Molecular analysis of PIP2 regulation of HERG and IKr.
We previously reported that cloned human ether-a-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 PIP2. The specific binding of radiolabeled PIP2 to both mutant channel proteins was nearly abolished. Stimulation of alpha1A-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 alpha-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 autonomic stimulation and localize a site of interaction to the COOH-terminal tail of the HERG K+ channel.
Bian
Jin-Song
JS
Department of Pharmacology, National University of Singapore.
Kagan
Anna
A
McDonald
Thomas V
TV
D000818:Animals; D002478:Cells, Cultured; D051668:Delayed Rectifier Potassium Channels; D000072237:ERG1 Potassium Channel; D004594:Electrophysiology; D051638:Ether-A-Go-Go Potassium Channels; D017353:Gene Deletion; D006801:Humans; D008297:Male; D009154:Mutation; D032383:Myocytes, Cardiac / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D020033:Protein Isoforms / Q000378:metabolism; D011817:Rabbits; D018340:Receptors, Adrenergic, alpha-1 / Q000378:metabolism; D013997:Time Factors
D051668:Delayed Rectifier Potassium Channels; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; D019269:Phosphatidylinositol 4,5-Diphosphate; D024642:Potassium Channels, Voltage-Gated; D020033:Protein Isoforms; D018340:Receptors, Adrenergic, alpha-1