Understanding the interactions that occur between KCNE1 and KCNQ1 : stoichiometry, gating and dynamic movements

The IKs current has an important role in repolarizing the cardiac action potential. KCNQ1 subunits form a tetrameric voltage-gated potassium channel, with which an accessory beta-subunit, KCNE1, can interact. KCNE1 binding to KCNQ1 remarkably alters channel kinetics by delaying activation, increasing current density and removing inactivation. There has always been confusion about how many KCNE1 subunits can associate with the KCNQ1 tetramer. Several groups have reported a strict fixed stoichiometry of two KCNE1 subunits to four KCNQ1 subunits. However, others have shown that the ratio varies depending on the concentration of KCNE1 subunits available. Using whole cell and single channel patch clamp recordings of tethered fusion constructs with different ratios of KCNE1:KCNQ1, as well as photo-crosslinking experiments, we show that up to four KCNE1 subunits can associate with the complex. Therefore, in vitro, IKs can have a variable stoichiometry. In further photo-crosslinking experiments we show that two adjacent residues in KCNE1 interact with KCNQ1 in different channel states, open and closed. Additionally, we show that these interactions are likely taking place with the pore domain of the channel. We also confirm what other groups had proposed, that KCNE1 moves within the cleft of KCNQ1 during channel activation, since the rates of crosslinking change when the channels are held at more depolarized holding potentials. Finally, we investigate whether or not all four voltage sensors have to activate and the complex undergo a concerted step in order for the pore to conduct current. We employ a mutation, E160R, which applies electrostatic repulsion to the positive charges of the voltage sensor and keeps it in a resting conformation. By locking one, two, three and four voltage sensors down we show, via whole cell and single channel recordings, that the channel can conduct when only one voltage sensor is free to move. From these experiments, we provide additional evidence that the IKs channel gates in an allosteric fashion, where each additional voltage sensor movement results in a progressively higher open probability. Additionally, we propose that there is cooperativity between KCNQ1 subunits, where movement of one voltage sensor facilitates movement of a neighboring voltage sensor.Medicine, Faculty ofAnesthesiology, Pharmacology and Therapeutics, Department ofGraduat