Figure 5. The Kv6.4 activation gate but not T1 limits the formation of Kv2.1:Kv6.4 2:2R heteromers. (A) Schematic diagram of the chimeric constructs used to test 2:2R formation by TIRF microscopy. The N-terminal T1 domain is labeled, and transmembrane domains S1–S6 are shown as rectangles, with a star next to the S6 activation gate. Sequences derived from Kv6.4 are shown in black, and sequences inserted from Kv2.1 are shown in blue. Chimera names are given below each diagram. (B) Percentages of 3:1R and 2:2R heteromers calculated from TIRF photobleaching assays for Kv2.1 coexpressed with GFP-tagged versions of WT Kv6.4 (magenta), Kv6.4-Kv2.1T1 (black), and Kv6.4-PIPIIV-Kv2.1CT (blue). The stoichiometry distribution for Kv6.4-Kv2.1T1 was unchanged, but Kv6.4-PIPIIV-Kv2.1CT had significantly fewer 3:1R heteromers and significantly more 2:2R heteromers (P < 0.05, χ2 test). Kv2.1 was expressed with Kv6.4-Kv2.1T1 and Kv6.4-PIPIIV-Kv2.1CT in a 1:80 and a 1:60 cRNA ratio, respectively, to eliminate Kv2.1 homomers and bias 2:2R heteromer formation. (C) Expression ratio dependence of 2:2R heteromer formation is shown for WT Kv6.4 and Kv6.4-PIPIIV-Kv2.1CT. 2:2R formation was significantly higher for both constructs at the highest concentration tested relative to lower concentrations (*, P < 0.05, χ2 test). Note 2:2R formation is higher for Kv6.4-PIPIIV-Kv2.1CT across a broad range of expression ratios. (D) Kv2.1:Kv6.4 2:2R heteromer percentages detected for all Kv6.4 chimeras tested in TIRF photobleaching assays at a highly Kv6.4-biased expression ratio. 2:2R formation was significantly increased relative to WT for the Kv6.4-PIPIIV-Kv2.1CT, Kv6.4-PIPIIV, and Kv6.4-Kv2.1T1-PIPIIV-Kv2.1CT constructs (*, P < 0.05, χ2 test). The P3T and V6F mutations in the Kv6.4-PIPIIV-Kv2.1CT background eliminate the increase in 2:2R formation. Bleaching step counts and expression ratios for the data in D were as follows (construct name, ratio, no. of one-steps, no. of two-steps): Kv6.4 WT, 1:200, 98, 23; Kv6.4-Kv2.1CT, 1:142, 87, 21; Kv6.4-PITIIF-Kv2.1CT, 1:154, 85, 13; Kv6.4-PITIIV-Kv2.1CT, 1:128, 64, 16; Kv6.4-PIPIIF-Kv2.1CT, 1:334, 67, 15; Kv6.4-PIPIIV-Kv2.1CT, 1:60, 72, 39; Kv6.4-PIPIIV, 1:185, 80, 39; and Kv6.4-Kv2.1T1-PIPIIV-Kv2.1CT, 1:78, 63, 32. We did not detect spots bleaching in three or four steps for any GFP-Kv6.4 chimera tested in this study.
Figure 5.

The Kv6.4 activation gate but not T1 limits the formation of Kv2.1:Kv6.4 2:2R heteromers. (A) Schematic diagram of the chimeric constructs used to test 2:2R formation by TIRF microscopy. The N-terminal T1 domain is labeled, and transmembrane domains S1–S6 are shown as rectangles, with a star next to the S6 activation gate. Sequences derived from Kv6.4 are shown in black, and sequences inserted from Kv2.1 are shown in blue. Chimera names are given below each diagram. (B) Percentages of 3:1R and 2:2R heteromers calculated from TIRF photobleaching assays for Kv2.1 coexpressed with GFP-tagged versions of WT Kv6.4 (magenta), Kv6.4-Kv2.1T1 (black), and Kv6.4-PIPIIV-Kv2.1CT (blue). The stoichiometry distribution for Kv6.4-Kv2.1T1 was unchanged, but Kv6.4-PIPIIV-Kv2.1CT had significantly fewer 3:1R heteromers and significantly more 2:2R heteromers (P < 0.05, χ2 test). Kv2.1 was expressed with Kv6.4-Kv2.1T1 and Kv6.4-PIPIIV-Kv2.1CT in a 1:80 and a 1:60 cRNA ratio, respectively, to eliminate Kv2.1 homomers and bias 2:2R heteromer formation. (C) Expression ratio dependence of 2:2R heteromer formation is shown for WT Kv6.4 and Kv6.4-PIPIIV-Kv2.1CT. 2:2R formation was significantly higher for both constructs at the highest concentration tested relative to lower concentrations (*, P < 0.05, χ2 test). Note 2:2R formation is higher for Kv6.4-PIPIIV-Kv2.1CT across a broad range of expression ratios. (D) Kv2.1:Kv6.4 2:2R heteromer percentages detected for all Kv6.4 chimeras tested in TIRF photobleaching assays at a highly Kv6.4-biased expression ratio. 2:2R formation was significantly increased relative to WT for the Kv6.4-PIPIIV-Kv2.1CT, Kv6.4-PIPIIV, and Kv6.4-Kv2.1T1-PIPIIV-Kv2.1CT constructs (*, P < 0.05, χ2 test). The P3T and V6F mutations in the Kv6.4-PIPIIV-Kv2.1CT background eliminate the increase in 2:2R formation. Bleaching step counts and expression ratios for the data in D were as follows (construct name, ratio, no. of one-steps, no. of two-steps): Kv6.4 WT, 1:200, 98, 23; Kv6.4-Kv2.1CT, 1:142, 87, 21; Kv6.4-PITIIF-Kv2.1CT, 1:154, 85, 13; Kv6.4-PITIIV-Kv2.1CT, 1:128, 64, 16; Kv6.4-PIPIIF-Kv2.1CT, 1:334, 67, 15; Kv6.4-PIPIIV-Kv2.1CT, 1:60, 72, 39; Kv6.4-PIPIIV, 1:185, 80, 39; and Kv6.4-Kv2.1T1-PIPIIV-Kv2.1CT, 1:78, 63, 32. We did not detect spots bleaching in three or four steps for any GFP-Kv6.4 chimera tested in this study.

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