Korean J Physiol Pharmacol.  2020 Nov;24(6):503-516. 10.4196/kjpp.2020.24.6.503.

The agonistic action of URO-K10 on Kv7.4 and 7.5 channels is attenuated by co-expression of KCNE4 ancillary subunit

• Lee JE
• Park CH
• Kang H
• Ko J
• Cho S
• Woo J
• Chae MR
• Lee SW
• Kim SJ
• Kim J
• So I

• KMID: 2507733
• DOI: http://doi.org/10.4196/kjpp.2020.24.6.503

Abstract

KCNQ family constitutes slowly-activating potassium channels among voltage-gated potassium channel superfamily. Recent studies suggested that KCNQ4 and 5 channels are abundantly expressed in smooth muscle cells, especially in lower urinary tract including corpus cavernosum and that both channels can exert membrane stabilizing effect in the tissues. In this article, we examined the electrophysiological characteristics of overexpressed KCNQ4, 5 channels in HEK293 cells with recently developed KCNQ-specific agonist. With submicromolar EC₅₀ , the drug not only increased the open probability of KCNQ4 channel but also increased slope conductance of the channel. The overall effect of the drug in whole-cell configuration was to increase maximal whole-cell conductance, to prolongate the activation process, and left-shift of the activation curve. The agonistic action of the drug, however, was highly attenuated by the co-expression of one of the βancillary subunits of KCNQ family, KCNE4. Strong in vitro interactions between KCNQ4, 5 and KCNE4 were found through Foster Resonance Energy Transfer and co-immunoprecipitation. Although the expression levels of both KCNQ4 and KCNE4 are high in mesenteric arterial smooth muscle cells, we found that 1 μM of the agonist was sufficient to almost completely relax phenylephrine-induced contraction of the muscle strip. Significant expression of KCNQ4 and KCNE4 in corpus cavernosum together with high tonic contractility of the tissue grants highly promising relaxational effect of the KCNQspecific agonist in the tissue.

Keyword

KCNE4; KCNQ4; KCNQ5; Vasorelaxation

Figure

• 

  Fig. 1 Chemical nature, reference, purchase information of the agonist/antagonists used in the study. (1) URO-K10 was synthesized by Sundia MediTech Company (Sanghai, China) based on reaction scheme suggested by Seefeld et al. [24]. (2) ML-213 was purchased from Tocris Bioscience (Bristol, UK). (3) XE-991 was purchased from Sigma Aldrich (St. Louis, MO, USA).

• 

  Fig. 2 Electrophysiologic characteristics of overexpressed Kv7.4 and Kv7.5 channels in HEK293 cells. (A) Voltage clamp current traces of control (black) and Kv7.4 with URO-K10 (blue) are shown. (B) Corresponding I–V curve is plotted with steady-state currents measured from –100 mV to +100 mV at 10 mV step intervals. At +100 mV, Kv7.4-expressing cells showed 6.08 ± 1.60 nA whole-cell current in the presence of 1 μM URO-K10. (C) Boltzmann function-fitted conductance curves with URO-K10 show a leftward shift compared to the control (half-maximal voltage of –59.13 ± 6.33 mV). (D) Activation processes are slower with URO-K10 (blue), and (E) activation time constant values increased with the presence of the drug. (F) Voltage clamp traces of control (black) and Kv7.5 with URO-K10 (blue) are shown. (G) I–V curve of Kv7.5 expressing cells is shown. At +100 mV, Kv7.5-expressing cells showed 5.26 ± 2.19 nA whole-cell current in the presence of 1 μM URO-K10. (H) Boltzmann function-fitted steady-state conductance curve of Kv7.5 expressing cells is shown (half-maximal voltage of –74.93 ± 8.64 mV). (I, J) Activation time constant values of Kv7.5-expressing cells in 1 μM URO-K10 are plotted against voltage. In (H) and (J), both regression of conductance-voltage data to Boltzmann function and regression of current-time data to single exponential function were deliberately disregarded in Kv7.5-expressing cells without 1μM URO-K10 (control) (see manuscript for detailed description).

• 

  Fig. 3 Dose-dependent action of URO-K10 on Kv7.4 and Kv7.5. (A) Kv7.4 current traces are obtained at +50 mV with URO-K10 concentrations ranging from 1 pM to 10 μM. At each concentration, drug-affected current traces at +50 mV was subtracted from drug-free current traces at the same cell and at the same membrane potential (Idiff). (B) Dose-response curve in a logarithmic scale shows EC50 value of 210.8 nM for Kv7.4. (C, D) Dose-dependent action of URO-K10 on Kv7.5 is also shown in parallel. Dose-response curve in a logarithmic scale shows EC50 value of 142.0 nM for Kv7.5. In both (A) and (C), black inset lines indicate corresponding voltage-clamp step.

• 

  Fig. 4 URO-K10 increases both open probability and slope conductance of KCNQ4 channel. (A) Current traces from cell-attached single channel recordings at given commanding potentials. Traces in blue lines indicate pipette solution with URO-K10 (1 μM) while traces in black lines indicate control. At each commanding potential, dotted lines and full lines indicate closed states (C) and open states (O), respectively. (B) Open probabilities of KCNQ4 channel in 30 sec recording interval. In both traces, –160 mV of commanding potential was applied. URO-K10 (1 μM) significantly increased mean open probability of KCNQ4 channel (0.15 vs. 0.46). (C) Slope conductances calculated from unitary current amplitude at given commanding potential. 1 μM URO-K10 significantly increased slope conductance of KCNQ4 channel (4.77 pS vs. 18.85 pS).

• 

  Fig. 5 Kv7.4 and Kv7.5 have a molecular interaction with ancillary subunit KCNE4. (A) Fluorescence imaging of KCNQ4-ECFP, KCNQ5-ECFP and KCNE4-EYFP. Both KCNQ4-ECFP and KCNQ5-ECFP showed strong cyan fluorescence in plasma membrane and inside the cell. KCNE4-EYFP, however, showed discrete, puncta-like YFP signal inside the cell while signal in plasma membrane was highly limited. (B) Fluorescence imaging of KCNQ4-ECFP+KCNE4-EYFP and KCNQ5-ECFP+KCNE4-EYFP. When co-expressed with KCNQ4 or KCNQ5, prominent localization of YFP signal in plasma membrane was observable. High effective FRET efficiency (EEFF, see Methods) between KCNQ4-ECFP and KCNE4-EYFP (11.3% ± 1.5%, n = 7), and between KCNQ5-ECFP and KCNE4-EYFP (12.5% ± 1.6%, n = 7) were also observable, suggesting strong in vitro interaction between each α subunit and KCNE4. (C) Co-immunoprecipitation blot shows interaction with KCNE4 at a molecular level. KCNQ4-EGFP band (104 kDa) and KCNQ5-EGFP band (130 kDa) is observed when pulled down with the anti-Flag antibody. Conversely, KCNE4-Flag (23 kDa) is observed when pulled down with anti-GFP.

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  Fig. 6 Electrophysiology of KCNQ4/KCNE4 and KCNQ5/KCNE4 complex. (A) KCNE4 suppresses the activity of KCNQ4, as shown in the whole-cell potassium current traces. Whole-cell potassium current in KCNQ4/KCNE4 cells is 1.66 ± 0.72 nA at +100 mV. (B, C) I–V and conductance (G–V) curves are shown for control (KCNQ4/E4, black) and with 1 μM URO-K10 (blue). Half-maximal voltage in the presence of the drug is –67.35 ± 5.50 mV. (D–F) Electrophysiological characteristics of KCNQ5/KCNE4 are also shown in parallel. In 1 μM URO-K10, whole cell potassium current in KCNQ5/KCNE4 was 2.51 ± 1.29 nA at +100 mV and the half-maximal voltage was –72.73 ± 7.09 mV. (G) Maximum steady-state conductances of KCNQ4/E4 and KCNQ5/E4 are compared. KCNE4 suppresses both agonist-activated and intrinsic current of KCNQ4. KCNE4 suppresses the agonist-activated current of KCNQ5, but its effects on intrinsic currents are not shown due to the small current size indistinguishable from the background HEK293 cell current. (H) Half maximal voltages of KCNQ4/E4 and KCNQ5/E4 are compared. KCNE4 exerts a negligible effect on the voltage-dependency of KCNQ4 and KCNQ5 channels. **p < 0.05.

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  Fig. 7 Effect of URO-K10 onto mesenteric arterial vasorelaxation. (A) Isometric pressure myograph shows almost complete relaxation of phenylephrine-induced contraction upon administration of 1 μM URO-K10 (blue) (11.42% ± 2.06%, n = 4). (B) This relaxation effect is abolished in 80 mM potassium extracellular concentration (86.88% ± 3.05%, n = 6). (C) Bar graph summarizes percentages of contraction with and without 1 μM URO-K10 in 5 mM or 80 mM potassium. (D) Various concentrations of URO-K10 (from 0.1 to 1 μM) was applied to measure EC50 in a tissue-specific scale. (E) Percentages of contraction is plotted against concentration at a logarithmic scale. EC50 is estimated to be 521.0 nM. ***p < 0.001.

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