Harry A. Fozzard

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

The cardiac sodium channel α subunit (RHI) is less sensitive to tetrodotoxin (TTX) and saxitoxin (STX) and more sensitive to cadmium than brain and skeletal muscle (�l) isoforms. An RHI mutant, with Tyr substituted for Cys at position 374 (as in �l) 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.

Increase in intracellular sodium ion activity during stimulation in mammalian cardiac muscle.

Changes in stimulation rate alter the electrical and mechanical characteristics of myocardial cells. We have investigated the possibility that intracellular sodium activity (aNa) changes with stimulation and correlates with changes in contraction strength. Two kinds of liquid membrane Na+-selective microelectrodes were used to measure aNa in guinea pig and sheep ventricular muscle and in sheep Purkinje strands. Stimulation produced a rate- and time-dependent elevation of aNa. Small increases in aka were seen at stimulation rates as slow as 0.2 Hz, and faster rates of stimulation elevated aNa by over 30%. The changes seen in Purkinje strands and ventricular muscle were similar. Following a period of stimulation, aiNa and Vm returned to their pre-stimulus levels with the same time courses. This is consistent with the suggestion that the post-stimulation hyperpolarization is the result of an increased rate of electrogenic Na+ extrusion. The effects of stimulation on a L and tension were compared with those of ouabain. The comparison suggests that rapid stimulation could produce increased contraction strength as the result of a substantial gain in intracellular calcium via a Na-Ca exchange mechanism, but that this is only one of several factors determining the forcefrequency relationship.

Influence of Extracellular K+ Concentration on Cable Properties and Excitability of Sheep Cardiac Purkinje Fibers

Conduction velocity was increased from 3.5 m/sec to 4.1 m/sec in sheep cardiac Purkinje fibers when the superfusing Tyrode solution was changed from 2.7 mM K+ to 4.0 mM K+. Further increase to 7.0 mM K+ resulted in a fall in conduction velocity to 3.3 m/sec. To understand the nature of the cellular change responsible for this effect, cable analyses were made. With increases in extracellular K+ concentration membrane resistance decreased and membrane capacitance did not change. Resistance of the myoplasm tended to fall in 7.0 mM [K]o. Since these effects did not explain the change in conduction velocity, excitability was studied by strength-duration curves using intracellular micropipettes for current passage and recording. Increase in K+ from 2.7 to 4.0 mM resulted in a shift of the entire curve to the left, with a fall in rheobasic current from 121 to 104 nanoamperes and a fall in the time constant from 2.79 to 2.4 msec. Normalized plotting of stimulating current over rheobasic current (I/IRh) against duration of stimulating current over the time constant (t/τ) suggested that the curves were not basically different. The increase in K+ from 2.7 to 4.0 mM was associated with a depolarization of the resting membrane, consistent with alteration in the potassium equilibrium potential, but no change in the absolute membrane potential for threshold. In this way, the changes in membrane voltage and charge necessary for excitation were reduced in 4.0 mM [K]o.

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 EADs, and cellular Ca overload is not related. These afterdepolarizations have characteristics that suggest their etiological role in certain arrhythmias found in heart failure.

The positive dynamic current and its inactivation properties in cardiac Purkinje fibres

1. The positive dynamic current in sheep cardiac Purkinje fibres was studied using ‘voltage clamp’ technique. The major portion of this current was carried by Cl− ions.

Strength—duration curves in cardiac Purkinje fibres: effects of liminal length and charge distribution

1. Strength—duration curves for excitation in long point‐stimulated sheep cardiac Purkinje fibres, where the charge distribution varies along the length of the fibre, are characterized by (a) a time constant which is short relative to the membrane time constant, (b) an apparent fall in charge threshold for short duration stimuli, (c) an apparent rise in voltage threshold as measured by an electrode at the point of current passage (see Dominguez & Fozzard, 1970).

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.

Differences in saxitoxin and tetrodotoxin binding revealed by mutagenesis of the Na+ channel outer vestibule.

The marine guanidinium toxins, saxitoxin (STX) and tetrodotoxin (TTX), have played crucial roles in the study of voltage-gated Na+ channels. Because they have similar actions, sizes, and functional groups, they have been thought to associate with the channel in the same manner, and early mutational studies supported this idea. Recent experiments by. Biophys. J. 67:2305-2315) have suggested that the toxins bind differently to the isoform-specific domain I Phe/Tyr/Cys location. In the adult skeletal muscle Na+ channel isoform (microliter), we compared the effects on both TTX and STX affinities of mutations in eight positions known to influence toxin binding. The results permitted the assignment of energies contributed by each amino acid to the binding reaction. For neutralizing mutations of Asp400, Glu755, and Lys1237, all thought to be part of the selectivity filter of the channel, the loss of binding energy was identical for the two toxins. However, the loss of binding energy was quite different for vestibule residues considered to be more superficial. Specifically, STX affinity was reduced much more by neutralizations of Glu758 and Asp1532. On the other hand, mutation of Tyr401 to Cys reduced TTX binding energy twice as much as it reduced STX binding energy. Kinetic analysis suggested that all outer vestibule residues tested interacted with both toxins early in the binding reaction (consistent with larger changes in the binding than unbinding rates) before the transition state and formation of the final bound complex. We propose a revised model of TTX and STX binding in the Na+ channel outer vestibule in which the toxins have similar interactions at the selectivity filter, TTX has a stronger interaction with Tyr401, and STX interacts more strongly with the more extracellular residues.

Mechanism of cAMP-dependent modulation of cardiac sodium channel current kinetics.

beta-Adrenergic modulation is one of the most important regulatory mechanisms of ion channel function. Only recently, however, have beta-adrenergic effects on cardiac Na+ channel activity been recognized, and some diversity of effects has been reported in different preparations. We report studies of protein kinase A-dependent phosphorylation effects on cardiac Na+ current using the macropatch on-cell mode voltage-clamp technique to maintain cytoplasmic composition intact. During the first 5 minutes after addition of 8-(4-chlorophenylthio)cAMP to the bath, the midpoints of both voltage-dependent availability and conductance shifted in the hyperpolarizing direction an average of -7.5 +/- 2.8 mV (n = 31). Moreover, these effects were not species specific; similar results were obtained in canine, rabbit, and guinea pig myocytes, and a similar shift occurred after exposure to 5 microM isoproterenol. Maximum conductance did not change, nor did single-channel conductance. The shifts of conductance and voltage-dependent availability that were induced by protein phosphorylation were distinct from and independent of the slow background shift in kinetics. We measured the background shift to be less than 0.3 mV/min and to be restricted to the channels within the patch. Pretreatment of cells with a blocker of protein kinase, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide (H-89), prevented the effect of 8-(4-chlorophenylthio)cAMP while not affecting the background shift in kinetics. Although clearly not the result of addition of a negatively charged phosphate to the inside face of the channel, cAMP-dependent phosphorylation affects the voltage-dependent kinetics, as expected, by an electrostatic interaction with the voltage sensor.

Sodium current in voltage clamped internally perfused canine cardiac Purkinje cells

Study of the excitatory sodium current (INa) intact heart muscle has been hampered by the limitations of voltage clamp methods in multicellular preparations that result from the presence of large series resistance and from extracellular ion accumulation and depletion. To minimize these problems we voltage clamped and internally perfused freshly isolated canine cardiac Purkinje cells using a large bore (25-microns diam) double-barreled flow-through glass suction pipette. Control of [Na+]i was demonstrated by the agreement of measured INa reversal potentials with the predictions of the Nernst relation. Series resistance measured by an independent microelectrode was comparable to values obtained in voltage clamp studies of squid axons (less than 3.0 omega-cm2). The rapid capacity transient decays (tau c less than 15 microseconds) and small deviations of membrane potential (less than 4 mV at peak INa) achieved in these experiments represent good conditions for the study of INa. We studied INa in 26 cells (temperature range 13 degrees-24 degrees C) with 120 or 45 mM [Na+]o and 15 mM [Na+]i. Time to peak INa at 18 degrees C ranged from 1.0 ms (-40 mV) to less than 250 microseconds (+ 40 mV), and INa decayed with a time course best described by two time constants in the voltage range -60 to -10 mV. Normalized peak INa in eight cells at 18 degrees C was 2.0 +/- 0.2 mA/cm2 with [Na+]o 45 mM and 4.1 +/- 0.6 mA/cm2 with [Na+]o 120 mM. These large peak current measurements require a high density of Na+ channels. It is estimated that 67 +/- 6 channels/micron 2 are open at peak INa, and from integrated INa as many as 260 Na+ channels/micron2 are available for opening in canine cardiac Purkinje cells.