Functional characterization of the C‐terminus of the human ether‐à‐go‐go‐related gene K+ channel (HERG)

The human eag (ether-a-go-go)-related gene (HERG) encodes a component of cardiac delayed rectifier potassium current IKr (Sanguinetti et al. 1995; Trudeau et al. 1995) and is critical for the maintenance of normal rhythmicity in the human heart (Curran et al. 1995). IKr mediates the terminal repolarization of the ventricular action potential (Sanguinetti & Jurkewicz, 1990), and the loss of this current due to inherited mutations in HERG or drug block leads to a prolongation of the action potential and an associated susceptibility to arrhythmia. Being a member of the eag family of voltage-gated K+ channels, HERG shares the structural characteristics of this family (Warmke & Ganetzky, 1994). Overall sequence similarity between Eag- and Shaker-type channels is quite low except for the pore (H5) region. A much higher degree of homology is found between eag-type channels and plant inwardly rectifying K+ channels (AKT1 and KAT1) (Anderson et al. 1992; Sentenac et al. 1992), as well as cyclic nucleotide-gated (CNG) channels. In addition, eag channels have a region of unknown function within the C-terminus that is homologous to the cyclic nucleotide-binding domain (CNBD) of CNG channels. It is presumed that eag-type channels are tetramers like members of the CNG family of ion channels (Kaupp et al. 1989; Liu et al. 1996). Ludwig et al. (1997) have identified a C-terminal domain (CAD, residues 897-937) that is important for rat eag (r-eag) subunit interactions. Similarly, Daram et al. (1997) have identified two regions in the AKT1 channel that are important for subunit interaction. The first of these regions is located in the putative CNBD of the channel and interacts with the region between amino acid residues 371 and 516. The second region is between the last 81 amino acid residues of the protein and a region lying between the channel hydrophobic core and the putative CNBD. The functional role of the C-terminus of HERG and other members of the eag family of channels is poorly understood. Studies of other voltage-gated K+ channels have demonstrated roles for the C-terminus in channel gating. For example, the N- and C-termini act in concert to mediate inactivation in mKv4.1 channels (mouse Shal) by a mechanism distinct from the ‘ball-and-chain’ mechanism first described for Shaker channels (Hoshi et al. 1991; Demo & Yellen, 1991; Perozo et al. 1992; Jerng & Covarrubias, 1997). In rat drk-1 channels, deletions of the C-terminus cause a hyperpolarizing shift of the voltage dependence of activation, but normal activation is either restored or compensated for by the additional deletion of the N-terminus (VanDongen et al. 1990). Miller & Aldrich (1996) showed that inward rectification could be induced in Shaker K+ channels by mutations that shift activation to more negative voltage ranges. This increases the inactivation at resting potentials and, when the membrane potential is stepped to more negative potentials, causes HERG-like tail currents that first recover from inactivation prior to deactivating. Mutations causing a more extreme shift of activation result in currents that recover from inactivation but do not deactivate, producing maintained inward currents like those of KAT-1 inward rectifier currents from plants. The authors suggested on the basis of these mutant phenotypes that the voltage-dependent ‘activation’ of KAT-1 channels is actually the recovery from inactivation as the channels enter the open state. Marten & Hoshi (1997) showed that the voltage dependence of this process is altered by deletions of the extreme C-terminus. Thus, sequences in the C-terminus may be important in regulating inactivation in KAT-1. In the present study, C-terminal deletions of HERG have been constructed in order to: (i) delineate the minimum amount of C-terminus necessary to form a functional ion channel, (ii) investigate the possible contribution of the CNBD to channel function and (iii) identify other regions in the C-terminus of HERG that may contribute to HERG channel function. Additionally, deletions in the C-terminus were coupled with N-terminal deletions to distinguish between N- and C-terminal effects that are cooperative or independent.