Disease-linked mutations alter the stoichiometries of HCN-KCNE2 complexes

Abstract

The four hyperpolarization-activated cylic-nucleotide gated (HCN) channel isoforms and their auxiliary subunit KCNE2 are important in the regulation of peripheral and central neuronal firing and the heartbeat. Disruption of their normal function has been implicated in cardiac arrhythmias, peripheral pain, and epilepsy. However, molecular details of the HCN-KCNE2 complexes are unknown. Using single-molecule subunit counting, we determined that the number of KCNE2 subunits in complex with the pore-forming subunits of human HCN channels differs with each HCN isoform and is dynamic with respect to concentration. These interactions can be altered by KCNE2 gene-variants with functional implications. The results provide an additional consideration necessary to understand heart rhythm, pain, and epileptic disorders.

Figure 1

PIF automated subunit analysis of HCN-KCNE2 complexes. (A) Spots of interest are automatically selected from a user-defined region of interest (outlined in blue) that encloses the cell. (B) (top) The fluorescence intensities of three spots are shown as examples. (Bottom) Following the filtering and step detection algorithms, traces are idealized, assessed against 5 quality control criteria, are accepted/rejected, and then steps are counted. Distributions of KCNE2-msfGFP in complex with HCN1 (C), HCN2 (D), HCN3 (E) or HCN4 (F) expressed in CHO-K1 cells were fit to a “Poisson distribution of a binomial distribution” function of n^th order (1 ≤ n ≤ 4 KCNE2 subunits) (Eq. 1) that accounts for the maturation of msfGFP (0.55), the number of unique channel complexes, complexes containing only KCNE2, and complexes that overlap on the same spot. KCNE2 in complex with HCN1 and HCN3 channels are best fit with an 2^nd order function (2 KNCE2’s per complex), while HCN2 and HCN4 are best fit with a 3^rd order function (3 KCNE2’s per complex). This distribution did, however, have a large residual between the fits and the number of spots observed containing >4 photobleaching steps (insets of C&E). Colocalization parameters (p_col) for the best fits where 0.41, 0.17, 0.37, and 0.17 for HCN1, HCN2, HCN3, and HCN4 respectively.

Figure 2

Photobleaching steps analysis using a linear superposition of distributions function. Step distributions of KCNE2-msfGFP in complex with HCN1 (A), HCN2 (B), HCN3 (C) or HCN4 (D) expressed in CHO-K1 cells were fit to a using a linear superposition of distributions that permitted a mix of complexes containing 1–4 KCNE2 subunits per HCN tetramer. The fraction of complexes for n = 1–4 KCNE2 subunits (blue circles) is also shown. (E) The affinity parameter from each fit is plotted for each HCN isoform. (F) We observe a concentration dependence of the 4:4 complex as we increase the ratio of HCN4:KCNE2 transfected into CHO-K1 cells.

Figure 3

Effects of KCNE2 gene variants on HCN channel function determined by whole-cell patch clamp recordings. (A) Current-Voltage relationships (B) Steady-state activation curves and (C) voltage dependencies of activation and deactivation kinetics for HCN1 alone (open square; n = 10), HCN1 + WT KCNE2 (closed squares; n = 10), HCN1 + Q9E KCNE2 (green triangles; n = 8), and HCN1 + V14I (blue circles; n = 6). Similarly, (D) Current-Voltage relationships (E) Steady-state activation curves and (F) voltage dependencies of activation and deactivation kinetics for HCN2 alone (open square; n = 9), HCN2 + WT KCNE2 (closed squares; n = 10), HCN2 + Q9E KCNE2 (green triangles; n = 8), and HCN2 + V14I (blue circles; n = 9) were determined. Also, (G) Current-Voltage relationships (H) Steady-state activation curves and (I) voltage dependencies of activation and deactivation kinetics for HCN4 alone (open square; n = 8), HCN4 + WT KCNE2 (closed squares; n = 5), HCN4 + Q9E KCNE2 (green triangles; n = 5), and HCN4 + V14I (blue circles; n = 6).

Figure 4

The effects of KCNE2 gene variants on HCN-KCNE2 stoichiometry. The binding affinity parameters (left) and fraction of 4:n HCN:KCNE2 complexes(right) we calculated from step distributions of KCNE2-msfGFP gene variants Q9E, V14I, and M54T in complex with HCN1 (A), HCN2 (B), HCN3 (C) or HCN4 (D). It is evident that these disease-linked KCNE2 mutations alter the stoichiometries within the HCN-KCNE2 complexes.