MinK-KvLQT1 fusion proteins, evidence for multiple stoichiometries of the assembled I-sK channel

I-sK, a slowly activating delayed rectifier K+ current through channels for med by the assembly of two channel proteins KvLQT1 and MinK, modulates the repolarization of cardiac action potentials, Mutations that map to the KvLQ T1 and minK genes account for more than 50% of an inherited cardiac disorde r, the Long QT syndrome (Splawski, I., Tristani-Firouzi, Ra,, Lehmann, M. H ., Sanguinetti, M. C., and Keating, M. T. (1997) Nat. Genet. 17, 338-340), Despite the importance of these channels to human cardiac function, the mol ecular basis of their uniquely slow gating properties as well as the stoich iometry and interaction sites of these two subunits are still unclear. We h ave constructed several fusion channel proteins to begin investigating the stoichiometry of these two subunits and the role of voltage-dependent subun it assembly in channel gating, Functional properties of these constructs we re measured using whole cell patch clamp recordings of transiently transfec ted Chinese hamster ovary cells, The constructs we tested are as follows: M K24 (C terminus of MinK linked to N terminus of KvLQT1); KK40 (a tandem hom odimer of KvLQT1); and MKK44 (C terminus of MinK linked to N terminus of KK 40), In control experiments (no DNA, control DNA, or only MinK), no ti:me-d ependent K+ current was observed. Expression of KvLQT1 or KK40 produced cur rents that activate and inactivate in a voltage-dependent manner as reporte d by others for KvLQT1. In contrast, expression of MK24 and MKK44 elicited current with activation kinetics and voltage dependence very similar to nat ive I,,and identical to currents expressed by cells co-transfected with ind ependent MinK and KvLQT1 cDNA, Expression of MK24 plus additional MinK sign ificantly slows current kinetics. Our data raise the possibility 1) of mult iple MinK/KvLQT1 stoichiometries and 2) indicate that uniquely slow kinetic s of I-sK channels is due to voltage-dependent conformational changes of th e channel protein and not to assembly of channel subunits.