Potential independent action of sigma receptor ligands through inhibition of the Kv2.1 channel

Abstract

The sigma-1 receptor (σ1-R) and sigma-2 receptor (σ2-R) are potential drug targets for treatment of cancer, pain, depression, retinal degeneration and other neuronal diseases. Previous reports show that sigma-1 receptor modulates the activities of multiple channels. We are interested in possible sigma receptor modulation of Kv2.1, a K⁺ channel abundant in retinal photoreceptors. We tested the effect of established sigma receptor ligands on Kv2.1 channels which were stably expressed in HEK293 cells. Surprisingly, σ1-R antagonists inhibited Kv2.1 currents in both wild type and σ1-R knockout HEK293 cells that we engineered using the CRISPR/Cas9 technology. Moreover, PB28, a σ1-R antagonist and also σ2-R agonist, inhibited Kv2.1 in σ1-R knockout cells, but this action was not blocked by the σ2-R antagonists that did not have an effect on Kv2.1. We also observed inhibition of electroretinogram by PB28 in wild type as well as σ1-R knockout mice. Thus, the results in this study indicate that the Kv2.1-inhibiting function of the sigma ligands is not sigma receptor dependent, suggesting a direct effect of these ligands on the Kv2.1 channel.

Keywords: CRISPR/Cas9; Kv2.1 channel; electroretinogram; patch-clamp; sigma receptor ligands.

Figure 1. Only s1-R antagonists inhibit Kv2.1 channel current

(A) Representative current traces from a cell expressing Kv2.1 channel before (HR; black trace) and during treatment with σ1-R receptor agonist PRE084 (10 μM, red trace). Inset voltage pulse to 50 mV from a holding potential of -60 mV. (B) Representative Kv2.1 channel current traces from a cell treated with σ1-R antagonist BD1047 (50 μM, red trace) compared to total current (black trace). (C) Comparison of Kv2.1 current traces before (HR, black trace) and during treatment with σ1-R antagonist NE100 (50 μM, red trace). (D) Time course of current amplitude at +60 mV. Recordings during Pre84 treatment is represented as red filled circles. (E) Filled circles (red) showing current amplitude time course during treatment of Kv2.1 expressing cells with BD1047. (F) Kv2.1 outward current amplitude as in D in presence of NE100 (50 μM, red filled circles). Both in E and F the solid line represents single exponential curve fit. (G) Average Kv2.1 whole-cell normalized current-voltage plot obtained in control solution (black squares) and during application of Pre084. (H) Comparison of average normalized current-voltage curve in presence of control bath solution and BD1047. (I) Average normalized current-voltage curve determined in control solution (black squares) and NE100 (red circles). (J) The G-V relationships of the control (solid black square), and in presence of BD1047 (blue triangle) or NE100 (red circle) are illustrated. (K) Normalized G-V curves as in J were fitted with the Boltzmann function. Data points in G-K are mean ± SEM (n=5). * P < 0.05 compared to control.

Figure 2. Antagonists inhibits Kv2.1 current after CRISPR/Cas9 mediated σ1-R receptor knockout

(A) Western blot confirmed protein levels for σ1-R, Kv2.1, and ß–actin. Lane 1 represents HEK293 cells and lanes 2-5 represent HEK-Kv2.1 cells. Three CRISPR/Cas9 mediated σ1-R KO cell samples are marked as 1, 2, and 3. (B) Normalized current-voltage curve before (black squares) and after (red circles) treatment of cells, as in lane 3(A), with BD1047. (C) Normalized current-voltage curve for no drug control and with 50 M NE100. Data points in B and C are mean ± SE of the mean from at least 5 independent recordings and *P<0.05.

Figure 3. σ2-R agonist PB28 but not antagonists inhibits Kv2.1 current in σ1-R knockout HEK 293 cells expressing Kv2.1 channel

(A) Representative cell showing current amplitude time course over 10 minutes with duration of PB28 application shown as red circles. (B) A plot of average normalized current-voltage curve in control (black trace) and after PB28 treatment (red trace). (C) Time course of current amplitude for a cell during the treatment of progesterone 20 μM (blue circles) or during the treatment of progesterone 10 μM and PB28 10 μM (red circles). (D) Average normalized current-voltage plot showing the effect of progesterone (blue trace) and progesterone plus PB28 (red trace) on Kv2.1 current. (E) In a representative cell current amplitude time course during the application of CM398 20 μM alone (blue circle) or CM398 10 μM plus PB28 10 μM (red circle). (F) Average plot of normalized current-voltage plot comparing Kv2.1 channel current responses to CM398 (blue trace) of CM398 plus PB28 (red trace) treatment.

Figure 4. Summary of σ1 and 2-R ligands inhibition of Kv2.1 current

(A) Bar graph summary of average % inhibition of Kv2.1 channel in HEK293 cells (open bar) or after σ1-R knockout (black bar) in HEK 293 cells in presence of various σ1- and σ2-R ligands. Data is mean ± SEM (n = 4 at least) and *P < 0.05. Concentrations of the ligands are presented in the parentheses under the bar graph. (B) σ1-R and σ2-R binding affinities, functions, and chemical structures of σ1-R ligands.

Figure 4. Summary of σ1 and 2-R ligands inhibition of Kv2.1 current

(A) Bar graph summary of average % inhibition of Kv2.1 channel in HEK293 cells (open bar) or after σ1-R knockout (black bar) in HEK 293 cells in presence of various σ1- and σ2-R ligands. Data is mean ± SEM (n = 4 at least) and *P < 0.05. Concentrations of the ligands are presented in the parentheses under the bar graph. (B) σ1-R and σ2-R binding affinities, functions, and chemical structures of σ1-R ligands.

Figure 5. σ-R ligand PB28 attenuate mouse ERG possibly through Kv2.1 inhibition

(A) Micrograph of mouse retina showing localization of Kv2.1 (red) and σ1-R (green). Nuclear layer is shown as blue DAPI staining. In the lower panel two enlarged ganglion cells marked with a white box showing Kv2.1 (red) and σ1-R (green) immunostaining. Scale bar 15 μM. (B) Average response of a- and b-wave amplitude in relation to light flash intensity. Vehicle injected eye response is shown as black (a-wave) and red (b-wave) traces. PB28 injected eye is shown as blue (a-wave) and purple (b-wave) traces. (C) Average ERG a- and b-wave responses from σ1-R knock out mice after saline or PB28 injection. Color representation as in B. Data is represented as mean ± SEM from at least 5 observations for each point and *P < 0.05 defines significance compared to control.