The Gβ1 and Gβ3 Subunits Differentially Regulate Rat Vascular Kv7 Channels

Figures

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  FIGURE 1. Gβγ enhances Kv7.4 currents in CHO expression system. Top panels show representative traces of currents generated in cells expressing Kv7.4 either with the control intracellular solution (left) or with 250 ng/ml Gβγ purified from bovine brain (right). Zero current is indicated by the dashed line. Mean IV relationship of Kv7.4 currents in control (n = 7) or with 250 ng/ml Gβγ (n = 4) is shown in bottom panel. Data was analyzed by Bonferroni post hoc test following a two-way ANOVA. p < 0.01 is denoted (∗∗) and p < 0.005 is denoted (∗∗∗).

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  FIGURE 2. Gβ1 and Gβ3 positively modulate Kv7.4 currents. (Ai) Diagram of voltage step protocol used to examine IV relationships. (Aii–iv) Representative traces of currents generated in cells expressing Kv7.4 with an empty vector (EV) (ii), Gβ1 (iii) or Gβ3 (iv). Zero current is indicated by the dashed line and point used for determination of voltage dependence of activation is indicated by the arrow. Mean data is shown in panel (B) (Gβ1, n = 14) and panel (C) (Gβ3, n = 13). (D) Voltage dependence of activation to determine V1/2 of Kv7.4 with EV (−2.4 ± 2.3 mV), Gβ1 (−9.12 ± 3.9 mV), and Gβ3 (−5.9 ± 3.3 mV). (E) Time dependence of activation of Kv7.4 currents when expressed with EV, Gβ1, and Gβ3 from −20 mV to 40 mV. Data was analyzed by Bonferroni post hoc test following a two-way ANOVA. p < 0.05 is denoted (∗), p < 0.01 is denoted (∗∗) and p < 0.005 is denoted (∗∗∗).

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  FIGURE 3. Gβ2, Gβ4, and Gβ5 effects on Kv7.4 currents. IV relationships of Kv7.4 currents in empty vector (EV) or when co-transfected with Gβ2 (A, n = 12), Gβ4 (B, n = 10) or Gβ5 (C, n = 12). (D) Representative traces of currents cells expression Kv7.4 + EV (upper panel) or with Gβ5 (lower panel). Zero current is indicated by the dashed line and point used for determination of voltage dependence of activation is indicated by the arrow. (E) Voltage dependence of activation curves to determine V1/2 of Kv7.4 with EV (−2.4 ± 2.3 mV) and Gβ5 (−8.1 ± 3 mV). (F) Time dependence of activation of Kv7.4 currents when expressed with EV or Gβ5 from −20 mV to 40 mV. Data was analyzed by Bonferroni post hoc test following a two-way ANOVA. p < 0.05 is denoted (∗), p < 0.01 is denoted (∗∗), and p < 0.005 is denoted (∗∗∗).

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  FIGURE 4. Immunofluorescence of specific Gβ subunits in rat renal artery myocytes. Representative images showing staining in isolated rat renal artery myocytes of Gβ1 (A), Gβ3 (B), and Gβ5 (C), all in red pseudocolour. Nuclear staining by DAPI is shown in blue pseudocolour, while membrane staining by Wheat Germ Agglutinin (WGA) is shown in green pseudocolour. Scale bar: 10 μm.

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  FIGURE 5. Proximity Ligation Assay detection of Kv7.4 interactions with specific Gβ subunits. (A) Representative images of renal artery myocytes investigated for interactions between Kv7.4 and Gβ1, Gβ3 or Gβ5 in control (upper panels) or after treatment with 50 μmol/L gallein (lower panels). (B) Mean data of number of PLA puncta/cell before and after 50 μmol/L gallein treatment for Kv7.4- Gβ1 (i, n = 30–31, N = 5), Kv7.4- Gβ3 (ii, n = 15–33, N = 3–4), and Kv7.4- Gβ1 (iii, n = 11–12, N = 2). “n” = number of cells and “N” = number of animals. Scale bars represent 10 μm. PLA data was analyzed by one-way ANOVA with Bonferroni post hoc analysis where p < 0.005 is denoted (∗∗∗).

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  FIGURE 6. K+ currents in renal artery myocytes – effects of linopirdine and β1 or β3 morpholino. (A) Representative images of whole cell K+ currents recorded in rat renal artery myocytes in control (i) or in the presence of 10 μm/L linopirdine (ii), and mean data [iii, n = 17 N = 4 (control), n = 9 N = 4 (linopirdine)]. Zero current is indicated by the dashed line. (B) Representative images of whole cell K+ currents recorded in rat renal artery myocytes after morpholino knockdown of Gβ1 (i) or Gβ3 (ii) and mean data [iii, n = 10 N = 4 (Gβ1), n = 8 N = 4 (Gβ3)]. Zero current is indicated by the dashed line. “n” = number of cells and “N” = number of animals. Data was analyzed by Bonferroni post hoc test following a two-way ANOVA. p < 0.05 is denoted (∗), p < 0.01 is denoted (∗∗), and p < 0.005 is denoted (∗∗∗).

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  FIGURE 7. Gβ1 knockdown effects on renal artery myocytes. (A) Mean data of whole cell K+ currents recorded in rat renal artery myocytes in Gβ1 morpholino in control (n = 10, N = 4) or in the presence of 10 μm/L linopirdine (n = 8, N = 4). Mean fluorescence intensity of (B) Gβ1 (n = 20–22, N = 4) (C) Gβ3 (n = 15, N = 3) or (D) Kv7.4 (n = 14, N = 3) in renal artery myocytes in control morpholino or Gβ1 morpholino. “n” = number of cells and “N” = number of animals. Electrophysiology data was analyzed by Bonferroni post hoc test following a two-way ANOVA. IF data was analyzed by one-way ANOVA with Bonferroni post hoc analysis. p < 0.01 is denoted (∗∗) and p < 0.005 is denoted (∗∗∗).

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  FIGURE 8. Gβ3 knockdown effects on renal artery myocytes. (A) Mean data of whole cell K+ currents recorded in rat renal artery myocytes in Gβ3 morpholino in control (n = 8, N = 4) or in the presence of 10 μm/L linopirdine (n = 5, N = 4). Mean fluorescence intensity of (B) Gβ3 (n = 20–23, N = 4) (C) Gβ1 (n = 20–21, N = 4) or (D) Kv7.4 (n = 15, N = 3) in renal artery myocytes in control morpholino or Gβ3 morpholino. “n” = number of cells and “N” = number of animals. Electrophysiology data was analyzed by Bonferroni post hoc test following a two-way ANOVA. IF data was analyzed by one-way ANOVA with Bonferroni post hoc analysis. p < 0.01 is denoted (∗∗).