Kv2.1 channels play opposing roles in regulating membrane potential, Ca ²⁺ channel function, and myogenic tone in arterial smooth muscle

Figures

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  Fig. 1.. Female mesenteric myocytes express more Kv2.1 protein and have larger ionic Kv2.1 currents than male myocytes. (A) IKv2.1 records (+50 mV) from representative WT and Kv2.1−/− male and female smooth muscle cells. IKv2.1 traces were obtained by subtracting the currents after the application of ScTx1 from control currents (i.e., IK). (B) Current–voltage relationship of IK2.1. (C) Images of immunoblots of Kv2.1 and β-actin using WT and Kv2.1−/− mesenteric arteries. (D) The bar plot shows the intensities of the Kv2.1 band in all arteries examined. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate mean ± SEM.

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  Fig. 2.. Most Kv2.1 channels in the sarcolemma of male and female arterial myocytes are nonconductive. (A) Kv2.1 gating currents (GxTx1E-sensitive) from representative WT and Kv2.1−/− male and female mesenteric smooth muscle cells. (B) Bar plot of the total charge associated with Qon,Kv2.1 gating currents. (C) Plots of the voltage dependencies of Qon,Kv2.1/Qmax (dashed lines) and Kv2.1 G/Gmax (solid lines) in WT male (Left) and female (Right) myocytes. *P < 0.05. Error bars indicate mean ± SEM.

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  Fig. 3.. Differential effects of acute and chronic loss of Kv2.1 channel function in male and female arteries. (A) Sharp-electrode records of the membrane potential of smooth muscle in pressurized (80-mmHg) WT female and male arteries. (B) Bar plot of the mean ± SEM membrane potential in WT and Kv2.1−/− male and female arteries. (C) Bar plot of the ScTx1-induced depolarization on male and female pressurized (80 mmHg) arteries. (D) Two-dimensional [Ca2+]i images from representative pressurized (80-mmHg) WT and Kv2.1−/− female and male arteries. (Scale bars, 25 µm.) (D, Right) Bar plot showing [Ca2+]i in WT and Kv2.1−/− female and male arteries at intravascular pressures ranging from 20 to 120 mmHg. (E) Pressure–tone relationships of WT and Kv2.1−/− female and male arteries. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars indicate mean ± SEM.

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  Fig. 4.. Expression of Kv2.1 increases the CaV1.2 channel currents in female but not in male arterial myocytes. (A, Left) ICa records (+10 mV) from representative WT and Kv2.1−/− male and female myocytes. (A, Right) Voltage dependence of ICa at membrane potentials ranging from −80 to +60 mV. (B) Voltage dependence of the G/Gmax of ICa in WT and Kv2.1−/− male and female myocytes. (C, Top) Image of a representative CaV1.2 immunoblot. (C, Bottom) Bar plot showing the CaV1.2 band intensity in WT and Kv2.1−/− male and female arteries. (D, Top) CaV1.2 gating current records (nitrendipine-sensitive) from representative WT and Kv2.1−/− female and male mesenteric smooth muscle cells. (D, Bottom) Bar plot showing the Qon,Cav1.2 gating currents. (E) Plots of the voltage dependencies of Qon,Cav1.2/Qmax in WT and Kv2.1−/− male (Top) and female (Bottom) myocytes. Error bars indicate mean ± SEM.

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  Fig. 5.. CaV1.2 channels have a higher open probability and longer open times in female than in male myocytes. (A) Elementary CaV1.2 current records at −30, −10, and +10 mV from representative WT and Kv2.1−/− male and female myocytes. (B) Amplitude of iCa at voltages ranging from −30 to +20 mV. Solid lines are linear fits to the data. (C) Bar plot of the open probability (Po) of single CaV1.2 channel currents at +10 mV in WT and Kv2.1−/− male and female myocytes. *P < 0.05, ***P < 0.001. Error bars indicate mean ± SEM.

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  Fig. 6.. Kv2.1 channel expression increases CaV1.2 channel cluster size in female myocytes to a larger extent than in male myocytes. Superresolution GSD images of regions of interest from representative Kv2.1−/− male and female myocytes. The bar plot shows the mean ± SEM CaV1.2 cluster area per cell. *P < 0.05.

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  Fig. 7.. Kv2.1 channels colocalize with CaV1.2 channels, promoting CaV1.2–CaV1.2 channel interactions. (A) PLA images of representative Kv2.1 and CaV1.2 in WT male and female myocytes. (Scale bars, 15 µm.) (B) TIRF images of Venus fluorescence reconstitution in HEK-293 cells expressing CaV1.2-VN and CaV1.2-VC or CaV1.2-VN, CaV1.2-VC, and Kv2.1P404W-dsRed. (B, Insets) Representative images of dimerization of CaV1.2-VN and CaV1.2-VC. ROI, region of interest. (C) Bar plot showing the mean Venus fluorescence (F/F0) ± SEM after depolarization. (D) ICa currents from the HEK-293 cells expressing CaV1.2-VN and CaV1.2-VC or CaV1.2-VN, CaV1.2-VC, and Kv2.1. (E) I–V relationships of mean ± SEM ICa. G/Gmax sigmoidal curves clearly show a shift in the activation of CaV1.2 channels after the protocol, which is more marked in the cells transfected with CaV1.2-VN, CaV1.2-VC, and Kv2.1 (∼10 mV). *P < 0.05.