Homomeric and heteromeric assembly of KCNQ (Kv7) K+ channels assayed by total internal reflection fluorescence/fluorescence resonance energy transfer and patch clamp analysis

Abstract

M-type K(+) channels, consisting of KCNQ1-5 (Kv7.1-7.5) subunits, form a variety of homomeric and heteromeric channels. Whereas all the subunits can assemble into homomeric channels, the ability of the subunits to assemble into heteromultimers is highly variable. KCNQ3 is widely thought to co-assemble with several other KCNQ subtypes, whereas KCNQ1 and KCNQ2 do not. However, the existence of other subunit assemblies is not well studied. To systematically explore the heteromeric assembly of KCNQ channels in individual living cells, we performed fluorescence resonance energy transfer (FRET) between cyan fluorescent protein- and yellow fluorescent protein-tagged KCNQ subunits expressed in Chinese hamster ovary cells under total internal reflection fluorescence microscopy in which excitation light only penetrates several hundred nanometers into the cell, thus isolating membrane events. We found significant FRET between homomeric subunits as expected from their functional expression in heterologous expression systems. Also as expected from previous work, robust FRET was observed between KCNQ2 and KCNQ3. KCNQ3 and KCNQ4 also showed substantial FRET as did KCNQ4 and KCNQ5. To determine functional assembly of KCNQ4/KCNQ5 heteromers, we performed two types of experiments. In the first, we constructed a mutant tetraethylammonium ion-sensitive KCNQ4 subunit and tested its assembly with KCNQ5 by patch clamp analysis of the tetraethylammonium ion sensitivity of the resulting current; however, those data were not conclusive. In the second, we co-expressed a KCNQ4 (G285S) pore mutant with KCNQ5 and found the former to act as a dominant negative, suggesting co-assembly of the two types of subunits. These data confirm that among the allowed assembly conformations are KCNQ3/4 and KCNQ4/5 heteromers.

FIGURE 1.

Positive and negative controls for FRET under TIRF microscopy. A, images of CHO cells expressing CFP-tagged KCNQ2 and YFP-tagged ENaC. Panels show CFP (left) and YFP (right) emissions before and after selective photobleaching of YFP. Note the lack of increase in CFP emission after YFP photobleaching, indicating minimal FRET. B, CHO cells expressing a membrane-targeted tandem construct of CFP and YFP (Rho-pYC) before and after selective photobleaching of YFP. Panels show CFP (left) and YFP (right) emissions before and after selective photobleaching of YFP. The strong increase in CFP emission after YFP photobleaching indicates robust FRET. C, bars show summarized FRET efficiency data calculated as the percent increase in CFP emission after YFP photobleaching. Error bars show standard errors.

FIGURE 2.

Homomeric interaction between KCNQ1–5 subunits assayed by TIRF/FRET. CHO cells were co-transfected with homomeric CFP- and YFP-tagged KCNQ1 (A), KCNQ2 (B), KCNQ3 (C), KCNQ4 (D), or KCNQ5 (E) subunits. Panels show CFP (left) and YFP (right) emissions before and after selective photobleaching of YFP. F, bars show summarized FRET efficiency data calculated as the percent increase in CFP emission after YFP photobleaching. Error bars show standard errors.

FIGURE 3.

Heteromeric interaction between KCNQ subunits assayed by TIRF/FRET. CHO cells were co-transfected with CFP-tagged KCNQ1 + YFP-tagged KCNQ4 (A), CFP-tagged KCNQ2 + YFP-tagged KCNQ5 (B), KCNQ2-CFP and KCNQ3-YFP (C), KCNQ3-CFP and KCNQ4-YFP (D), or CFP-tagged KCNQ4 and YFP-tagged KCNQ5 (E). The panels show CFP and YFP emission images before and after selective photobleaching of YFP. F, bars indicate the summarized FRET efficiency data calculated as the percent increase in CFP emission after YFP photobleaching (^**, p < 0.01; ^***, p < 0.001). Error bars show standard errors.

FIGURE 4.

TEA dose-response relationship for KCNQ4, KCNQ4 (T290Y), and KCNQ5 channels. CHO cells were transfected with the indicated subunits, and the current was studied under perforated patch voltage clamp. A, representative currents from the indicated transfections with zero (control) or the indicated concentrations of TEA in the bath. The voltage protocol used is depicted in the inset. B, plotted are the summarized data of the percent block of the indicated current as a function of [TEA]. The KCNQ current was measured as the amplitude of the holding current at 0 mV. The data for wild-type KCNQ4, KCNQ5, and KCNQ4 (T290Y) were fit by single Hill equations (see “Experimental Procedures”). The data from co-expression of KCNQ4 (T290Y) + KCNQ5 were fit either by a double Hill equation with the K_½ values taken from the fits of the corresponding homomeric currents (dashed line) or by the sum of five Hill equations corresponding to the five populations of channels predicted by the binomial relation with the K_½ for each population calculated according to energy additivity of each subunit (see text) and the K_½ values for KCNQ4 (T290Y) and KCNQ5 taken from the fits of the corresponding homomeric currents (dotted line). For all the fits, except KCNQ4, the minimum and maximum were constrained to be 0 and 100%, respectively, and the Hill coefficient was constrained to be unity.

FIGURE 5.

The KCNQ4 (G285S) pore mutant indicates functional assembly with KCNQ5. CHO cells were transfected with wild-type KCNQ5, KCNQ4 (G285S), wild-type KCNQ4, both wild-type KCNQ5 and KCNQ4 (G285S), or both wild-type KCNQ4 and KCNQ4 (G285S) and studied under perforated patch voltage clamp. A and B, shown are representative currents from the indicated transfections either using a traditional or “classic” M current protocol (depicted in the insets). C, bars show summarized current densities from the five groups of cells. The KCNQ current was measured as the amplitude of the time-dependent relaxation at -60 mV. pF, picofarad.