Structural determinants of M-type KCNQ (Kv7) K+ channel assembly

Abstract

The ability of KCNQ (Kv7) channels to form hetero-oligomers is of high physiological importance, because heteromers of KCNQ3 with KCNQ2 or KCNQ5 underlie the neuronal M-current, which modulates neuronal excitability. In KCNQ channels, we recently identified a C-terminal subunit interaction (si) domain that determines their subunit-specific assembly. Within this si domain, there are two motifs that comprise approximately 30 amino acid residues each and that exhibit a high probability for coiled-coil formation. Transfer of the first or the second coiled-coil (TCC) domain from KCNQ3 into the KCNQ1 scaffold resulted in chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3, both of which coimmunoprecipitated with KCNQ2. However, only KCNQ1(TCC2)Q3 enhanced KCNQ2 currents and surface expression or exerted a strong dominant-negative effect on KCNQ2. Deletion of TCC2 within KCNQ2 yielded functional homomeric channels but prevented the current augmentation measured after coexpression of KCNQ2 and KCNQ3. In contrast, deleting TCC1 within KCNQ2 did not give functional homomeric KCNQ2 or heteromeric KCNQ2/KCNQ3 channels. Mutations that disrupted the predicted coiled-coil structure of TCC1 in KCNQ2 or KCNQ3 abolished channel activity after expressing these constructs singly or in combination, whereas helix-breaking mutations in TCC2 of KCNQ2 gave functional homomeric channels but prevented the heteromerization with KCNQ3. In contrast, KCNQ3 carrying a coiled-coil disrupting mutation in TCC2 hetero-oligomerized with KCNQ2. Our data suggest that the TCC1 domains of KCNQ2 and KCNQ3 are required to form functional homomeric as well as heteromeric channels, whereas both TCC2 domains facilitate an efficient transport of heteromeric KCNQ2/KCNQ3 channels to the plasma membrane.

Figure 1.

Sequence alignment and helical wheel projection. A, Alignment of C-terminal sequences of KCNQ1, KCNQ2, and KCNQ3 according to Schroeder et al. (2000a). The si domains from KCNQ1, KCNQ2, and KCNQ3 are indicated by light gray boxes, and the TCC domains within the si domains are shown as dark gray boxes, respectively. Conserved amino acids within the TCC domains are shown in white. The conserved leucine residues within the TCC domains, which were substituted for prolines, are marked with asterisks. The X indicates the last amino acid in the truncated KCNQ2-TCC1-X construct, whereas Y and the Ω indicate the first amino acids of the TCC2-CT-KCNQ2 and dCT-KCNQ2 construct, respectively. To generate the KCNQ2(NT-TCC2) construct, we used the truncated KCNQ2-TCC1-X construct and inserted, after the start methionine, the TCC2 fragment of KCNQ2 shown in italics. B, Schematic helical wheel projection for coiled-coils. Heptad positions are labeled a–g. Residues of the first two helical turns are boxed (positions a and d) or circled (positions b, c, e, f, and g).

Figure 2.

Exchange of both TCC sequences within the si domain. A, Schematic illustration of the chimeras KCNQ1-sid_Q3 and KCNQ1(TCC1+TCC2)Q3. B, C, Representative current recordings from Xenopus oocytes injected with cRNAs of KCNQ2 plus KCNQ1-sid_Q3 (B) and KCNQ2 plus KCNQ1(TCC1+TCC2)Q3 (C). D, Current–voltage (I/V) curves of KCNQ2 (n = 20), KCNQ2 plus KCNQ1 (n = 24), KCNQ2 plus KCNQ1(TCC1+TCC2)Q3 (n = 55), and KCNQ2 plus KCNQ1-sid_Q3 (n = 45). Steady-state current levels measured at the end of a 2-s-long voltage pulse are shown. E, Bar graph of current levels obtained at the end of a 2-s-long voltage pulse to +40 mV. Currents from different oocyte batches were normalized to the mean KCNQ2 plus KCNQ1-sid_Q3 current at +40 mV.

Figure 3.

Exchange of individual TCC sequences within the si domain. A, Schematic illustration of the chimeras KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3. B, C, Representative current recordings from oocytes injected with cRNAs of KCNQ2 plus KCNQ1(TCC1)Q3 (B) and KCNQ2 plus KCNQ1(TCC2)Q3 (C). D and E, Current–voltage relationships of KCNQ2 (n = 20), KCNQ2 plus KCNQ1 (n = 24), KCNQ2 plus KCNQ1(TCC1)Q3 (n = 38), and KCNQ2 plus KCNQ1-sid_Q3 (n = 45) (D) and of KCNQ2 (n = 20), KCNQ2 plus KCNQ1 (n = 24), KCNQ2 plus KCNQ1(TCC2)Q3 (n = 43), and KCNQ2 plus KCNQ1-sid_Q3 (n = 45) (E). Steady-state current levels measured at the end of a 2-s-long voltage pulse are shown. Currents from different oocyte batches were normalized to the level of KCNQ2 plus KCNQ1-sid_Q3 currents at +40 mV. F, G, Bar diagrams of mean current levels obtained at +40 mV from currents depicted in D and E, respectively. Analysis of dominant-negative effects (H) and surface expression (I) by exchange of individual TCC sequences within the si domain. H, Effect of coexpressing pore mutants KCNQ1-G314S (n = 24), KCNQ1(TCC1)Q3-G314S (n = 44), KCNQ1(TCC2)Q3-G314S (n = 55), KCNQ1(TCC1+TCC2)Q3-G314S (n = 31), and KCNQ3-G318S (n = 20) on KCNQ2 (n = 20) currents. Average current levels at the end of a test pulse to +40 mV from oocytes (co-)injected with cRNAs as indicated are shown. I, Surface expression determined from luminescence measurements of oocytes coinjected with HA-tagged KCNQ2 and nontagged KCNQ2 (n = 35), KCNQ1(TCC1)Q3 (n = 39), KCNQ1(TCC2)Q3 (n = 39), or KCNQ3 (n = 42). Data were normalized to the value for the KCNQ2(HA) plus KCNQ3 coinjection. Oocytes expressing nontagged KCNQ2 (n = 40) served as a background control. Values are means ± SE. J, Coassembly of the HA-tagged KCNQ1(TCC1)Q3 and KCNQ1(TCC2)Q3 constructs with FLAG-tagged KCNQ2. Protein complexes obtained from Cos-7 cells expressing various KCNQ subunits were immunoprecipitated with anti-HA antibody, separated by SDS-PAGE, and detected by Western blot analysis using an anti-FLAG antibody. The arrowhead denotes the molecular weight of FLAG-tagged KCNQ2 proteins, and the asterisk represents the weight of the heavy chain of the anti-HA antibody. Left, Molecular weight marker. K, Summary of three independent coimmunoprecipitation experiments. The corresponding Western blots were analyzed by densitometric analysis. The band intensities were normalized to the Q3HA/Q2FLAG value in each experiment and combined.

Figure 4.

Effect of the KCNQ2(TCC2)Q1 chimera and of KCNQ2 constructs with individually deleted TCC domains. A, B, Representative current recordings from oocytes (co-)injected with cRNA(s) of KCNQ2(TCC2)Q1 (A) and KCNQ2(TCC2)Q1 plus KCNQ3 (B). C, Bar diagram of current levels recorded at the end of a 2-s-long test pulse to +40 mV from oocytes expressing KCNQ2(TCC2)Q1 (n = 32), KCNQ2(TCC2)Q1 plus KCNQ3 (n = 42), and KCNQ2 plus KCNQ3 (n = 42). Currents were normalized to the value obtained from (KCNQ2 plus KCNQ3)-expressing oocytes. D, E, Representative current recordings from oocytes (co-)injected with cRNA(s) of KCNQ2-ΔTCC2 (D) and KCNQ2-ΔTCC2 plus KCNQ3 (E). F, Bar diagram of current levels from oocytes (co-)expressing KCNQ2 (n = 19), the deletion construct KCNQ2-ΔTCC2 (n = 18), KCNQ2-ΔTCC2 plus KCNQ3 (n = 25), or KCNQ2 plus KCNQ3 (n = 22). Currents were normalized to the value obtained from oocytes coexpressing KCNQ2 plus KCNQ3.

Figure 5.

Effects of disrupting the coiled-coil probability within the TCC domains of KCNQ2. Coiled-coil probabilities within the si domain of KCNQ2 wild-type (A), KCNQ2-L585P (D), and KCNQ2-L637P (G), determined with the program Coils (version 2.2). Representative current recordings from oocytes injected with cRNAs of KCNQ2, KCNQ2-L585P, and KCNQ2-L637P are shown in B, E, and H and from coexpressions of these constructs with KCNQ3 in C, F, and I, respectively. J shows I/V curves, and K shows a bar diagram of current levels recorded at the end of a 2-s-long test pulse to +40 mV for KCNQ2 (n = 36), KCNQ3 (n = 42), KCNQ2 plus KCNQ3 (n = 45), KCNQ2-L585P (n = 38), KCNQ2-L585P plus KCNQ3 (n = 40), KCNQ2-L637P (n = 37), and KCNQ2-L637P plus KCNQ3 (n = 37) (co-)injection schemes. Data in J and K were normalized to KCNQ2 plus KCNQ3 currents.

Figure 6.

Effects of disrupting the coiled-coil probability within the TCC domains of KCNQ3. Coiled-coil probabilities within the si domain of KCNQ3 wild-type (A), KCNQ3-L564P (D), and KCNQ3-L636P (G), determined with the program Coils (version 2.2). Representative current recordings from oocytes injected with cRNAs of KCNQ3, KCNQ3-L564P, and KCNQ3-L636P are shown in B, E, and H and from coexpressions of these constructs with KCNQ2 in C, F, and I, respectively. J shows I/V curves, and K shows a bar diagram of current levels recorded at the end of a 2-s-long test pulse to +40 mV for KCNQ2 (n = 36), KCNQ3 (n = 42), KCNQ2 plus KCNQ3 (n = 45), KCNQ3-L564P (n = 24), KCNQ3-L564P plus KCNQ2 (n = 28), KCNQ3-L636P (n = 31), and KCNQ3-L636P plus KCNQ2 (n = 47) (co-)injection schemes, as indicated. Data in J and K were normalized to current levels obtained from oocytes expressing KCNQ2 plus KCNQ3.

Figure 7.

Effect of different KCNQ2 channel fragments on KCNQ3 currents. A, Schematic illustration of the KCNQ2 constructs. The N terminus and the transmembrane region of KCNQ2 are shown as light gray boxes, and the C-terminus is shown as dark gray boxes. The TCC domains are indicated in black. B, Comparison of current levels after 2 s at +40 mV recorded from oocytes (co-)injected with KCNQ3 (n = 55), KCNQ2 plus KCNQ3 (n = 27), KCNQ2-TCC1-X plus KCNQ3 (n = 28), KCNQ2-TCC1-X plus TCC2-CT-KCNQ2 plus KCNQ3 (n = 41), TCC2-CT-KCNQ2 plus KCNQ3 (n = 74), and dCT-KCNQ2 plus KCNQ3 (n = 26) cRNA(s), respectively. Data were normalized to TCC2-CT-KCNQ2 plus KCNQ3 currents. C, Representative current recording from an oocyte coinjected with cRNAs of KCNQ2-TCC1-X and KCNQ3 measured 5 d after injection. D, Surface expression determined from luminescence measurements of oocytes expressing either the HA-tagged KCNQ3 (n = 47) alone or coinjected with TCC2-CT-KCNQ2 (n = 47) and dCT-KCNQ2 (n = 47). Data were normalized to the value for the KCNQ3(HA) plus TCC2-CT-KCNQ2 coinjection. Oocytes expressing nontagged KCNQ3 (n = 44) served as a background control. E, TEA sensitivity of KCNQ2, KCNQ3, KCNQ2 plus KCNQ3, TCC2-CT-KCNQ2 plus KCNQ3, and dCT-KCNQ2 plus KCNQ3 (n = 10 for each data set). F, Western blot analysis probing expression of KCNQ2 and N-terminal KCNQ2 fragments performed on pooled oocytes that had been used for measurements of TEA sensitivity as shown in E. Antibody KCNQ2(N-19) directed against an N-terminal epitope of KCNQ2 was used for detection. The arrowhead denotes the molecular weight of KCNQ2 proteins, and the asterisk represents the weight of the truncated KCNQ2-TCC1-X proteins. Right, Molecular weight marker. G, Comparison of current levels after 2 s at +40 mV recorded from oocytes (co-)injected with KCNQ3 (n = 23), KCNQ2 (n = 38), KCNQ2 plus KCNQ3 (n = 37), KCNQ2(NT-TCC2) plus KCNQ3 (n = 44), and KCNQ2(NT-TCC2;ΔTCC1) plus KCNQ3 (n = 29) cRNA(s), respectively. Data were normalized to the mean of KCNQ2(NT-TCC2) plus KCNQ3 currents. All values are means ± SE