Korean J Physiol Pharmacol.  2019 Sep;23(5):381-392. 10.4196/kjpp.2019.23.5.381.

Onion peel extract and its constituent, quercetin inhibits human Slo3 in a pH and calcium dependent manner

• Wijerathne TD
• Kim JH
• Kim MJ
• Kim CY
• Chae MR
• Lee SW
• Lee KP

• KMID: 2455814
• DOI: http://doi.org/10.4196/kjpp.2019.23.5.381

Abstract

Sperm function and male fertility are closely related to pH dependent K⁺ current (KSper) in human sperm, which is most likely composed of Slo3 and its auxiliary subunit leucine-rich repeat-containing protein 52 (LRRC52). Onion peel extract (OPE) and its major active ingredient quercetin are widely used as fertility enhancers; however, the effect of OPE and quercetin on Slo3 has not been elucidated. The purpose of this study is to investigate the effect of quercetin on human Slo3 channels. Human Slo3 and LRRC52 were co-transfected into HEK293 cells and pharmacological properties were studied with the whole cell patch clamp technique. We successfully expressed and measured pH sensitive and calcium insensitive Slo3 currents in HEK293 cells. We found that OPE and its key ingredient quercetin inhibit Slo3 currents. Inhibition by quercetin is dose dependent and this degree of inhibition decreases with elevating internal alkalization and internal free calcium concentrations. Functional moieties in the quercetin polyphenolic ring govern the degree of inhibition of Slo3 by quercetin, and the composition of such functional moieties are sensitive to the pH of the medium. These results suggest that quercetin inhibits Slo3 in a pH and calcium dependent manner. Therefore, we surmise that quercetin induced depolarization in spermatozoa may enhance the voltage gated proton channel (Hv1), and activate non-selective cation channels of sperm (CatSper) dependent calcium influx to trigger sperm capacitation and acrosome reaction.

Keyword

KSper; LRRC52; Phosphoinositides; Quercetin; Slo3

MeSH Terms

Acrosome Reaction
Calcium*
Fertility
HEK293 Cells
Humans*
Hydrogen-Ion Concentration*
Male
Onions*
Phosphatidylinositols
Protons
Quercetin*
Sperm Capacitation
Spermatozoa
Calcium
Phosphatidylinositols
Protons
Quercetin

Figure

• 

  Fig. 1 Slo3 currents in HEK293 cells are inhibited by onion peel extract (OPE) in a pH dependent manner. Representative step pulse recordings (A, top) and IV curves (A, bottom) measured from cells transfected with different combinations of Slo3 and leucine-rich repeat-containing protein 52 (LRRC52) as indicated. (B) Current densities as calculated from non-transfected (n = 20), LRRC52 transfected (n = 8), Slo3 transfected (n = 8) and Slo3+LRRC52 co-transfected (n = 21) cells. (C) The percentage current remaining after the exposure to 0.1% DMSO (n = 15), 100 µM progesterone (n = 7), 20 µM tetraethylammonium (TEA) (n = 5), 100 nM Iberiotoxin (bTX) (n = 5), 25 mM 4AP 4-aminopyridine (4-AP) (n = 10), and 10 mM NH4Cl (n = 6) for 60 sec. (D) Current density plot of non-transfected (n = 7), Slo3 transfected (n = 6) and Slo3+LRRC52 transfected (n = 6) cells before and after 60 sec of exposure to 100 µg/ml OPE. (E) Continuous recording indicating the changes in Slo3+LRRC52 current upon exposure to100 µg/ml OPE perfused with external solution. (F) The inhibition of Slo3 currents by OPE at concentrations of 1 µg/ml (n = 5), 2.5 µg/ml (n = 5), 10 µg/ml (n = 6), 50 µg/ml (n = 5), 75 µg/ml (n = 5), 100 µg/ml (n = 6), 200 µg/ml (n = 5), 400 µg/ml (n = 5), was dose-dependent that could be fitted to a single dose (continuous line) or double-dose response (dotted line). (G) Representative voltage recording indicating 100 µg/ml OPE depolarize the membrane potential in Slo3 transfected cells in a reversible manner. (H) Average membrane potential shift due to OPE (n = 9) in non-transfected and Slo3 transfected cells. (I) Quercetin dependent inhibition of Slo3 diminishes with increasing internal pH. (J) Relative conductivity plot of Slo3 currents at mentioned internal pH values before and after exposure to the OPE. (K) Half activation voltages as calculated from GV curves at plate J at internal pH of 6 (n = 5) 7.3 (n = 5), 8 (n = 5) and 10 (n = 10). CTRL, control.

• 

  Fig. 2 Quercetin is the main component of onion peel extract (OPE) that inhibits Slo3 in a pH dependent manner. (A) Representative step pulses before (top left) and after (bottom left) exposure to 50 µM quercetin. (Right) IV curve generated from step pulses show at left. (B) Dose response of quercetin on Slo3 calculated at doses of 1 µM (n = 5), 3.12 µM (n = 5), 10 µM (n = 5), 50 µM (n = 7), and 150 µM (n = 5) reveals a EC50 value of 10.87 ± 3.48 µM. Quercetin depolarizes the cell membrane in Slo3 over expressing cells as shown in continuous (C) recording and average membrane potential data (D) (n = 5). (E) Relative Slo3 current inhibition by quercetin at different internal pH levels of 6.0 (n = 8), 7.3 (n = 9), 8.0 (n = 7), and 9.0 (n = 7). (F) Normalized GV curves representing effect of quercetin on mentioned internal pH conditions. (G) Half activation voltages calculated from plate D, plotted against internal proton concentration. CTRL, control. *p < 0.05.

• 

  Fig. 3 Raising internal free calcium hinders the quercetin dependent inhibition of Slo3. Relative current remaining (A) and change in GV curves (B) after exposure to quercetin at internal free calcium concentrations of 0 µM (n = 8), 20 µM (n = 6), and 200 µM (n = 6) buffered by ethylene glycol bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid (EGTA). (C) Half activation voltage calculated from the GV curves in plate B. CTRL, control.

• 

  Fig. 4 Relative inhibition of Slo3 by quercetin is dependent on structural variations in quercetin. (A) Increasing in external pH decreases the inhibitory effects of quercetin. (C) Percentage current remaining after exposure to 50 µM quercetin (n = 7) compared to its structural analogues, 50 µM luteolin (n = 7) and 50 µM morin (n = 7). (B) Step recordings and IV curves representing the inhibition of current after exposure to quercetin (top), luteolin (middle) and morin (bottom). (D) Structure of each flavonoid with the different number of hydrogen bond acceptors in each chemical. Chemical structures and information on hydrogen bond acceptors of each chemical are adopted from IUPHAR/BPS Guide to PHARMACOLOGY [48]. CTRL, control. *p < 0.05.

• 

  Fig. 5 Protein kinase or phosphoinositide 3 kinase (PI3K) inhibitory action of quercetin alone cannot explain the inhibitory effect of quercetin on Slo3. (A) Representative step pulses prior to treatment (left), after exposure to protein kinase A (PKA) inhibitor H89 (middle), after exposing to quercetin with H89 (right) and representative IV curve generated from those step pulses. (B) Representative step pulses prior to treatment (left), after exposure to protein kinase C (PKC) inhibitor GF109203X (GFX) (middle), after exposing to quercetin with GFX (right) and representative IV curve generated from those step pulses. (C) Relative current remaining after treatment of 50 µM quercetin (n = 7) and 5 µM protein kinase inhibitors H89 (n = 6) and GFX (n = 5). (D) Relative inhibition of Slo3 currents by quercetin in cells pre-treated with protein kinase inhibitor (n = 6). (E) Representative continuous trace indicating the further inhibition of Slo3 currents by quercetin in the cells pretreated with protein kinase inhibitors. (F) Representative step pulses prior to treatment (left), after exposure to PI3K inhibitor wortmanin (middle), after exposing to quercetin with wortmanin (right) and IV curve generated from those step pulses. (G) Relative current remaining after treatment of 50 µM quercetin (n = 6) and 5 µM PI3K inhibitors wortmanin (n = 5). (H) Relative inhibition of Slo3 currents by quercetin in cells pre-treated with PI3K inhibitor (n = 5) compared to control (n = 6). (I) Representative continuous trace indicating the further inhibition of Slo3 currents by quercetin in the cells pretreated with PI3K inhibitor wortmanin. Compared to untreated (DMSO) condition. Ctrl, control. *p < 0.05.

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