Analysis of the interactions between the C-terminal cytoplasmic domains of KCNQ1 and KCNE1 channel subunits

Abstract

Ion channel subunits encoded by KCNQ1 and KCNE1 produce the slowly activating K+ current (IKs) that plays a central role in myocardial repolarization. The KCNQ1 alpha-subunit and the KCNE1 beta-subunit assemble with their membrane-spanning segments interacting, resulting in transformation of channel activation kinetics. We recently reported a functional interaction involving C-terminal portions of the two subunits with ensuing regulation of channel deactivation. In the present study, we provide evidence characterizing a physical interaction between the KCNQ1-CT (KCNE1 C-terminus) and the KCNE1-CT (KCNE1 C-terminus). When expressed in cultured cells, the KCNE1-CT co-localized with KCNQ1, co-immunoprecipitated with KCNQ1 and perturbed deactivation kinetics of the KCNQ1 currents. Purified KCNQ1-CT and KCNE1-CT physically interacted in pull-down experiments, indicating a direct association. Deletion analysis of KCNQ1-CT indicated that the KCNE1-CT binds to a KCNQ1 region just after the last transmembrane segment, but N-terminal to the tetramerization domain. SPR (surface plasmon resonance) corroborated the pull-down results, showing that the most proximal region (KCNQ1 amino acids 349-438) contributed most to the bimolecular interaction with a dissociation constant of approximately 4 microM. LQT (long QT) mutants of the KCNE1-CT, D76N and W87F, retained binding to the KCNQ1-CT with comparable affinity, indicating that these disease-causing mutations do not alter channel behaviour by disruption of the association. Several LQT mutations involving the KCNQ1-CT, however, showed various effects on KCNQ1/KCNE1 association. Our results indicate that the KCNQ1-CT and the KCNE1-CT comprise an independent interaction domain that may play a role in IKs channel regulation that is potentially affected in some LQTS (LQT syndrome) mutations.

Figure 1. KCNQ and KCNE family K channel subunits

(A) Sequence alignment of KCNE1 cytoplasmic domains with C-termini of the other KCNE family members. (B) Alignments of the partial amino acid sequences of KCNQ1 C-terminal region containing helix A compared to other KCNQ family members. (C) Schematic representation of recombinant constructs use in this study. Q1Cf refers to the full-length KCNQ1-C-terminus and Q1C1, Q1C2, Q1C3, Q1C1A, Q1C1B each refer to truncated Versions as illustrated. E1C refers to the KCNE1-C-terminus. Boldface residues are those subjected to mutagenesis. All recombinant proteins were expressed in E. coli BL21 (DE3) pLsS strains, and purified with a Ni-affinity column followed by Superdex 200 gel filtration chromatography. (Numbers indicate amino acid position; black and shaded boxes represent α-helical segments)

Figure 2. Functional interaction of free-KCNE1-CT with KCNQ1 channels

(A) Schematic Diagram of KCNQ1 and KCNE1 channel subunits with cylinders representing predicted α-helices. (B) Voltage clamp current traces (elicited by depolarizing voltage steps) of KCNQ1 alone, KCNQ1/KCNE1-CT, KCNQ1/KCNE1 and KCNQ1/KCNE1/KCNE1-CT channels expressed in CHO cells. (C) Deactivation rates plotted against voltage from CHO cells expressing KCNQ1 either without KCNE1, or with KCNE1, KCNE1-CT, or with both KCNE1 and KCNE1-CT. (D) Normalized voltage-dependence of activation curves from same cells as in panel C.

Figure 3

Co-immunoprecipitation of the C-terminal cytoplasmic regions of KCNQ1 and KCNE1 and co-localization of KCNE1 C-terminus and KCNQ1 in the surface of live HEK cells. (A) Immunoblot analysis from HEK 293 cells co-transfected with E1C (lane 2), Q1Cf (lane 3), or both (lanes 4–6). E1C was co-immunoprecipitated with Q1Cf by anti-KCNQ1 antibody (Santa Cruz) (lane 5). Lane 4 shows results from nonspecific antibody IP and lane 6 shows input lysate prior to IP demonstrating expression levels. (B) HEK 293 cell line stably expressing full-length KCNQ1 channels (top row of micrographs) or HEK 293 cells (bottom row of micrographs) were transfected with Flag-tagged KCNE1 C-terminus expression plasmids, and the subcellular distribution of the KCNE1 C-terminus was detected by immunofluorescence using confocal microscopy. KCNE1-CT staining is shown in green, KCNQ1 in red and cadherin (surface) in blue. (C) Graphical analysis of signal strength across co-transfected cells (indicated by solid white line in right panels of 3B) for KCNQ1 plus KCNE1-CT (on left) and KCNe1-CT without KCNQ1 (on right).

Figure 4

Association of purified, recombinant C-terminal cytoplasmic regions of KCNQ1-KCNE1. (A) Coomassie-stained SDS-PAGE of purified recombinant KCNQ1-C-terminal protein fragments, Q1C1, Q1C2, Q1C3, and Q1Cf (1–4). (B) Coomassie-stained SDS-PAGE of purified recombinant proteins, Q1C1, Q1C1B and Q1C1A (1–3). (C) Coomassie-stained SDS-PAGE of purified recombinant protein, KCNE1 (1^st lane) and KCNE1 C-terminus (2^nd lane). (D) Immunoblot of Co-IP analysis of the interaction between the full length C-termini of KCNQ1 and KCNE1. Reactions included KCNQ1-Cf (all lanes) and KCNE1-CT (lanes 2–4). Anti-KCNQ1 antibody was used for IP in lanes 1 & 2, control antibody in lane 3 and protein-G beads alone in lane 4 for controls. Immunoblots for FLAG-tagged KCNE1-CT and KCNQ1-Cf show that only IP of KCNQ1-Cf co-precipitated KCNE1-CT (arrows indicate KCNQ1-Cf and KCNE1-CT).

Figure 5

Interaction of KCNQ1 C-terminal cytoplasmic domains with KCNE1 C-terminus. (A) Coomassie-stained SDS-PAGE of purified MBP-fusion proteins, MBP- Q1Cf, MBP-Q1C1, MBP-Q1C2, and MBP-Q1C3. (B) Immunoblot analysis of association of KCNE1 C-terminus with KCNQ1-Cf. Pulldown of MBP-Q1Cf with immobilized amylose co-precipitated KCNE1-CT (lanes 1 & 3) however MBP alone did not pull down KCNE1-CT (lane 4). (C) Immunoblot analysis of MBP-pulldown reaction demonstrates KCNQ1-C-terminal fragments Q1Cf, Q1C2 and Q1C1 each co-precipitate KCNE1-CT but not KCNQ1-C3. (D) Immunoblot analysis of MBP-pulldown reaction demonstrates KCNQ1-C-terminal fragments Q1C1, Q1C1B and Q1C1A each co-precipitate KCNE1-CT.

Figure 6

Surface plasmon resonance analysis of the interactions of KCNQ1 C-terminal cytoplasmic domains with KCNE1 C-Terminus. SPR analysis was carried out as described in Materials and Methods. (A) Illustrates a typical sensorgram for the interaction of various concentrations of the Q1C1 fragment of KCNQ1 with E1C immobilized on a CI sensor chip. Each trace represents the response to increasing concentrations of Q1C1. Introduction of each concentration of Q1C1 is started at time=0 and washout at 750 seconds. (The concentration ranges used were 0.5–10 μM for KCNQ1 C-terminal fragments.) (B) A typical plot of sensorgram data is show for a Q1C1-E1C interaction experiment. The data were fitted separately for each experiment to the two-state binding model where A + B ↔ AB* ↔ AB, as described in the Methods section. (C) The graph shows maximum response values (RUmax) for the highest concentration (10μM) of various KCNQ1-C-terminal fragments and LQT1 mutants. These data were used to derive Kd calculations shown in Table 1.