| The cardiac delayed rectifier K+ current (IKS) is carried by a complex of KCNQ1 (Q1) subunits, containing the voltage-sensing domains and the pore, and auxiliary KCNE1 (E1 subunits, required for the characteristic IKS voltage dependence and kinetics. To locate the transmembrane helix of E1 (E1TM) relative to the Q1 TM helices (S1-S6), we mutated, one at a time, the first four residues flanking the extracellular ends of S1-S6 and El-TM to Cys, co-expressed all combinations of Q1 and E1 Cys-substituted mutants in CHO cells, and determined the extents of spontaneous disulfide bond formation. Cys flanking E1-TM readily formed disulfides with Cys flanking S1 and S6, much less so with the S3-S4 linker, and not at all with S2 or S5. These results imply that the extracellular flank of the E1-TM is located between S1 and S6 from two diametrically opposed Q1 subunits. The function of two crosslinking pairs from S1 to E1 altered deactivation: a disulfide from S1 I145C to E1 K41C strongly slowed deactivation, and one from S1 I145C to E1 L42C sped deactivation. Crosslinking from S6 to E1 altered activation and steady-state channel equilibrium: a disulfide from S6 V324C to E1 K41C slowed activation and increased the V1/2, and one from S6 V324C to E1 L42C locked the channel in the open state. Furthermore, a rotational orientation where El K41C crosslinks to S1 and E1 L42C crosslinks to S6 favors the open state while the approximately opposite orientation favors the closed state.;Sympathetic nervous system stimulation enhances IKS, resulting in shortening of the action potential duration. Such regulation requires assembly of a macromolecular signaling complex composed of Q1, E1, the A-kinase anchoring protein (AKAP) Yotiao, PKA, and PP1. Yotiao, like other AKAPs, binds the RII regulatory subunit of PKA (PKARII) through a well conserved amphipathic alpha helix. We present the isolation of two PKARII binding sites on Yotiao. Mutation of both sites ablates binding of PKARII and eliminates the functional effect of cAMP in the regulation of the IKS channel. |
