Large conductance voltage- and calcium-dependent K+ channel, a distinct member of voltage-dependent ion channels with seven N-terminal transmembrane segments (S0-S6), an extracellular N terminus, and an intracellular (S9-S10) C terminus

Abstract

Large conductance voltage- and Ca2+-dependent K+ (MaxiK) channels show sequence similarities to voltage-gated ion channels. They have a homologous S1-S6 region, but are unique at the N and C termini. At the C terminus, MaxiK channels have four additional hydrophobic regions (S7-S10) of unknown topology. At the N terminus, we have recently proposed a new model where MaxiK channels have an additional transmembrane region (S0) that confers beta subunit regulation. Using transient expression of epitope tagged MaxiK channels, in vitro translation, functional, and "in vivo" reconstitution assays, we now show that MaxiK channels have seven transmembrane segments (S0-S6) at the N terminus and a S1-S6 region that folds in a similar way as in voltage-gated ion channels. Further, our results indicate that hydrophobic segments S9-S10 in the C terminus are cytoplasmic and unequivocally demonstrate that S0 forms an additional transmembrane segment leading to an exoplasmic N terminus.

Figure 1

Hydrophobicity analysis and alternative Models for MaxiK channel topology. (A) Hydrophobicity analysis of Hslo and Dslo using GCG-program pepplot (window size of nine amino acids). Hydrophobic regions (S0 to S4) are according to the model in C (16). Numbers at arrows indicate amino acids that were removed from the protein sequence. Bars above S8, S9, and S10 in Dslo correpond to 16 amino acids length (minimum requirement for a protein α helix to span a lipid bilayer of 2.5 nm thickness). (B) Previous model with intracellular N terminus, and unknown topology of the C terminus (S7-S10). (C) Proposed membrane topology of MaxiK channels. (B and C) Flags, numbered from 1–4 in both models, mark positions where c-myc epitopes were introduced. ψ, consensus sites for N-linked glycosylation (NXS/T) in bovine slo, regardless of being extra- or intracellular.

Figure 2

MaxiK channels have an exoplasmic N terminus with an additional membrane spanning segment (S0). (A) Immunocytochemistry of c-myc tagged MaxiK channels (HF1, HF2 and HF3) expressed in COS-M6 cells. Cells were incubated with anti-c-myc mAb under nonpermeabilizing and permeabilizing conditions. Antibody binding was visualized using beads coated with secondary antibodies or with FITC-labeled secondary antibodies. Confocal images (two right panels) are from sections at the middle of the cells. Experiments were performed at least three times for each construct (also in Fig. 4) with similar results. (B) Functional expression of c-myc tagged clones in oocytes measured in inside-out patches. (Left) Voltage activation curves at 10 μM intracellular Ca²⁺, [Ca²⁺]_i. Values for half activation potentials (V_1/2) in 10 μM [Ca²⁺]_i are (in mV): 12 ± 18 (n = 64) for wild-type Hslo (Wt); 5 ± 9 (n = 3) for HF1; 14 ± 8 (n = 6) for HF2; 98 ± 7 (n = 4) for HF3; 13 ± 6 (n = 3) for HF4 (see Fig. 4). (Right) V_1/2 as a function of [Ca²⁺]_i.

Figure 4

Topology of S7 and S8 regions. (A) Immunocytochemistry of Hslo epitope tagged between S7 and S8 (HF4). Visualization with both magnetic beads and FITC-labeled antibodies. (B) In vitro translation of HS7S8 and DS7S8 containing hydrophobic regions S7 and S8. P, pellet or microsomal fraction; S, soluble fraction; M, protein marker.

Figure 3

Linker between S5 and S6 forms the pore region of MaxiK channels. (A) IbTx sensitivity of Hslo. IbTx (100 nM) applied to the external side of an outside-out patch, completely blocks ionic currents. Holding potential = 0 mV; test potential = 80 mV. (Right): Time course of IbTx blockade at different IbTx concentrations. (Insert) Dose-response curve, K_d = 0.72 nM, Hill coefficient near one. (B) Dslo currents are insensitive to 100 nM IbTx. (C) Chimeric construct HDP carries the pore of Dslo in the Hslo backbone. Current traces show that the pore of Dslo makes Hslo insensitive to 100 nM IbTx.

Figure 5

Cytosolic C terminus tail (containing S9-S10). (A) In vitro translation of the tail region containing S9 and S10. Hslo-tail and Dslo-tail cRNAs were in vitro translated (see Materials and Methods). The calculated molecular weights are: Hslo-tail, 48.2 kDa; Dslo-tail, 52.2 kDa. Pellet (P) or membrane fraction and soluble fraction (S). (B) Coexpression of core and tail in the same oocytes produce functional channels (17). Currents in cell-attached mode with test pulses from −50 mV to +136 mV every 6 mV (Vh = 0 mV). (C) Assembly of functional MaxiK channels by cross-cramming: (Left) Cell attached patch in a core injected oocyte does not show any current. (Right) Functional channels assemble within few minutes when the cell-attached patch was excised and crammed into a tail injected oocyte. Currents were elicited using the same protocol as in B. The larger K⁺ currents in the cross-cramming experiments compared with those in B, may be due to diffusion of external Ca²⁺ into the oocyte during cramming.