Hilary F. Brown

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.

Reciprocal Role of the Inward Currents i

Experiments and computations were done to clarify the role of the various inward currents in generating and modulating pacemaker frequency. Ionic currents in rabbit single isolated sino-atrial (SA) node cells were measured using the nystatin-permeabilized patch-clamp technique. The results were used to refine the Noble–DiFrancesco–Denyer model of spontaneous pacemaker activity of the SA node. This model was then used to show that the pacemaker frequency is relatively insensitive to the magnitude of the sodium-dependent inward background current ib, Na. This is because reducing ib, Na hyperpolarizes the cell and so activates more hyperpolarizing-activated current, if, whereas the converse occurs when ib, Na is increased. The result is that if and ib, Na replace one another and so stabilize nodal pacemaker frequency.

_{\text{b,Na}}

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

and i

Permeabilized patch whole‐cell voltage clamp methods were used to investigate the effects of isoprenaline (ISO) on total delayed rectifier potassium current, iK, in rabbit sino‐atrial (SA) node pacemaker cells; total iK is composed of the rapidly activating iKr and the slowly activating iKs, but predominantly iKr in this species. ISO (20 nM) increased the amplitude of total iK and caused a negative shift of approximately 10 mV in the activation curve for iK, both in the absence and in the presence of 300 nM nisoldipine to block the L‐type Ca2+ current, iCa,L. The same concentration (20 nM) of ISO increased the spontaneous pacemaker rate of SA node pacemaker cells by 16%. In addition to increasing the amplitude of iK, ISO (20‐50 nM) also increased the rate of deactivation of this current. The stimulation of iK by ISO was reversed by 10 μM H‐89, a selective protein kinase A inhibitor, but not by 200 nM bisindolymaleimide I, a selective protein kinase C inhibitor. It therefore appears that the mechanisms by which β‐adrenoceptor agonists increase pacemaking rate in sinoatrial node pacemaker cells include an increase in the rate of deactivation of iK in addition to the well‐documented augmentation of iCa,L and the positive shift of the activation curve for the hyperpolarization‐activated inward current, if. The observations are also consistent with a role for protein kinase A in the stimulation of iK by ISO in SA node cells.

_{\text{f}}

The delayed rectifier current was studied in rabbit isolated sino‐atrial (SA) node cells using the whole‐cell voltage clamp technique with amphotericin‐permeabilized patches. The envelope of tails test indicated that in SA node cells the decay of IK (IK.tall) comprises two distinct current components similar to the specific fast and slow components of IK (IKr and JKs) that have been found in atrial and ventricular myocytes. Dofetilide, a Class III antiarrhythmic agent and a selective blocker of IKr. separated the delayed rectifier current into drug‐sensitive current (IKr) and drug‐insensitive current (IKs). The dofetilide‐sensitive current activated rapidly and it showed two components of deactivation, the larger of which was very slow, while the dofetilide‐insensitive current activated more slowly and deactivated quickly. The effects of propofol, an IKs blocker, on the delayed rectifier current were also investigated. The results show that IKs contributes to IK.tall and that the more positive the membrane potential, the more the contribution of IKs. The ratio of IKs to IKr in tail currents at ‐40 mV after a 1 s clamp pulse to +40 mV was 0.3‐0.4:1. Dofetilide slowed spontaneous activity, suggesting that IKr contributes to the pacemaker activity of the SA node cell.

in Controlling and Stabilizing Pacemaker Frequency of Rabbit Sino-Atrial Node Cells

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

The ionic selectivity of the hyperpolarizationactivated inward current (if) channel to monovalent cations was investigated in single isolated sinoatrial node cells of the rabbit using the whole-cell patch-clamp technique. With a 140 mM K+ pipette, replacement of 90% external Na+ by Li+ caused a −24.5 mV shift of the fully activated current/voltage I/V curve without a significant decrease of the slope conductance. With a 140 mM Cs+ pipette, the if current decreased almost proportionally to the decrease in external [Na+]o as Li+ was substituted. These responses are practically the same as those observed with N-methyl glucamine (NMG+) substitution, suggesting that the relative permeability of Li+ compared with Na+ for the if channel is as low as that of NMG+. When Cs+ or Rb+ was substituted for internal K+, the fully activated I/V relationship for if showed strong inward rectification with a positive reversal potential, indicating low permeability of the if channel for Cs+ and Rb+. These results show that the if channel is highly selective for Na+ and K+ and will not pass the similar ions Li+ and Rb+. Such a high degree of selectivity is unique and may imply that the structure of the if channel differs greatly from that of other Na+ and K+ conducting channels.

Modulation of delayed rectifier potassium current, iK, by isoprenaline in rabbit isolated pacemaker cells.

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.

Two components of the delayed rectifier potassium current, IK, in rabbit sino‐atrial node cells

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

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.