© 2001 by European Society of Cardiology
Copyright © 2001, European Society of Cardiology
Transient outward K+ current, ito, in the sinoatrial node
aDepartment of Physiology, School of Biomedical Sciences, University of Leeds, Leeds LS2 9JT, UK
bResearch Institute of Environmental Medicine, Nagoya University, Nagoya 464-8601, Japan
cUniversity Laboratory of Physiology, University of Oxford, Oxford OX1 3PT, UK
* Corresponding author. Tel.: +44-113-233-4298; fax: +44-113-233-4224 m.r.boyett{at}leeds.ac.uk
Received 17 September 2001; accepted 17 September 2001
The transient outward K+ current, ito, is a major current in the heart. It is responsible for the early phase of repolarization (phase 1) in some regions of the heart and it can control action potential duration, e.g. Refs. [1,2]. We and others have argued that it contributes to the regional differences in electrical activity in different regions of the heart, such as across the ventricular wall [3] and in the sinoatrial (SA) node [4]. It also contributes to electrical remodelling, e.g. Ref. [5].
We have measured ito in rabbit SA node cells [6,7]. In their letter, A.O. Verkerk and A.C.G. van Ginneken point out that the recordings of ito during depolarizing pulses from rabbit SA node at a holding potential of –80 mV from our laboratory (published by Lei et al. [7]) may be contaminated by tail currents of the hyperpolarization-activated current, if. These tail currents are the result of the deactivation of if during depolarizing pulses. They also point out that the use of 4-AP by Lei et al. [7] to isolate ito is hampered by the non-specificity of 4-AP, including an effect of 4-AP on if.
The presence of ito in rabbit SA node cells is certain. First Nathan [8] and then later Denyer and Brown [9] and Ito and Ono [10] reported the presence of ito in rabbit SA node cells. Denyer and Brown [9] commented that ito was rarely present in rabbit SA node cells, but in their study they used 5 µM D600 to block the L-type Ca2+ current, iCa,L, and D600 is known to partially block ito [11]. Nathan [8], Denyer and Brown [9] and Ito and Ono [10] used holding potentials of –70 or –50, –65 and –60 mV, respectively; at these holding potentials, there is unlikely to be significant activation of if (e.g. Ref. [12]) and, therefore, there is unlikely to be if tail currents during depolarizing voltage clamp pulses applied to measure ito. All three of these early studies showed ito to be abolished by 4-AP [8–10]. None of these early reports were a systematic study of ito in rabbit SA node cells. However, since these early reports, there have been three systematic studies of ito in rabbit SA node cells: that of Lei et al. [7] (the subject of the letter from A.O. Verkerk and A.C.G. van Ginneken), the earlier study of Honjo et al. [6] from our laboratory and the study of Uese et al. [13] from another laboratory. The holding potential was –60 mV in the studies of Honjo et al. [6] and Uese et al. [13], but the more negative potential of –80 mV in the study of Lei et al. [7]. In all of these studies, ito was shown to be abolished by 4-AP. In the early studies as well as the later systematic studies, the reported characteristics of ito are qualitatively similar in terms of current density, kinetics, voltage-dependence of activation and inactivation and block by 4-AP. For example, Uese et al. [13] (holding potential, –60 mV) reported a half inactivation potential of –35 mV and an EC50 for 4-AP of 0.28 mM, whereas Lei et al. [7] (the study in question; holding potential, –80 mV) reported values of –49 mV and 0.33 mM.
The first of the concerns of A.O. Verkerk and A.C.G. van Ginneken is the use of a holding potential of –80 mV in the study of Lei et al. [7] at which there may be activation of if. As argued above, there can be no doubt of the existence of ito in rabbit SA node cells. Furthermore, as shown above, the characteristics of ito as recorded by Lei et al. [7] are similar to those recorded by others. Although an if blocker was not used in the study of Lei et al. [7], various lines of evidence show that there was unlikely to have been significant contamination of ito by if despite the use of a holding potential of –80 mV. In our earlier study [6], we commented that the 4-AP-sensitive current (including ito) was essentially the same at holding potentials of –80 and –60 mV. In our laboratory, we have reported minimal activation of if at –80 mV [12]. Furthermore, any activation of if at –80 mV will occur very slowly (the time constant at –80 mV can be calculated to be 4.3 s using the equations of Yanighara and Irisawa [14]) and, therefore, simply based on the kinetics of if, with the interpulse interval of 1.8 s (as used by Lei et al. [7]) there should be minimal activation of if. Furthermore, as pointed out by A.O. Verkerk and A.C.G. van Ginneken, there is no evidence of activation of if in the records of Lei et al. [7]: the holding currents immediately before and after depolarising pulses (during which if will be deactivated) are about the same (whereas, if there was significant activation of if during the interpulse interval, current prior to the pulse should be more inward than that after the pulse). We conclude that if contamination of ito was unlikely to have been significant.
A.O. Verkerk’s and A.C.G. van Ginneken’s second concern is the non-specificity of 4-AP. This undoubtedly is a concern — the likely effects of 4-AP on various ionic currents have been reviewed by our laboratory [6,7]. Despite the non-specificity of 4-AP, in the absence of a more specific blocker, 4-AP is widely used to block ito as shown above (in addition to the examples above, there are many other examples, too numerous to quote, concerned with other tissues in the heart). None of the reported characteristics of ito mentioned above (current density, kinetics, voltage-dependence of activation and inactivation) depend on the use of 4-AP. In every case above, 4-AP sensitivity was used as an indication of the identity of the transient outward current. van Bogaert and Snyders [15] reported a 17-mV depolarizing shift of the if activation curve by 5 mM 4-AP in sheep Purkinje fibres. The shift of the if activation curve by 4-AP, if it occurs in rabbit SA node cells, is expected to result in an increase in if at the holding potential — there is no evidence of this in the records of Lei et al. [7]. Even if such an effect was to occur, it would result in an increase in if tail current during a depolarizing pulse. In the records of Lei et al. [7], 4-AP abolished transient outward current.
We conclude that the concerns of A.O. Verkerk and A.C.G. van Ginneken did not significantly effect the recordings of Lei et al. [7] and do not effect the conclusions of that study. We can turn the question around: are measurements of if tail currents contaminated by ito? Could the if activation curve derived from if tail currents be affected by the ito inactivation curve?
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