Drug-induced block of the cardiac hERG (human Ether-à-go-go-Related Gene) potassium channel delays cardiac repolarization and increases the risk of Torsade de Pointes (TdP), a potentially lethal arrhythmia. A positive hERG assay has been embraced by regulators as a non-clinical predictor of TdP despite a discordance of about 30%. To test whether assaying concomitant block of multiple ion channels (Multiple Ion Channel Effects or MICE) improves predictivity we measured the concentration-responses of hERG, Nav1.5 and Cav1.2 currents for 32 torsadogenic and 23 non-torsadogenic drugs from multiple classes. We used automated gigaseal patch clamp instruments to provide higher throughput along with accuracy and reproducibility. Logistic regression models using the MICE assay showed a significant reduction in false positives (Type 1 errors) and false negatives (Type 2 errors) when compared to the hERG assay. The best MICE model only required a comparison of the blocking potencies between hERG and Cav1.2.

Small-conductance calcium-activated potassium channels (SK channels) are gated solely by intracellular calcium ions and their activity is responsible for the slow afterhyperpolarization (AHP) that follows an action potential in many excitable cells. Brain slice studies commonly employ a methyl derivative of bicuculline (bicuculline-m), a GABAA (gamma-aminobutyric acid) receptor antagonist, to diminish the tonic inhibitory influences of GABAergic synapses, or to investigate the role of these synapses in specialized neural networks. However, recent evidence suggests that bicuculline-m may not be specific for GABAA receptors and may also block the slow AHP. Therefore, the effects of bicuculline-m on cloned apamin-sensitive SK2 and apamin-insensitive SK1 channels were examined following expression in Xenopus oocytes. The results show that at concentrations employed for slice recordings, bicuculline-m potently blocks both apamin-sensitive SK2 currents and apamin-insensitive SK1 currents when applied to outside-out patches. Apamin-insensitive SK1 currents run down in excised patches. The potency of bicuculline-m block also decreases with time after patch excision. Site-directed mutagenesis that changes two residues in the outer vestibule of the SK1 pore that confers apamin sensitivity also reduces run down of the current in patches, and endows stable sensitivity to bicuculline-m indistinguishable from SK2. Therefore, the use of bicuculline-m in slice recordings may mask apamin-sensitive slow AHPs that are important determinants of neuronal excitability. In addition, bicuculline-m-insensitive slow AHPs may indicate that the underlying channels have run down.

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Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels are activated by hyperpolarizations that cause inward movements of the positive charges in the fourth transmembrane domain (S4), which triggers channel opening. If HCN channels are held open for prolonged times (Ͼ50 ms), HCN channels undergo a mode shift, which in sea urchin (spHCN) channels induces a Ͼ50 mV shift in the midpoint of activation. The mechanism underlying the mode shift is unknown. The mode shift could be attributable to conformational changes in the pore domain that stabilize the open state of the channel, which would indirectly shift the voltage dependence of the channel, or attributable to conformational changes in the voltage-sensing domain that stabilize the inward position of S4, thereby directly shifting the voltage dependence of the channel. We used voltage-clamp fluorometry to detect S4 movements and to correlate S4 movements to the different activation steps in spHCN channels. We here show that fluorophores attached to S4 report on fluorescence changes during the mode shift, demonstrating that the mode shift is not simply attributable to a stabilization of the pore domain but that S4 undergoes conformational changes during the mode shift. We propose a model in which the mode shift is attributable to a slow, lateral movement in S4 that is triggered by the initial S4 gating-charge movement and channel opening. The mode shift gives rise to a short-term, activitydependent memory in HCN channels, which has been shown previously to be important for the stable rhythmic firing of pacemaking neurons and could significantly affect synaptic integration.