Nakajo K et al. (2011), KCNQ1 subdomains involved in KCNE modulation re...

XB-ART-44110

J Gen Physiol 2011 Nov 01;1385:521-35. doi: 10.1085/jgp.201110677.

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KCNQ1 subdomains involved in KCNE modulation revealed by an invertebrate KCNQ1 orthologue.

Nakajo K , Nishino A , Okamura Y , Kubo Y .

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KCNQ1 channels are voltage-gated potassium channels that are widely expressed in various non-neuronal tissues, such as the heart, pancreas, and intestine. KCNE proteins are known as the auxiliary subunits for KCNQ1 channels. The effects and functions of the different KCNE proteins on KCNQ1 modulation are various; the KCNQ1-KCNE1 ion channel complex produces a slowly activating potassium channel that is crucial for heartbeat regulation, while the KCNE3 protein makes KCNQ1 channels constitutively active, which is important for K(+) and Cl(-) transport in the intestine. The mechanisms by which KCNE proteins modulate KCNQ1 channels have long been studied and discussed; however, it is not well understood how different KCNE proteins exert considerably different effects on KCNQ1 channels. Here, we approached this point by taking advantage of the recently isolated Ci-KCNQ1, a KCNQ1 homologue from marine invertebrate Ciona intestinalis. We found that Ci-KCNQ1 alone could be expressed in Xenopus laevis oocytes and produced a voltage-dependent potassium current, but that Ci-KCNQ1 was not properly modulated by KCNE1 and totally unaffected by coexpression of KCNE3. By making chimeras of Ci-KCNQ1 and human KCNQ1, we determined several amino acid residues located in the pore region of human KCNQ1 involved in KCNE1 modulation. Interestingly, though, these amino acid residues of the pore region are not important for KCNE3 modulation, and we subsequently found that the S1 segment plays an important role in making KCNQ1 channels constitutively active by KCNE3. Our findings indicate that different KCNE proteins use different domains of KCNQ1 channels, and that may explain why different KCNE proteins give quite different outcomes by forming a complex with KCNQ1 channels.

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Species referenced: Xenopus laevis
Genes referenced: kcne1 kcne3 kcnq1 kcnq2 tbx2

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	Figure 1. Amino acid sequences of Ci-KCNQ1 and human KCNQ1 (hKCNQ1) are aligned for comparison. Same and similar amino acid residues are marked with asterisks and dots, respectively. Transmembrane regions (S1–S6) are boxed in green. The S5-P connector and pore helix are underlined. Red and blue arrowheads indicate the mutated amino acid residues in this study.
	Figure 2. Ci-KCNQ1 is not properly modulated by KCNE1. (A) Representative current traces in the absence and presence of KCNE1 or KCNE3 for human KCNQ1 (hKCNQ1) and Ci-KCNQ1. The membrane potential was stepped up from −120 or −100 to 60 mV in 20-mV increments and subsequently stepped to −30 mV for tail currents. (B) G-V relationships for human KCNQ1 (top) and Ci-KCNQ1 (bottom) in the absence and presence of KCNE1 or KCNE3.
	Figure 3. Second half of the transmembrane region from hKCNQ1 is necessary for the modulation by KCNE1. (A) The half-maximal potential (V1/2) without (light gray bars) and with (dark gray bars) KCNE1 for human KCNQ1 (hKCNQ1; left end), Ci-KCNQ1 (right end), and their chimeras are plotted. Asterisks indicate the significant reduction in the level of V1/2 with KCNE1 compared with that of hKCNQ1 with KCNE1 (Dunnett’s test). The design for each chimera is depicted at the bottom of the bar graphs; red regions are from human KCNQ1, and blue regions are from Ci-KCNQ1. (B) Representative current traces of chimera ciQ1hQ1(S4–S6) in the absence and presence of KCNE1. The membrane potential was stepped from −100 to +60 mV in 20-mV increments and subsequently stepped to −30 mV for tail currents. (C) G-V relationships for ciQ1hQ1(S4–S6) in the absence and presence of KCNE1. Plots are fitted with the Boltzmann equation (red curves).
	Figure 4. Amino acid residues in the pore region of KCNQ1 have a large impact on the KCNE1 modulation. (A) V1/2 without (light gray bars) and with (dark gray bars) KCNE1 for point mutants of the ciQ1hQ1(S5P) chimera and Ci-KCNQ1 are plotted. Asterisks indicate the significant increase in the level of V1/2 with KCNE1 compared with those of ciQ1hQ1(S5P) or Ci-KCNQ1 with KCNE1 (Dunnett’s test). The locations of the mutations are indicated by yellow stars in the inset. (B) Representative current traces for the A191G/I258V double mutant of ciQ1hQ1(S5P) in the absence (left) and presence (right) of KCNE1. The membrane potential was stepped up from −100 to +40 mV in 10-mV increments for KCNE1-less current and from −100 to +60 mV in 20-mV increments for KCNE1 current. (C) G-V relationships for the point mutants in the absence (closed circles) and presence (open circles) of KCNE1.
	Figure 5. Human KCNQ1 mutants confirm that Gly272, Val324, and Val334 of KCNQ1 are important for KCNE1 modulation. (A) V1/2 values without (light gray bars) and with (dark gray bars) KCNE1 for hKCNQ1 (hQ1) point mutants are plotted. Asterisks indicate the significant reduction in the level of V1/2 with KCNE1 compared with that of hKCNQ1 with KCNE1 (Dunnett’s test). (B) Representative current traces for hKCNQ1 G272A/V334I double mutant in the absence (left) and presence (right) of KCNE1. The membrane potential was stepped up from −100 to +60 mV in 20-mV increments. (C) G-V relationships for the hQ1 G272A/V334I mutant in the absence (open circles) and presence (closed red circles) of KCNE1. The G-V relationships of WT hKCNQ1 in the absence (black dotted curve) and presence (red dotted curve) of KCNE1 are superimposed.
	Figure 6. The S1 segment, not the pore region of hKCNQ1, is important for the modulation by KCNE3. (A) Representative current traces of chimera ciQ1hQ1(S4–S6) in the absence and presence of KCNE3. The membrane potential was stepped up from −100 to +60 mV in 20-mV increments and subsequently stepped to −30 mV for tail currents. Insets are the expanded tail current traces after depolarization to +20, +40, and +60 mV. Expanded areas are indicated by red circles below. Bars in the insets indicate 0.1 s and 0.1 µA, respectively. The “hook” tail current, which reflects de-inactivation and is the characteristic feature of the homomeric KCNQ1 channel, was only seen in the homomeric ciQ1hQ1(S4–S6) channel, indicating that KCNE3 abolished inactivation. (B) G-V relationships for the ciQ1hQ1(S4–S6) in the absence (closed circles) and presence (blue triangles) of KCNE3. The G-V relationships of ciQ1hQ1(S4–S6) in the presence of KCNE1 (red dotted curve) is superimposed for comparison. (C) The constitutive activity indices (G−80mV/G+20mV) with KCNE3 for human KCNQ1 (hKCNQ1; left end), Ci-KCNQ1 (right end), and their chimeras are plotted. Asterisks indicate the significant reduction in the level of G−80mV/G+20mV, with KCNE3 compared with that of hKCNQ1 with KCNE3 (Dunnett’s test). The design for each chimera is depicted at the bottom of the bar graphs; red regions are from human KCNQ1, and blue regions are from Ci-KCNQ1.
	Figure 7. The V46F mutation with a human S5P connector changes Ci-KCNQ1 into a KCNE3-sensitive channel. (A) The constitutive activity index (G−80mV/G+20mV) with KCNE3 for the point mutants of the ciQ1hQ1(S2–S3,S5P) chimera. Asterisks indicate the significant increase in the level of G−80mV/G+20mV, with KCNE3 compared with that of ciQ1hQ1(S2–S3,S5P) with KCNE3 (Dunnett’s test). The locations of the mutations are indicated by yellow stars in the inset. (B) Representative current traces of ciQ1hQ1(S5P) V46F in the absence and presence of KCNE3. The membrane potential was stepped up from −100 to +60 mV and subsequently stepped to −30 mV for tail currents. (C) G-V relationships for Ci-KCNQ1 V46F and ciQ1hQ1(S5P) V46F in the absence (open symbols) and presence (closed symbols) of KCNE3. Plots are fitted with the Boltzmann equation.
	Figure 8. The human KCNQ1 F127V mutant confirms that the S1 segment of KCNQ1 is important for the KCNE3 modulation. (A) Representative current traces for the hKCNQ1 F127V mutant in the absence (left) and presence (right) of KCNE3. The membrane potential was stepped up from −100 to +60 mV in 20-mV increments. (B) G-V relationships for the hQ1 F127V mutant in the absence (closed circles) and presence of KCNE1 (red squares) or KCNE3 (blue triangles). The G-V relationships of WT hKCNQ1 in the absence (black dotted curve) and presence of KCNE1 (red dotted curve) or KCNE3 (blue dotted curve) are superimposed. (C) Representative current traces for the hKCNQ1 F127V mutant in the presence of KCNE3 from the same oocyte at different holding potentials (−80 and −120 mV). 2-s depolarizing pulses were applied at every 10 s. (D) G-V relationships for the hQ1 F127V mutant with KCNE3 at a holding potential of −80 mV (open circles) and −120 mV (closed circles).
	Figure 9. Phenylalanine residues on the S1 segment of KCNQ1 play a role in KCNE3 modulation. (A) Location of phenylalanine residues on the hKCNQ1 S1 segment. Trp120 is not on the S1 segment and therefore is not depicted. (B) G-V relationships for hQ1 F(W)-to-A mutants in the absence (left) and presence of KCNE3 (right). All but WT KCNQ1, W120A, and F123A with KCNE3 are fitted with the Boltzmann equation. For the WT KCNQ1, W120A, and F123A with KCNE3, plots were connected by dotted lines, as they could not be fitted with the Boltzmann equation.
	Figure 10. KCNE3 sensitivities are lost in the S1 phenylalanine double mutants. (A) Representative current traces of the hKCNQ1 F127L/F130L mutant in the absence (black) and presence of KCNE1 (red) or KCNE3 (blue) are shown. The membrane potential was stepped up from −100 to +60 mV in 20-mV increments. (B) G-V relationships for the human KCNQ1 WT and phenylalanine double mutants in the absence (black) and presence of KCNE1 (red) or KCNE3 (blue). All plots are fitted with the Boltzmann equation except the hKCNQ1 WT with KCNE3, which could not be fitted with it.
	Figure 11. Sequence comparisons among different species and other KCNQ channels. KCNQ1 amino acid sequences of part of the S1 segment (122–135), S5 segment (269–275), S5-P connector (286–298), and the upper half of the S6 segment (324–334) from various species are aligned. Other types of KCNQ channels (KCNQ2-5) from humans are also aligned. Conserved Phe123, Phe127, Phe130, Gly272, Val324, and Val334 are colored in red. Conserved amino acid residues are highlighted in bold.
	Figure 12. Locations of important amino acid residues determined in this study and a putative binding site of the KCNE protein. The structural model for the open state of human KCNQ1 (Smith et al., 2007) is used to locate Phe123 (hidden below Phe127 in the top view), Phe127 and Phe130 (hidden behind Phe127 in the side view) on the S1 segment (orange), Gly272 on the S5 segment (green), and Val324 and Val334 on the S6 segment (yellow). S5-P connectors are circled in the side view (left). All four α subunits are shown, and each subunit is in a different color. The hypothetical location for the KCNE protein (“KCNEx”) is indicated as a light green circle in the top view (right).

References [+] :

Barhanin, K(V)LQT1 and lsK (minK) proteins associate to form the I(Ks) cardiac potassium current. 1996, Pubmed, Xenbase