Molecular analysis of PIP₂ regulation of HERG and I_Kr

We previously reported that cloned human ether-à-go-go-related gene (HERG) K⁺ channels are regulated by changes in phosphatidylinositol 4,5-bisphosphate (PIP₂) concentration. Here we investigated the molecular determinants of PIP₂ interactions with HERG channel protein. To establish the molecular nature of the PIP₂-HERG interaction, we examined a segment of the HERG COOH terminus with a high concentration of positively charged amino acids (nos. 883-894) as a possible site of interaction with negatively charged PIP₂. When we excised deletion-HERG (D-HERG) or mutated methionine-substituted-HERG (M-HERG) this segment of HERG to neutralize the amino acid charge, the mutant channels produced current that was indistinguishable from wild-type HERG. Elevating internal PIP₂, however, no longer accelerated the activation kinetics of the mutant HERG. Moreover, PIP₂-dependent hyperpolarizing shifts in the voltage dependence of activation were abolished with both mutants. PIP₂ effects on channel-inactivation kinetics remained intact, which suggests an uncoupling of inactivation and activation regulation by PIP ₂. The specific binding of radiolabeled PIP₂ to both mutant channel proteins was nearly abolished. Stimulation of α_1AA-adrenergic receptors produced a reduction in current amplitude of the rapidly activating delayed rectifier K⁺ current (the current carried by ERG protein) from rabbit ventricular myocytes. The α-adrenergic-induced current reduction was accentuated by PKC blockers and also unmasked a depolarizing shift in the voltage dependence of activation, which supports the conclusion that receptor activation of PLC results in PIP₂ consumption that alters channel activity. These results support a physiological role for PIP₂ regulation of the rapidly activating delayed rectifier K⁺ current during autonomie stimulation and localize a site of interaction to the COOH-terminal tail of the HERG K ⁺ channel.