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The low-dielectric plasma membrane provides an energy barrier hindering transmembrane movement of charged particles. The positively charged, voltage-sensing fourth transmembrane domain (S4) of voltage-gated ion channels must surmount this energy barrier to initiate channel activation, typically necessitating both membrane depolarization and interaction with membrane lipid phospho-head groups (MLPHGs). In contrast, and despite containing S4, the KCNQ1 K+ channel α subunit exhibits predominantly constitutive activation when in complexes with transmembrane β subunits, MinK-related peptide (MiRP) 1 (KCNE2) or MiRP2 (KCNE3). Here, using a 2-electrode voltage clamp and scanning mutagenesis of channels heterologously expressed in Xenopus laevis oocytes, we discovered that 2 of the 8 MiRP2 extracellular domain acidic residues (D54 and D55) are important for KCNQ1-MiRP2 constitutive activation. Double-mutant thermodynamic cycle analysis revealed energetic coupling of D54 and D55 to R237 in KCNQ1 S4 but not to 10 other native or introduced polar residues in KCNQ1 S4 and surrounding linkers. MiRP2-D54 and KCNQ1-R237 also similarly dictated susceptibility to the inhibitory effects of MLPHG hydrolysis, whereas other closely situated polar residues did not. Thus, by providing negative charge near the plasma membrane extracellular face, MiRP2 uses a lipomimetic mechanism to constitutively stabilize the activated KCNQ1 voltage sensor. [ABSTRACT FROM AUTHOR]

MinK-related peptide 2 (MiRP2) and Kv3.4 subunits assemble in skeletal muscle to create subthreshold, voltage-gated potassium channels. MiRP2 acts on Kv3.4 to shift the voltage dependence of activation, speed recovery from inactivation, suppress cumulative inactivation and increase unitary conductance. We previously found an R83H missense mutation in MiRP2 that segregated with periodic paralysis in two families and diminished the effects of MiRP2 on Kv3.4. Here we show that MiRP2 has a single, functional PKC phosphorylation site at serine 82 and that normal MiRP2-Kv3.4 function requires phosphorylation of the site. The R83H variant does not prevent PKC phosphorylation of neighboring S82; rather, the change shifts the voltage dependence of activation and endows MiRP2-Kv3.4 channels with sensitivity to changes in intracellular pH across the physiological range. Thus, current passed by single R83H channels decreases as internal pH is lowered (pKa ∼7.3, consistent with histidine protonation) whereas wild-type channels are largely insensitive. These findings identify a key regulatory domain in MiRP2 and suggest a mechanistic link between acidosis and episodes of periodic paralysis. [ABSTRACT FROM AUTHOR]