na+ channels.

We describe the expression of functional Na+ channels in Xenopus oocytes injected with cRNA transcribed from the rat heart I cDNA clone. The expressed rat heart I Na+ currents show kinetic properties and resistance to tetrodotoxin and saxitoxin which are characteristic of native cardiac Na+ currents. The primary amino acid sequence of the rat heart I alpha-subunit is therefore sufficient for expression of tetrodotoxin resistance, and the rat heart I clone is likely to account for the tetrodotoxin-resistant phenotype of cardiac and denervated skeletal muscle.

Animals↗

PubMed1990

Kinetic analysis of single sodium channels from canine cardiac Purkinje cells.

Single sodium channel events were recorded from cell-attached patches on single canine cardiac Purkinje cells at 10-13 degrees C. Data from four patches containing two to four channels and one patch with one channel were selected for quantitative analysis. The channels showed prominent reopening behavior at voltages near threshold, and the number of reopenings declined steeply with depolarization. Mean channel open time was a biphasic function of voltage with the maximum value (1-1.5 ms) occurring between -50 and -40 mV and lower values at more and at less hyperpolarized levels. Inactivation without opening was also prominent near threshold, and this occurrence also declined with depolarization. The waiting time distributions and the probability of being open showed voltage and time dependence as expected from whole-cell current studies. The results were analyzed in terms of a five-state Markovian kinetic model using both histogram analysis and a maximum likelihood method to estimate kinetic parameters. The kinetic parameters of the model fits were similar to those of GH3 pituitary cells (Horn, R., and C. A. Vandenberg. 1984. Journal of General Physiology. 84:505-534) and N1E115 neuroblastoma cells (Aldrich, R. W., and C. F. Stevens. Journal of Neuroscience. 7:418-431). Both histogram and maximum likelihood analysis implied that much of the voltage dependence of cardiac Na current is in its activation behavior, with inactivation showing modest voltage dependence.

Animals↗

PubMed1990

Gating currents associated with Na channels in canine cardiac Purkinje cells.

Gating currents (Ig) were recorded in single canine cardiac Purkinje cells at 10-12 degrees C. Ig characteristics corresponded closely to macroscopic INa characteristics and appeared to exhibit little contamination from other voltage-gated channels. Charge density predicted by peak INa was 0.14-0.22 fC micron -2 and this compared well with the measured value of 0.19 +/- 0.10 fC micron -2 (SD; n = 28). The charge-voltage relationship rose over a voltage similar to the peak INa conductance curve. The midpoints of the two relationships were not significantly different although the conductance curve was 1.5 +/- 0.3 (SD; n = 9) times steeper. Consistent with this observation, which predicted that a large amount of the gating charge would be associated with transitions close to the open state, an analysis of activation from Hodgkin-Huxley fits to the macroscopic currents showed that tau m corresponded well with a prominent component of Ig. Ig relaxations fitted two exponentials better than one over the range of voltages in which Na channels were activated. When the holding potential was hyperpolarized, relaxation of Ig during step depolarizations to 0 mV was prolonged but there was no substantial increase in charge, further suggesting that early closed-state transitions are less in charge, further suggesting that early closed-state transitions are less voltage dependent. The single cardiac Purkinje cell appears to be a good candidate for combining Ig and single-channel measurements to obtain a kinetic description of the cardiac Na channel.

Animals↗

PubMed1989

Modulation of the cardiac transient outward current by catecholamines.

We studied modulation of the transient outward current in single canine Purkinje cells that were voltage clamped under Ca2+-free conditions using the patch pipette. The current showed two exponential time constants of inactivation (48, 352 ms at +58 mV and 53, 325 ms at +78 mV). Norepinephrine or isoproterenol modified the inactivation kinetics of this current without affecting the activation kinetics. The half maximum dose for norepinephrine effect was 1.9 x 10(-8) M and the effect was saturated at 10(-6) M. Norepinephrine or isoproterenol reduced the amplitude of the fast time constant component of inactivation, while increasing the amplitude of the slow component, without changing their time constants. They also increased the amplitude of a time-independent current component. The beta-antagonist, sotalol, blocked the norepinephrine effect on the transient outward current. On the other hand, both activation of adenyl cyclase by forskolin and increase of intracellular cAMP concentration produced the same effect as exposure to norepinephrine. Intracellular perfusion with the catalytic subunit of the cAMP-activated protein kinase reproduced the modulation of the current. These results suggest a role for neurotransmitter regulation of the transient outward current in cardiac cells, perhaps by channel phosphorylation.

Animals↗

PubMed1989

Inactivation properties of T-type calcium current in canine cardiac Purkinje cells.

The kinetic behavior of T-type Ca2+ current (ICa-T) was studied in canine cardiac Purkinje cells using a single suction-pipette whole-cell voltage clamp method. ICa-T was studied without contamination of conventional L-type Ca2+ current (ICa-L). Ca2+, Sr2+, or Ba2+ were used as the charge carrier. During maintained depolarization ICa-T decayed rapidly, and under most conditions the decay showed a voltage-dependent single exponential time course that did not depend on the species of charge carrier. The development of inactivation did not depend on Ca2+, but the time course required more than a single exponential process. Just negative to the threshold voltage for activating ICa-T, inactivation slowly developed and there was a delay in its onset. The time course of recovery from inactivation was dependent on the protocol used to measure it. As the duration of an inactivating voltage step was increased, recovery slowed markedly and there was a delay in its onset. The time course of recovery could be fit as a biexponential. The fast and slow time constants of recovery were relatively constant, however, the relative amplitudes were dependent on the duration of the inactivating voltage step. Recovery was not dependent on Ca2+, and it was slower at a less negative voltage. These results suggest that the T-type Ca2+ channel in cardiac Purkinje cells follows a complex kinetic scheme dependent only on voltage. This behavior can be accounted for by incorporating into a Markovian model several inactivated and closed states.

Animals↗

PubMed1989

Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje cells.

Two types of Ca2+ currents were recorded in single dialyzed canine cardiac Purkinje cells using a whole cell voltage clamp technique. T-type current was easily separated from L-type current, because its voltage dependence of inactivation and activation was more negative and it decayed rapidly. L-type current was available at more depolarized holding potentials, activated at more positive voltages, and decayed slowly. In 2 mM extracellular Ca2+ concentration [( Ca]o), the average peak T- and L-type current density was 1.70 and 2.87 pA/pF, respectively. T-type current was relatively insensitive to modification by Ca2+, nifedipine, Cd2+, BAY K 8644, or isoproterenol. T-type current was more sensitive to block by Ni2+ and amiloride. Replacement of Ca2+ by Ba2+ or Sr2+ did not increase T-type current. Changes in the Ca2+ or Ba2+ concentration caused parallel shifts in the voltage dependence of several kinetic parameters for L- and T-type current. In 2 mM [Ca]o, the V1/2 (Boltzmann fit) for inactivation of T-type current was -68 mV with a slope of 3.9, and for L-type current the V1/2 was -31 mV with a slope of 5.5. Recovery from inactivation of L- and T-type current was voltage dependent, and for similar conditions L-type current recovered from inactivation more rapidly than T-type current. These findings show that T- and L-type currents are large in cardiac Purkinje cells, and they can easily be separated by their voltage, kinetic, and pharmacological differences. Both may have important physiological roles.

Animals↗