Modeling Of The Adrenergic Response Of The Human IKs Current (hKCNQ1/hKCNE1) Stably Expressed In HEK-293 Cells

Adrenergic enhancement of the slowly activating delayed rectifier current (IKs) in cardiac myocytes constitutes a critical “repolarization reserve”. Stable co-expression of human (h)KCNQ1 and hKCNE1 in HEK-293 cells reconstitutes a native-like IKs current (HEK-IKs), allowing s-adrenergic modulation of the current by stimulation of endogenous signalling pathways in the host cell line. HEK-IKs currents were enhanced two- to fourfold by bath application of isoproterenol (EC50 =13 nM), forskolin (10 μM), or 8-(4-chlorophenylthio) adenosine 3′,5′-cyclic monophosphate (50 μM), indicating an intact cAMP dependent ion channel-regulating pathway analogous to that observed in native cardiac myocytes. We made use of the robust modulation of the IKs current to model in detail the effects of adrenergic modulation on IKs gating kinetics. Activation kinetics of HEK-IKs were accurately fit with a novel modified 2nd order Hodgkin-Huxley (H-H) gating model incorporating a fast and a slow gate, each independent of each other in scale and adrenergic response, or a “heterodimer” model. Macroscopically, β-adrenergic enhancement shifted HEK-IKs current activation to more negative potentials and accelerated activation kinetics, while leaving deactivation kinetics relatively unaffected. Modeling of the current in response to 10 μM forskolin indicated that the observed changes in gating could be largely explained by modulation of the opening rate of the fast gate of the H-H model. Rate-dependent accumulation of IKs at high pulsing rates had two phases, an initial staircaselike effect, followed by a slower, incremental accumulation phase. These phases are readily interpreted in the context of a heterodimeric H-H model with two independent gates with differing closing rates. These results indicate the HEK-293 line serves as an attractive host for studies of the effects of pharmacological and genetic manipulations upon the adrenergic modulation of IKs.