Susan Noble

Sodium‐calcium exchange during the action potential in guinea‐pig ventricular cells.

1. Slow inward tail currents attributable to electrogenic sodium‐calcium exchange can be recorded by imposing hyperpolarizing voltage clamp pulses during the normal action potential of isolated guinea‐pig ventricular cells. The hyperpolarizations return the membrane to the resting potential (between ‐65 and ‐88 m V) allowing an inward current to be recorded. This current usually has peak amplitude when repolarization is imposed during the first 50 ms after the action potential upstroke, but becomes negligible once the final phase of repolarization is reached. The envelope of peak current tail amplitudes strongly resembles that of the intracellular calcium transient recorded in other studies. 2. Repetitive stimulation producing normal action potentials at a frequency of 2 Hz progressively augments the tail current recorded immediately after the stimulus train. Conversely, if each action potential is prematurely terminated at 0.1 Hz, repetitive stimulation produces a tail current much smaller than the control value. The control amplitude of inward current is only maintained if interrupted action potentials are separated by at least one full ‘repriming’ action potential. These effects mimic those on cell contraction (Arlock & Wohlfart, 1986) and suggest that progressive changes in tail current are controlled by variations in the amplitude and time course of the intracellular calcium transient. 3. When intracellular calcium is buffered sufficiently to abolish contraction, the tail current is abolished. Substitution of calcium with strontium greatly reduces the tail current. 4. The inward tail current can also be recorded at more positive membrane potentials using standard voltage clamp pulse protocols. In this way it was found that temperature has a large effect on the tail current, which can change from net inward at 22 degrees C to net outward at 37 degrees C. The largest inward currents are usually recorded at about 30 degrees C. It is shown that this effect is attributable predominantly to the temperature sensitivity of activation of the delayed potassium current, iK, whose decay can then mask the slow tail current at high temperatures. 5. Studies of the relationship between the tail current and the membrane calcium current, iCa, have been performed using a method of drug application which is capable of perturbing iCa in a very rapid and highly reversible manner. Partial block of iCa with cadmium does not initially alter the size of the associated inward current tail. When iCa is increased by applying isoprenaline, the percentage augmentation of the associated tail current is much greater but occurs more slowly.(ABSTRACT TRUNCATED AT 400 WORDS)

A model of sino-atrial node electrical activity based on a modification of the DiFrancesco-Noble (1984) equations

DiFrancesco & Noble’s (1984) equations (Phil. Trans. R. Soc. Lond. B (in the press.)) have been modified to apply to the mammalian sino-atrial node. The modifications are based on recent experimental work. The modified equations successfully reproduce action potential and pacemaker activity in the node. Slightly different versions have been developed for peripheral regions that show a maximum diastolic potential near — 75 mV and for central regions that do not hyperpolarize beyond —60 to —65 mV. Variations in extracellular potassium influence the frequency of pacemaker activity in the s.a. node model very much less than they do in the Purkinje fibre model. This corresponds well to the experimental observation that the node is less sensitive to external [K] than are Purkinje fibres. Activation of the N a-K exchange pump in the model by increasing intracellular sodium can suppress pacemaker activity. This phenomenon may contribute to the mechanism of overdrive suppression.

Membrane currents underlying activity in frog sinus venosus

1. The spontaneous electrical activity of small strips of muscle from the sinus venosus region of the heart of Rana catesbeiana was investigated using the double sucrose gap technique. The voltage clamp was used to record the ionic currents underlying the pace‐maker depolarization and the action potential.

The ionic currents underlying pacemaker activity in rabbit sino-atrial node: experimental results and computer simulations

The membrane currents underlying the pacemaker depolarization have been investigated in rabbit s.a. node preparations using the twomicroelectrode voltage clamp technique. Many of the experimental results have been simulated using a computer model of s.a. node electrical activity. Changes of three time-dependent membrane currents which could contribute to pacemaker depolarization are found to occur in the relevant potential range: decay of the potassium current, iK, and activation of the inward current, it,and of the slow inward current isi

The slow inward current, iSi, in the rabbit sino-atrial node investigated by voltage clamp and computer simulation

The properties of the slow inward current, isi in the sino-atrial (s.a.) node of the rabbit have been investigated using two microelectrodes to apply voltage clamp to small, spontaneously beating, preparations. Many of the experimental results can be closely simulated using the computer model of s.a. node electrical activity (Noble & Noble 1984) which has been developed from models of Purkinje fibre activity (Noble 1962; DiFrancesco & Noble 1984). Comparison of the computed reconstructions with experimental results provides a test of the validity of the modelling. Experiments using paired depolarizing clamp pulses show that inactivation of isi is calcium -entry dependent although, unlike the inactivation of Ca2+ currents in some other systems, it also shows some voltagedependence. Re-availability (recovery from inactivation) of isi in s.a. node is much slower than inactivation at the same potential, showing that isi is not controlled by a single first order process. This very slow recovery from inactivation of isi in the s.a. node and the slow time course of its activation and inactivation a t voltages near threshold ( — 40 to —50 mV) can be closely modelled by assuming that there are two components of ‘total isi ’: a fast inward current, iCa f, representing the ‘gated’ fraction and a second, slower, inward current component, iNaCa which, we propose, is caused by the sodium -calcium exchange that ensues when the initial Ca2+-entry triggers the release of stored intracellular Ca2+. When repetitive trains of clamp pulses are given, a ‘staircase’ of isi magnitude is seen which can be increasing (‘positive’) or decreasing (‘negative’) according to the potential level and frequency of the pulse train given. W hen computer reconstructions of such staircases are made, it is found that the positive staircases (which, in contrast to negative staircases, imply that more complex processes than simple inactivation are present) can be closely simulated by a model which incorporates slower processes (suggested Na-Ca exchange current) in the total isi in addition to the gated current component. Further evidence for such additional components of isi is provided firstly by its slow time course (ca. 75 ms to peak) near threshold; second, by a change in time course of the isi recorded after progressively more negative conditioning hyperpolarizations (a result which is hard to account for using equations for a single gated channel) and, third, by the occasional appearance of a double-peaked isi record, both when isi is recorded after hyperpolarizations and in ‘ staircases ’ during trains of repetitive clamp pulses. In this latter case, the two components of isi show a different pattern of change one from another as the staircase progresses.

The influence of non‐uniformity on the analysis of potassium currents in heart muscle.

1. A method is described for determining the space constant gamma of heart muscle strips using a sucrose gap technique. 2. The average value of gamma for frog atrial trabeculae was found to be nearly 700 mum. This value is nearly twice the length of the test gap (400 mum). Near the resting potential, the voltage non‐uniformity should be about 10%. This was confirmed experimentally by comparing the membrane voltages recorded across the current‐passing and voltage‐recording sucrose gaps. 3. The non‐uniformity during large depolarizations was calculated using a computer model. This model includes the inward‐going rectification displayed by iK1 and the delayed rectification that occurs following depolarizations beyond ‐40mV. A single component of delayed rectification was included. 4. It is shown that even very large non‐uniformities have relatively small effects on the shape of the activation curve and on the time course of onset or decay of current. 5. It is comcluded that the fast component of current decay described in a previous paper (Brown, Clark & Noble, 1976b) is not attributable to a non‐uniformity artifact.

The Relative Contributions of Various Time-Dependent Membrane Currents to Pacemaker Activity in the Sino Atrial Node

The successful application of the voltage clamp technique to the mammalian sino-atrial node has already given much information about the membrane currents in nodal pacemaking cells. In particular, an apparently new time-dependent current, if (or ih) has been found on voltage clamping SA node cells negative to -50 mV (Brown et al., 1979; Yanagihara and Irisawa, 1980a). It now seems clear that this current, which has been shown to be a developing inward current (DiFrancesco and Ojeda, 1980) is closely related to the current iK2 which underlies pacemaking in Purkinje fibres. IK2, whose kinetics were first described by Noble and Tsien (1968) was for many years thought to be an outward current which decays in the pacemaker range and which is carried by potassium ions: hence its name. Recent experiments, using barium to block the background current, iK2, in Purkinje fibres have shown that IK2 is in reality a developing inward current which cannot be carried by potassium ions alone (see DiFrancesco, this volume). It probiably has the same ionic composition as if (ih) in the SA node and is carried by both sodium and potassium ions (DiFrancesco, 1980; Brown et al., 1980). This being the case, both currents should have the same name, but since no universal agreement has yet been reached as to whether this should be if or ih we here use our original nomenclature if.

The contribution of potassium accumulation to outward currents in frog atrium.

1. Voltage‐clamp experiments on frog atrial muscle were designed to distinguish effects due to K accumulation in extracellular spaces from those due to activation of K conductance mechanisms in the membrane. 2. The set of instantaneous current‐voltage relations obtained at various external K concentrations following depolarization to about ‐10 mV for several seconds was found to be quite different from that obtained before the depolarization. Hence the process of increasing the extracellular K concentration cannot account for all the time‐dependent changes in outward current during depolarization. 3. Although the instantaneous current‐voltage relations obtained at different values of external K concentration before prolonged depolarization show the cross‐over phenomenon (Noble, 1965), those obtained at the end of the depolarization did not show this feature. It is concluded that the current‐voltage relations for the channels conducting the time‐dependent K current do not show cross‐over. 4. These results were used to construct a model involving both K activation and K accumulation. This model successfully reproduces the appearance of a very slow component in outward current decay tails which, when subtracted by semi‐exponential curve‐stripping leaves a component with the real time constant of conductance change. The model does not however reproduce the appearance of a fast decaying component without adding a second conductance mechanism, or assuming non‐exponential decay of a single conductance mechanism. 5. It is therefore suggested that i chi, fast is not a perturbation of i chi, slow or of iK1 by the process of K accumulation. This conclusion is reinforced by the results of experiments showing that the relative magnitude of i chi, fast is not greatly changed by substantially increasing the external K concentration in order to reduce the proportionate effect of K accumulation on the K concentration.

Background Inward Current in Ventricular and Atrial Cells of the Guinea-Pig

Atrial and ventricular myocytes were exposed to Ca2+- and K+-free solutions containing blockers of gated channel and exchange currents. Replacement of external sodium by large organic cations revealed a background sodium current ib, Na. In atrial cells, the average conductance was 5.0 pS pF-1. In ventricular cells the conductance was 2.3 pS pF-1. Together with previous results, these figures reveal a strong gradient of background current density: sinus > atrium > ventricle. Replacement of sodium with inorganic cations showed that the channel selectivity behaves like an Eisenman group III/IV sequence, in agreement with previous results. The permeability of the channel to TMA was found to be pH dependent, suggesting that protonation of the channel is a factor determining permeation in addition to ionic size. The values of gb, Na obtained from these experiments are very similar to those assumed in computer modelling of cardiac cell electrical activity.

The effects of sodium substitution on currents determining the resting potential in guinea‐pig ventricular cells

It has recently been shown that a sodium background current, ib,Na, exists in cardiac muscle cells whose effect is to depolarize the membrane so that the resting potential, Vm, is positive to the potassium equilibrium potential, EK. In ventricular cells, where ib,Na is smallest, Vm is about 10 mV positive to EK (EK = ‐87 mV at 37 degrees C). Yet, replacement of Na+ ions by large impermeant cations does not cause the expected hyperpolarization. We have studied this problem in guinea‐pig myocytes using a single microelectrode recording technique in combination with a rapid external solution switch. Cells depolarized < or = 0.5 mV from potentials between ‐80 and ‐73 mV and hyperpolarized up to 5 mV from potentials between ‐73 and ‐64 mV when 70 mM choline chloride or N‐methyl‐D‐glucamine chloride were used to replace 70 mM Na+ in the bathing solution. Replacement by 70 mM lithium chloride, however, only caused hyperpolarization in very depolarized cells when the voltage change was much smaller. The changes were complete almost as soon as the solution change, i.e. within 250 ms, indicating that the actions are attributable to the external solution change rather than to secondary changes in intracellular concentrations. Patch clamp recording was used to investigate the mechanism involved. These experiments showed that the presence or absence of the inward rectifier current iK1 determines in which direction Na+ removal acts. In the absence of iK1 the changes are attributable to removal of ib,Na, whereas in the presence of iK1 the changes resemble the i(V) relation for iK1, implying that Na+ regulates iK1 in a way that can mask the changes in ib,Na. These results explain why removal of Na+ does not lead to hyperpolarization in ventricular cells as would be expected if changes in ib,Na were solely responsible. Computer reconstruction shows that the effects may be attributed to actions of sodium removal on the conductance and gating of iK1.