Considerations in studying the transient outward K+ current in cells exhibiting the hyperpolarization-activated current

Recently, we focussed our research on cellular electrophysiological changes involved in the modulated sinoatrial (SA) node function during heart failure [1] and aging [2]. Heart failure and age-dependent changes of ionic membrane currents, especially the transient outward K+ current (Ito1), are well documented in working myocardium [3,4]. Therefore, we read with interest the paper in Cardiovascular Research by Lei et al. [5], on the characterization of Ito1 in rabbit SA node cells. In their paper, Lei et al. [5] determined (1) the properties of Ito1, (2) the relationship between the Ito1 density and cell size, and (3) the physiological relevance of Ito1 in SA node cell electrophysiology. Based on their findings we hypothesized that changes in Ito1 may also play a role in the altered SA node cycle length during heart failure and aging. In a first set of experiments, we aimed at measuring Ito1 in SA node cells isolated from control and failing rabbit hearts using the voltage-clamp protocols of Lei et al. [5]. Our experiments, however, indicate that accurate measurement of Ito1 is hampered by the presence of the hyperpolarization-activated current (If) in these cells. A well-known feature of mammalian SA node cells is that they exhibit If, which plays a role in pacemaker activity [6,7]. If, carried by Na+ and K+ ions, is typically studied by hyperpolarizing voltage-clamp steps from a holding potential of −40 mV. If is activated at potentials negative to −50 mV, reflected as a time-dependent increase in inward current. When If is activated (by hyperpolarization) and subsequently depolarized, it shows a tail current due to deactivation. This tail current reflects the fully activated current and is often used for studying the reversal potential of If[6]. A typical voltage-clamp protocol for measuring the reversal potential is clamping a cell at −80 mV or at more negative potentials, for activating If, followed by depolarizing voltage-clamp steps in a range between −40 and +50 mV. Noteworthy, a comparable voltage-clamp protocol is frequently used for studying activation characteristics of Ito1 and was also used in the study of Lei et al. [5]. Therefore, it is likely that If tail currents interfere with Ito1 in SA node cells. Lei et al. [5] measured membrane currents evoked by 200-ms depolarizing pulses from a holding potential of −80 mV. In these experiments, 300 μM CdCl2 was present to block Ca2+ currents, but no attempts were made to rule out a contribution of If. An interference of If with the Ito1 measurements of Lei et al. [5] (e.g. see their Fig. 1) is suspected by their findings that at potentials negative to −20 mV the current decays in the outward direction, while at potentials positive to −20 mV the current decays in the inward direction. The reversal potential of If was found to be around −20 mV [6]. It remains puzzling, however, that in these SA node cells holding current immediately before and after the depolarizing steps is similar (see their Figs. 1 and 3–7). One would expect that immediately before the step If is activated to some extent. It might be that Lei et al. [5] used blockers of If without mentioning. However, blockade of If by drugs is voltage-dependent. CsCl, for example, better blocks If at negative than at positive potentials [6]. Therefore, If tail currents will still hinder Ito1 measurements. Another possibility is that If is not present in the cells used by Lei et al. [5]. Previous work shows that, while a variety of cells from the SA node region exhibit If, those from the crista terminalis region do not [6–8]. An important feature of cells isolated from the crista terminalis is the presence of a large Ito1, while a small percentage (5%) of these cells exhibited spontaneous activity [8]. Incorporation of cells from the crista terminalis in the study of Lei et al. [5] might explain the presence of large Ito1 while other reports suggests that Ito1 in freshly isolated SA node cells is rarely observed [9], very small [10], or absent unless the cells were derived from the most peripheral portions of the node [11]. On the other hand, the experiments of Lei et al. [5] were carried out on spindle and/or spider-shaped cells, typical for SA node cells, while cells from the crista terminalis are rod-shaped [8]. In the last part of the study, Lei et al. [5] used 4-aminopyridine (4-AP) as a blocker of Ito1 to study the relevance of Ito1 in SA node cell electrophysiology. They found that 4-AP affected both the action potential and pacemaker activity. However, 4-AP is not a selective blocker of Ito1. In many cardiac cell types including SA node cells, 4-AP not only blocks the transient current but also a sustained outward current (see their Fig. 2). The nature of this 4-AP-sensitive sustained outward current is unclear, but it could either represent: (1) a non-inactivating component of Ito1[12], (2) the rapid delayed rectifier current [13], (3) the ultra-rapid delayed rectifier current [14]. In addition, 4-AP results in a depolarizing shift of If activation [15]. Thus the use of 4-AP seems neither valid to measure accurately the role of Ito1 in SA node cell electrophysiology nor a valid alternative to determining the presence of Ito1 in spontaneous active SA node cells. In conclusion, we think that an accurate measurement of the contribution of Ito1 in cells that exhibit If is hampered by current tails of If or specificity of 4-AP.