Troubleshooting problems with in vitro screening of drugs for QT interval prolongation using HERG K⁺ channels expressed in mammalian cell lines and Xenopus oocytes

Abstract

The majority of drugs associated with QT interval prolongation share an ability to inhibit ionic currents passed by HERG potassium channels. One method of screening new chemical entities (NCEs) for QT prolonging potential is therefore to use heterologous systems expressing HERG channels. Such systems are also of value in the understanding of the function, kinetics, sorting, pharmacological sensitivities, and important molecular determinants of the HERG potassium channel. The methods for incorporating the HERG potassium channel into cells and measuring the consequent current are a mixture of techniques that are standard (for heterologous expression of most ion channels) and individualised to HERG. This review presents a selection of the most commonly used methods for examining heterologous HERG currents, as well as introducing some of the technical problems that may be encountered and their solutions. In mammalian cell lines, problems such as fragile membranes, high leak currents, inability to form a gigaseal, diminished HERG current, endogenous transient outward current, altered kinetics, and even occasional run down can interfere with measurements. In Xenopus oocytes, endogenous chloride currents, insufficient superfusate flow, diminished HERG current and HERG current ‘run up’ may create difficulties.

Introduction

HERG is a potassium (K⁺) channel α subunit that comprises the conducting pore mediating the current in ventricular myocytes known as the ‘rapid’ delayed rectifier current (I_Kr), and pharmacological blockade of this current or mutation of the channel can lead to long QT syndrome (LQTS), a disorder of the cardiac rhythm in which the sufferers, who can be otherwise healthy, are subject to a risk of fainting or sudden arrhythmogenic death Tseng, 2001, Witchel & Hancox, 2000. LQTS derives its name from its defining clinical feature: on the electrocardiogram the duration of time between the Q wave and the completion of the T wave is prolonged. When the HERG protein was successfully expressed heterologously, the characteristics of the observed potassium ion currents fit relatively well with what was known of I_Kr in ventricular myocytes Sanguinetti et al., 1995, Trudeau et al., 1995. Subsequently, a large number of mutations were found to occur in HERG in families suffering from LQTS Sanguinetti et al., 1996, Splawski et al., 2000. In addition to mutations, pharmacological inhibition of this channel was shown to be associated with LQTS and could also lead to a risk of sudden death (Sanguinetti et al., 1995). Because the HERG channel has been associated mechanistically with the risk of sudden death for pharmaceutical agents from a wide variety of drug classes, it has attracted a lot of attention in the pharmaceutical industry, among clinical guidelines, and particularly with government regulators Committee for Proprietary Medicinal Products, 1997, Crumb & Cavero, 1999, Food and Drug Administration USA, 2002, Haverkamp et al., 2000, Redfern et al., 2003. Ultimately, this has resulted in the withdrawal or reclassification of drugs that had already been approved for the USA and European markets (e.g., Committee on Safety of Medicines, 2001).

Much effort has been made to exploit the heterologous expression of HERG channels to evaluate in vitro the risk of potential drug-induced repolarisation anomalies. Although there have been a variety of efforts to incorporate high-throughput strategies into drug screening processes for HERG blockade, using either fluorescence (Netzer, Ebneth, Bischoff, & Pongs, 2001) or radioligand binding (Finlayson, Turnbull, January, Sharkey, & Kelly, 2001) based assays, the vast majority of the work has involved electrophysiological measurements of currents from single cells, and as of this date, the electrophysiological approach remains the “gold standard” for testing HERG channel blockade. These electrophysiological recordings are typically in the voltage-clamp mode, and include whole cell patch clamp, perforated patch clamp, two (sharp) electrode voltage clamp, and single-channel recordings.

In electrophysiological recordings in the voltage-clamp mode, in addition to conductance changes derived from actions of pharmacological agents, changes in channel state can be inferred from the current elicited by step depolarisations. For the purposes of interpreting current recordings, it can be helpful to think of K⁺ channels as a membrane spanning K⁺ selective pore with two doors or gates (the activation and inactivation gates) that open and close in a voltage- and time-dependent manner. For K⁺ ions to pass through the pore, generating a current flow across the membrane, both gates must be simultaneously open (see Fig. 1). Closure of either gate results in zero current flow. The gates are controlled by the membrane potential; upon depolarisation (i.e. the membrane potential changing to a more positive voltage), the activation gate opens (a process called activation), and upon repolarisation (i.e. the membrane potential changing to a more negative voltage approaching E_K, which is approximately −80 mV in physiological salt solutions), the activation gate closes (deactivation). By contrast, the inactivation gate closes with depolarisation (a process called inactivation), and opens with repolarisation (recovery from inactivation). In cell membranes with large numbers of ion channels, the speed of the gates' response to voltage changes (the kinetics of the channel) is what controls the time course and amplitude of the current. If the movement of the two gates were simultaneous, instantaneous, and occurred in all channels, there would never be any current flowing through the channel, because one of the gates would always be closed. However, typically, in A-type inactivating K⁺ channels, the activation gate responds to voltage changes faster than the inactivation gate, which results in a transient current upon membrane depolarisation. For the HERG channel, the inactivation gate responds to voltage changes faster than the activation gate, hence, it displays a current that is radically different from A-type K⁺ channels.

The voltage protocols used to identify HERG are designed to display the idiosyncratic currents associated with HERG, which are described as “resurgent” (Robertson, 2000). When the membrane is at depolarised potentials, much of the HERG channel population is in an inactivated state in which current does not flow through the channel; in the process of the membrane returning to a repolarised state, the HERG channels recover from inactivation and the channels linger in a highly stable open state before closing. The result of this transition is that the channels can transiently conduct more current as the membrane potential moves toward E_K (see Fig. 2A), a process that would otherwise be thought to reduce K⁺ current magnitude, due to the reduced electromotive driving force for outward K⁺ ion movement.

The cells that are used for these electrophysiological measurements can be those that naturally have HERG current in them (‘native’ I_Kr currents, presumably mediated by HERG-encoded channels), such as isolated ventricular cardiomyocytes (Jurkiewicz & Sanguinetti, 1993), atrial cell lines (e.g., mouse AT-1 cells; Busch et al., 1998), or neuroblastoma cell lines (e.g., SH-SY5Y cells; Arcangeli et al., 1998), but the majority of studies employ cells that have the HERG channel introduced into them using molecular biological methods, a process known as heterologous expression. One advantage of heterologous expression is that one can choose what cell type to add the HERG channel current to, and such investigations invariably choose cell types that are much less electrically active than the cells that have native HERG. This means that the cells that have heterologous HERG are virtually without other contaminating currents that will be evident during the electrophysiological examination, while cells with native HERG often have other current types that must be excluded pharmacologically, ionically, or by other electrophysiological means. This is of great value in the mechanistic investigation of drug–channel interactions, and is one reason why heterologous HERG expression is increasingly favoured over I_Kr from cardiomyocytes during drug screening. Also, native I_Kr is typically of comparatively small amplitude, and signal-to-noise levels are typically better in the heterologous systems used because heterologous systems allow for high levels of the channel gene to be expressed. The most important advantage that heterologous systems offer over cells with native HERG is that mutated versions of the HERG channel may be compared with the unmutated (wild type) version of the channel in the heterologous systems. The examination of the mutated channels allows for the examination of the molecular determinants of channel blockade, as well as for the investigation of the role of functional domains within the channel in state-dependent blockade (e.g., Milnes et al., 2003, Mitcheson et al., 2000).

While a complete discussion of the methods for heterologous expression and electrophysiology would be too long and outside the scope of a review such as this, good reviews can be found for the use of Xenopus oocytes (Wagner, Friedrich, Setiawan, Lang, & Broer, 2000), patch clamp (Cahalan & Neher, 1992), transfection (Schenborn, 2000), and mammalian cell line care (Pollard, 1997). This review will focus specifically on issues relating to the experimentation on the pharmacology of HERG, concentrating on the options that are used most commonly, and will discuss approaches for troubleshooting the problems that our laboratories have observed in this endeavour.