Interactions of the Kv1.1 Channel with Peptide Pore Blockers: A Fluorescent Analysis on Mammalian Cells

Figures

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  Figure 1. Fluorescent confocal images of Neuro2a cells expressing K-Kv1.1wt. (a,c) Typical distribution of K-Kv1.1wt in cells in the absence (a) or after incubation with 2 nM A-HgTx (c). (b,d) Distribution of fluorescence in the 500–530 nm range (the range of A-HgTx fluorescence) in the absence (b) or after incubation with 2 nM A-HgTx (d). Bar is 5 µm. The absence of K-Kv1.1wt expression in the plasma membrane is consistent with the absence of A-HgTx binding at the cell membrane.

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  Figure 2. Schematic structures of A-HgTx and its complex with the Kv1.1 channel. (a) Structure of HgTx1 (pdb: 1HLY) labeled with Atto488 at the N-terminus. Positions of amino acid residues that are discussed in the text are indicated. (b) Characteristic structure of Kv1 channels in the membrane region (based on pdb: 2R9R). The position of the binding site of A-HgTx and other peptide blockers is shown by a bracket. (c) Structure elements of the outer pore region of the pore domain of the Kv1 channel (based on pdb: 2R9R), where the binding site of peptide pore blockers is situated. Front and back α-subunits are removed for clarity. Pink balls are potassium ions in the selectivity filter. S5 and S6 are transmembrane helices forming the pore domain. Positions of amino acid residues that are discussed in the text are indicated.

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  Figure 3. Fluorescent confocal images of Neuro2a cells expressing K-Kv1.1. (a,c,e) Typical distribution of K-Kv1.1 in cells in the absence (a) or after the addition of 2 nM A-HgTx (c), or in the presence of 2 nM A-HgTx and 10 nM HgTx1 (e). (b,d,f) Distribution of fluorescence in the 500–530 nm range (the range of A-HgTx fluorescence) in the absence (b) or after the addition of 2 nM A-HgTx (d), or in the presence of 2 nM A-HgTx and 10 nM HgTx1 (f). Bar is 5 µm. The expression of K-Kv1.1 in the plasma membrane ensures the binding of A-HgTx at the cell membrane (d) and the competitive displacement of A-HgTx from complexes with K-Kv1.1 by an excess of non-fluorescent HgTx1 (f).

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  Figure 4. The whole-cell recording of currents in Neuro2a cells expressing K-Kv1.1 without (a) or with added 2 nM HgTx1 (b). The membrane potential was changed from −70 to +70 mV in increments of 20 mV using 200 ms pulses (as shown in the insert at the top of panel a) that were applied at 20 s intervals. The holding potential (−40 mV) was maintained before and after depolarizing pulses. Representative series of currents are shown. (c) Dependence of the transmembrane current (mean± SEM, n = 10) on the applied potential in the absence (black color) or after addition (red color) of 2 nM HgTx1.

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  Figure 5. The whole-cell recording of currents in Neuro2a cells expressing K-Kv1.1 in the absence (a) or after the addition of 5 nM A-HgTx (b). The membrane potential was changed from −70 to +70 mV in increments of 20 mV using 200 ms pulses (as shown in the insert at the top of panel (a)) that were applied at 20 s intervals. Finally, the potential returned to the holding potential (−40 mV). Representative series of currents are shown. (c) Dependence of inhibition of K-Kv1.1 current on the concentration of A-HgTx at +50 mV. Data are mean ± SEM (n = 6).

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  Figure 6. Confocal images showing distributions of K-Kv1.1 (a) and ER marker ERTG (b) in living Neuro2a cells. (c) Merged image. Yellow color shows the co-localization of K-Kv1.1 and ERTG in ER. Scale bar is 10 µm. (d–f) Profiles of ERTG and K-Kv1.1 fluorescence distribution along white lines marked in panel (c).

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  Figure 7. Confocal images showing distributions of K-Kv1.1 (a) and Golgi apparatus marker NCer (b) in living Neuro2a cells. (c) Merged image. Yellow color shows the co-localization of K-Kv1.1 and NCer in the trans-Golgi apparatus. Scale bar is 10 µm.

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  Figure 8. Quantitative analysis of K-Kv1.1 interactions with different peptide pore blockers. (a) Concentration-dependent binding of A-HgTx to K-Kv1.1, which was measured as the dependence of Rav on the concentration L of A-HgTx added to Neuro2a cells and normalized to the maximum Rav value (Rm). Measurements were performed in a complete medium or a buffer with either 150 mM KCl (K+ buffer) or 4 mM KCl and 150 mM NaCl (Na+ buffer). The complete medium and Na+ buffer maintains the closed state of the channels, while the K+ buffer favors the open state of the channels. (b,d–f) Competitive binding of A-HgTx (2 nM) and different peptide pore blockers to K-Kv1.1. C—concentration of non-labeled peptide pore blocker. Representative dependencies are shown. Measurements were performed in a complete medium (b,d–f) or in a K+ buffer that favored the open state of the channels (d–f). (c) ChTx (100 nM) is not able to compete with A-HgTx (2 nM) for binding to K-Kv1.1. Control—binding of A-HgTx (2 nM) to K-Kv1.1 in the absence of ChTx. (g) Competitive binding of A-HgTx (2 nM) and different peptide pore blockers (concentrations are indicated in the diagram) to K-Kv1.1 in a complete medium, K+ and Na+…