title: Properties of HERG channels stably expressed in HEK 293 cells studied at physiological temperature.
abstract: We have established stably transfected HEK 293 cell lines expressing high levels of functional human ether-a go-go-related gene (HERG) channels. We used these cells to study biochemical characteristics of HERG protein, and to study electrophysiological and pharmacological properties of HERG channel current at 35 degrees C. HERG-transfected cells expressed an mRNA band at 4.0 kb. Western blot analysis showed two protein bands (155 and 135 kDa) slightly larger than the predicted molecular mass (127 kDa). Treatment with N-glycosidase F converted both bands to smaller molecular mass, suggesting that both are glycosylated, but at different levels. HERG current activated at voltages positive to -50 mV, maximum current was reached with depolarizing steps to -10 mV, and the current amplitude declined at more positive voltages, similar to HERG channel current expressed in other heterologous systems. Current density at 35 degrees C, compared with 23 degrees C, was increased by more than twofold to a maximum of 53.4 +/- 6.5 pA/pF. Activation, inactivation, recovery from inactivation, and deactivation kinetics were rapid at 35 degrees C, and more closely resemble values reported for the rapidly activating delayed rectifier K+ current (I(Kr)) at physiological temperatures. HERG channels were highly selective for K+. When we used an action potential clamp technique, HERG current activation began shortly after the upstroke of the action potential waveform. HERG current increased during repolarization to reach a maximum amplitude during phases 2 and 3 of the cardiac action potential. HERG contributed current throughout the return of the membrane to the resting potential, and deactivation of HERG current could participate in phase 4 depolarization. HERG current was blocked by low concentrations of E-4031 (IC50 7.7 nM), a value close to that reported for I(Kr) in native cardiac myocytes. Our data support the postulate that HERG encodes a major constituent of I(Kr) and suggest that at physiological temperatures HERG contributes current throughout most of the action potential and into the postrepolarization period.
authors.affiliation: Department of Medicine (Cardiology), University of Wisconsin, Madison 53792, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Zhou
authors.forename: Q
authors.initials: Q
authors.lastname: Gong
authors.forename: B
authors.initials: B
authors.lastname: Ye
authors.forename: Z
authors.initials: Z
authors.lastname: Fan
authors.forename: J C
authors.initials: JC
authors.lastname: Makielski
authors.forename: G A
authors.initials: GA
authors.lastname: Robertson
authors.forename: C T
authors.initials: CT
authors.lastname: January
mesh_terms: D000200:Action Potentials / Q000502:physiology*; D000889:Anti-Arrhythmia Agents / Q000494:pharmacology; D027682:Cation Transport Proteins; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D004268:DNA-Binding Proteins; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; D006801:Humans; D007668:Kidney; D007700:Kinetics; D008564:Membrane Potentials / Q000187:drug effects; D018408:Patch-Clamp Techniques; D010880:Piperidines / Q000494:pharmacology; D015221:Potassium Channels / Q000096:biosynthesis / Q000187:drug effects / Q000502:physiology*; D024642:Potassium Channels, Voltage-Gated; D011725:Pyridines / Q000494:pharmacology; D011994:Recombinant Proteins / Q000096:biosynthesis; D013696:Temperature; D013997:Time Factors; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; D014162:Transfection
chemical_list: D000889:Anti-Arrhythmia Agents; D027682:Cation Transport Proteins; D004268:DNA-Binding Proteins; C072312:ERG protein, human; D000072237:ERG1 Potassium Channel; D051638:Ether-A-Go-Go Potassium Channels; C000606913:KCNH2 protein, human; C092509:KCNH6 protein, human; D010880:Piperidines; D015221:Potassium Channels; D024642:Potassium Channels, Voltage-Gated; D011725:Pyridines; D011994:Recombinant Proteins; D015534:Trans-Activators; D000071230:Transcriptional Regulator ERG; C063968:E 4031