Sequence alignment, phylogenetic tree, and percent sequence identity of Kv6.3, Kv10.1, and Kv11.1. (A) The amino acid sequences of Kv2.1, Kv6.1, Kv6.2, Kv6.3, Kv10.1, and Kv11.1 were aligned using megalign. For convenience, only the first 460 aa of Kv2.1 are shown. Gaps (indicated by dashes) were introduced in the sequence to maintain the alignment. Conserved amino acids are shaded in gray. The six putative transmembrane domains and the pore region are indicated by an overline. (B) The phylogenetic tree for the Kv family. (C) The percent sequence similarity based on the S1–S6 core.

Expression of Kv6.3, Kv10.1, Kv11.1, and Kv2.1 in human tissues. A PCR analysis was performed on a cDNA panel of the indicated human tissues with gene-specific primers for the subunits indicated on the left.

Whole-cell current recordings of Kv6.3, Kv10.1, and Kv11.1, and the cotransfections with Kv2.1. The top sections in each panel show typical recordings for untransfected Ltk⁻ cells (A), or for cells expressing Kv6.3 (B), Kv10.1 (C), and Kv11.1 (D). The holding potential was −80 mV and cells were depolarized in 20-mV increments from −80 mV to +60 mV, 500 ms in duration, followed by a repolarizing pulse at −25 mV, 850 ms in duration. The bottom sections of each panel show typical recordings from Ltk⁻ cells expressing Kv2.1 (A), Kv2.1 + Kv6.3 (B), Kv2.1 + Kv10.1 (C), and Kv2.1 + Kv11.1 (D). The holding potential was −80 mV and cells were depolarized by 10-mV increments from −60 mV to +70 mV, 500 ms in duration. Deactivating tails were recorded at −25 mV or −35 mV for 850 ms.

(A) Voltage dependence of activation. The activation curves of Kv2.1, Kv2.1 + Kv6.3, Kv2.1 + Kv10.1, and Kv2.1 + Kv11.1 were obtained from the normalized initial tail amplitude recorded at −25 mV for Kv2.1, Kv2.1 + Kv10.1, and Kv2.1 + Kv11.1 or at −50 mV for Kv2.1 + Kv6.3 after 500-ms prepulses ranging from −60 mV to 70 mV in 10-mV steps. The solid line represents the Boltzmann function fitted to the experimental data (see Experimental Procedures). (B) Voltage dependence of inactivation. The inactivation curves of Kv2.1, Kv2.1 + Kv6.3, Kv2.1 + Kv10.1, and Kv2.1 + Kv11.1 were obtained from the normalized peak currents recorded during a 250-ms test pulse to 50 mV as a function of the 5-s prepulse ranging from −50 mV to 10 mV for Kv2.1, Kv2.1 + Kv10.1, and Kv2.1 + Kv11.1 and from −80 mV to −20 mV for Kv2.1 + Kv6.3. Experimental data were fitted with a Boltzmann function (solid lines). (C) Kinetics of activation and deactivation of Kv2.1 and Kv2.1 + Kv6.3. Mean time constants ± SEM of activation and deactivation are plotted as a function of the test potential. To obtain the time constants for activation, test pulses were applied ranging from −10 mV to 70 mV for Kv2.1 and −30 mV to 70 mV for Kv2.1 + Kv6.3 in 10-mV steps, 500 ms in duration. To obtain the time constants for deactivation, a 200-ms prepulse to 50 mV was followed by test pulses ranging from −20 mV to −100 mV in 10-mV steps, 850 ms in duration. The experimental data were fitted with mono- or double-exponential functions, as appropriate. The slow component of activation and deactivation are shown as triangles, whereas the fast components are shown as circles. WT Kv2.1 gating kinetics are connected with a solid line. (D) Kinetics of activation of Kv2.1, Kv2.1 + Kv10.1, and Kv2.1 + Kv11.1. Mean time constants ± SEM of activation are shown as a function of the step potentials (−10 mV to 70 mV). The pulse protocol for Kv2.1 + Kv10.1 and Kv2.1 + Kv11.1 is the same as for Kv2.1 alone in C.

Interaction of Kv6.3, Kv10.1, and Kv11.1 with representative subunits of all Kv subfamilies. The intracellular N-terminal segment that contains the subfamily-specific NAB domain was used as bait (B) and/or target (T) in a yeast two-hybrid analysis.

Co-immunoprecipitation of Kv6.3GFP, Kv10.1GFP, and Kv11.1GFP with Kv2.1c-myc. Immunoprecipitation was done with anti-GFP antibodies, Western blot was performed with anti-c-myc. Lanes 3–5 show that Kv2.1c-myc was coprecipitated with Kv6.3GFP, Kv10.1GFP, and Kv11.1GFP. GFP-tagged Kv2.1 (lane 1) and Kv1.5 (lane2) were used as positive and negative controls, respectively.

Subcellular localization of the channel-GFP fusion proteins assessed by confocal imaging. The rows show images with GFP fusion proteins of Kv6.3, Kv10.1, and Kv11.1, respectively. The first three columns show the fluorescence of the channel subunits, the DsRed-ER localization vector, and the overlay of both, respectively. The last two columns show cells cotransfected with Kv2.1, DsRed-ER, and each of the subunits; the surface staining of the GFP-tagged subunits (fourth column) is obvious with minimal overlap with the DsRed-ER fluorescence (overlay of both in the fifth column). (Scale bar, 10 μm.)

Alignment of the S6 segment of the Kv potassium channels. One member of each subfamily is represented. Conserved amino acids are shaded in gray. Sequence numbering of Kv2.1 is shown on top.