1. The human eag-related potassium channel, HERG, gives rise to inwardly rectifying K+ currents when expressed in Xenopus oocytes. 2. The apparent inward rectification is caused by rapid inactivation. In extracellular Cs+ solutions, large outward currents can be recorded having an inactivation time constant at 0 mV of about 50 ms with an e-fold change every 37 mV. 3. HERG channel inactivation is not caused by an amino-terminal ball structure, as a deletion of the cytoplasmic amino terminus (HERGA2-373) did not eliminate inactivation. However, channel deactivation was accelerated about 12-fold at -80 mV. 4. Mutation of S631 to A, the homologous residue of eag channels, in the outer mouth of the HERG pore completely abolished channel inactivation. 5. Activity of HERG channels depended on extracellular cations, which are effective for channel activation, in the order Cs+ > K+ >> Li+ > Na+. The point mutation S631A strongly reduced this channel regulation. 6. By analogy to functional aspects of cloned voltage-gated potassium channels, rectification of HERG, as well as its kinetic properties during the course of an action potential, are presumably governed by a mechanism reminiscent of C-type inactivation.

Intracellular Ca(2+) inhibits voltage-gated potassium channels of the ether à go-go (EAG) family. To identify the underlying molecular mechanism, we expressed the human version hEAG1 in Xenopus oocytes. The channels lost Ca(2+) sensitivity when measured in cell-free membrane patches. However, Ca(2+) sensitivity could be restored by application of recombinant calmodulin (CaM). In the presence of CaM, half inhibition of hEAG1 channels was obtained in 100 nM Ca(2+). Overlay assays using labelled CaM and glutathione S-transferase (GST) fusion fragments of hEAG1 demonstrated direct binding of CaM to a C-terminal domain (hEAG1 amino acids 673-770). Point mutations within this section revealed a novel CaM-binding domain putatively forming an amphipathic helix with both sides being important for binding. The binding of CaM to hEAG1 is, in contrast to Ca(2+)-activated potassium channels, Ca(2+) dependent, with an apparent K(D) of 480 nM. Co-expression experiments of wild-type and mutant channels revealed that the binding of one CaM molecule per channel complex is sufficient for channel inhibition.

Ion selectivity of voltage-activated sodium channels is determined by amino-acid residues in the pore regions of all four homologous repeats. The major determinants are the residues DEKA (for repeats I-IV) which form a putative ring structure in the pore; the homologous structure in Ca-channels consists of EEEE. By combining site-directed mutagenesis of a non-inactivating form of the rat brain sodium channel II with electrophysiological methods, we attempted to quantify the importance of charge, size, and side-chain position of the amino-acid residues within this ring structure on channel properties such as monovalent cation selectivity, single-channel conductance, permeation and selectivity of divalent cations, and channel block by extracellular Ca2+ and tetrodotoxin (TTX). In all mutant channels tested, even those with the same net charge in the ring structure as the wild type, the selectivity for Na+ and Li+ over K+, Rb+, Cs+, and NH4+ was significantly reduced. The changes in charge did not correlate in a simple fashion with the single-channel conductances. Permeation of divalent ions (Ca2+, Ba2+, Sr2+, Mg2+, Mn2+) was introduced by some of the mutations. The IC50 values for the Ca2- block of Na+ currents decreased exponentially with increasing net negative charge of the selectivity ring. The sensitivity towards channel block by TTX was reduced in all investigated mutants. Mutations in repeat IV are an exception as they caused smaller effects on all investigated channel properties compared with the other repeats.

1 The antipsychotic drug haloperidol can induce a marked QT prolongation and polymorphic ventricular arrhythmias. In this study, we expressed several cloned cardiac K + channels, including the human ether-a-go-go related gene (HERG) channels, in Xenopus oocytes and tested them for their haloperidol sensitivity. 2 Haloperidol had only little e ects on the delayed recti®er channels Kv1.1, Kv1.2, Kv1.5 and I sK , the A-type channel Kv1.4 and the inward recti®er channel Kir2.1 (inhibition 56% at 3 mM haloperidol). 3 In contrast, haloperidol blocked HERG channels potently with an IC 50 value of approximately 1 mM. Reduced haloperidol, the primary metabolite of haloperidol, produced a block with an IC 50 value of 2.6 mM. 4 Haloperidol block was use-and voltage-dependent, suggesting that it binds preferentially to either open or inactivated HERG channels. As haloperidol increased the degree and rate of HERG inactivation, binding to inactivated HERG channels is suggested. 5 The channel mutant HERG S631A has been shown to exhibit greatly reduced C-type inactivation which occurs only at potentials greater than 0 mV. Haloperidol block of HERG S631A at 0 mV was four fold weaker than for HERG wild-type channels. Haloperidol a nity for HERG S631A was increased four fold at +40 mV compared to 0 mV. 6 In summary, the data suggest that HERG channel blockade is involved in the arrhythmogenic side e ects of haloperidol. The mechanism of haloperidol block involves binding to inactivated HERG channels.

Human ether à go‐go potassium channels (hEAG1) open in response to membrane depolarization and they are inhibited by Ca2+/calmodulin (CaM), presumably binding to the C‐terminal domain of the channel subunits. Deletion of the cytosolic N‐terminal domain resulted in complete abolition of Ca2+/CaM sensitivity suggesting the existence of further CaM binding sites. A peptide array‐based screen of the entire cytosolic protein of hEAG1 identified three putative CaM‐binding domains, two in the C‐terminus (BD‐C1: 674–683, BD‐C2: 711–721) and one in the N‐terminus (BD‐N: 151–165). Binding of GST‐fusion proteins to Ca2+/CaM was assayed with fluorescence correlation spectroscopy, surface plasmon resonance spectroscopy and precipitation assays. In the presence of Ca2+, BD‐N and BD‐C2 provided dissociation constants in the nanomolar range, BD‐C1 bound with lower affinity. Mutations in the binding domains reduced inhibition of the functional channels by Ca2+/CaM. Employment of CaM‐EF‐hand mutants showed that CaM binding to the N‐ and C‐terminus are primarily dependent on EF‐hand motifs 3 and 4. Hence, closure of EAG channels presumably requires the binding of multiple CaM molecules in a manner more complex than previously assumed.