A shared mechanism for lipid- and beta-subunit-coordinated stabilization of the activated K+ channel voltage sensor

Abstract

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 alpha subunit exhibits predominantly constitutive activation when in complexes with transmembrane beta 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.

Figure 1.

MiRP2 D54 and D55 stabilize the open state of KCNQ1. A) Sequences of the predicted extracellular and transmembrane domains of the human KCNE family, aligned gap-free by their transmembrane domains. MK, MinK; M1–M4, MiRPs 1–4. Numbering is for human M2. Charged residues are highlighted. Green boxes, positioning of α-helices according to NMR structure determination for MK or prediction using the PHD program for M1-M4 . Dashes, before start of methionine. B) Net extracellular domain charge for MinK (MK) and MiRPs 1–4 (M1–M4). C) Cartoons of voltage-dependent activation of homomeric KCNQ1 (left) vs. constitutive activation of MiRP2-KCNQ1 (right), indicating a hypothetical model for stabilization of KCNQ1 S4 in the activated state by some or all of the 8 MiRP2 extracellular domain acidic residues (blue minus symbols). Yellow, plasma membrane. For clarity, S4–S6 from only one KCNQ1 α subunit is shown. D) Exemplar current traces recorded in oocytes expressing KCNQ1 alone (Q1) or with wild-type (wt), EE44,45AA-MiRP2, DD54,55AA-MiRP2, or D54A-MiRP2 (M2) as indicated. Inset: currents were recorded by TEVC, using the standard voltage family protocol. Dashed line indicates 0 current level. E) Mean normalized conductance (G/G_max, measured at arrow in D); channels and symbols as in D. n = 36 (Q1); 44 (+wt M2); 15 (E44,45A); 10 (D54,55A); 13 (D54A). Error bars = se. F) Mean ΔΔG° for the closed to open transition, calculated from macroscopic conductance plots as in E and for other single and multiple MiRP2 (M2) mutants with KCNQ1; n = 8–44. Error bars = se.

Figure 2.

Combined mutagenesis of charged residues in MiRP2 and KCNQ1 S4. A) Exemplar current traces recorded in oocytes coexpressing wild-type (wt) and mutant KCNQ1 and MiRP2 as indicated, recorded by TEVC using the protocol shown. Dashed line indicates 0 current level. B) Mean normalized conductance (G/G_max, measured at arrow in A); for wild-type, D54A-MiRP2 and D55A-MiRP2 with wild-type, D242A-KCNQ1 or R237A-KCNQ1 as indicated; n = 7–44. C) Mean ΔΔG° for the closed to open transition, calculated from tail G/G_max plots as in B and for other KCNQ1 single mutants with wild-type, D54A-MiRP2, and D55A-MiRP2 (M2); n = 7–44.

Figure 3.

Double-mutant thermodynamic cycle analysis for MiRP2 and KCNQ1 S4. A) Exemplar double-mutant thermodynamic cycles for D54A-MiRP2 with D242A-KCNQ1 or R237A-KCNQ1. B) Mean coupling energies (ΔΔG°_coupling) for the closed to open transition, calculated from tail G/G_max plots as in Fig. 2B, using ΔΔG° values from Fig. 2C and double-mutant cycles as in A; n = 7–44. Solid bars indicate values of ΔΔG°_coupling > 1.5 kcal/mol. wt, wild-type.

Figure 4.

R237A-KCNQ1 activation is sensitive to the MiRP2 54,55 side chains. A) Exemplar current traces recorded in oocytes expressing R237A-KCNQ1 with DD54,55EE or NN MiRP2, using the protocol shown. Dashed line indicates 0 current level. B) Mean normalized conductance (measured at arrow in A) for currents generated by R237A-KCNQ1 alone (−) or with the MiRP2 variants shown; n = 7–46. wt, wild-type.

Figure 5.

R237-D54 interaction protects activated KCNQ1 S4 from the effects of membrane sphingomyelin hydrolysis. A) Hydrolysis of sphingomyelin by SMase. B) Cartoon of the K_v channel activated-S4-destabilizing effect of SMase. C) Exemplar traces before application and after washout of SMase for oocytes expressing KCNQ1 with MinK or MiRP2, using the voltage protocol shown. Dashed line indicates 0 current level. D, E) Rundown-normalized mean peak current at 0 mV before and during application and after washout of SMase, normalized to time 0 peak current, for oocytes expressing KCNQ1 and MiRP2 variants as indicated, using the protocol shown in C; n = 4–8/group. Red bar indicates SMase application. F) Hypothetical model indicating feasibility of R237-D54/55 interaction. Open and closed KCNQ1 S4 model coordinates are from ref. ; MiRP2 was modeled (SwissModel) from the NMR structure of full-length MinK . Side chains are shown only for polar residues. MiRP2 Y60 and T80 indicate boundaries of proposed transmembrane domain (Fig. 1A). Inset: K_v2.1 S4 with nearby lipid molecules; coordinates from the crystal structure of K_v1.2/ K_v2.1 paddle chimera in lipid . ext, extracellular; int, intracellular.