Korean J Physiol Pharmacol 2017;21(2):241-249 https://doi.org/10.4196/kjpp.2017.21.2.241
Original Article
Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BKCa activator LDD175 Tharaka Darshana Wijerathne1, Jihyun Kim1, Dongki Yang2, and Kyu Pil Lee1,* 1
Laboratory of Physiology, College of Veterinary Medicine, Chungnam National University, Daejeon 34134, 2Department of Physiology, College of Medicine, Gachon University, Incheon 21936, Korea
ARTICLE INFO Received November 24, 2016 Revised December 26, 2016 Accepted December 27, 2016
*Correspondence Kyu Pil Lee E-mail:
[email protected]
Key Words Ca2+-activated potassium channel hLRRC52 hSlo3 KSper LDD175 Sperm motility
ABSTRACT Plasma membrane hyperpolarization associated with activation of Ca2+-activated K+ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary g2subunit, LRRC52 (leucine-rich-repeat–containing 52), is known to mediate the pHsensitive, sperm-specific K+ current KSper in mice, the molecular identity of this channel in human sperm remains controversial. In this study, we tested the classical BKCa activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting g2 subunit, hLRRC52. As previously reported, Slo3 K+ current was unaffected by iberiotoxin or 4-aminopyridine, but was inhibited by ~50% by 20 mM TEA. Extracellular alkalinization potentiated hSlo3 K+ current, and internal alkalinization and Ca2+ elevation induced a leftward shift its activation voltage. NS1619, which acts intracellularly to modulate hSlo1 gating, attenuated hSlo3 K+ currents, whereas LDD175 increased this current and induced membrane potential hyperpolarization. LDD175-induced potentiation was not associated with a change in the half-activation voltage at different intracellular pHs (pH 7.3 and pH 8.0) in the absence of intracellular Ca2+. In contrast, elevation of intracellular Ca2+ dramatically enhanced the LDD175-induced leftward shift in the half-activation potential of hSlo3. Therefore, the mechanism of action does not involve pH-dependent modulation of hSlo3 gating; instead, LDD175 may modulate Ca2+-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.
Introduction
that mediate the sperm hyperactivation underlying male fertility. Catsper (cation channel sperm-associated 1) and mSlo3 (mouse slowpoke homologue 3; also known as Kcnu1 [potassium channel subfamily U member 1] and Kcnma3 [potassium Ca2+-activated channel subfamily M alpha 1]) in mice, and HVCN1 (hydrogen voltage-gated channel 1) in humans, are ion channels localized to the principal piece of sperm flagellum that are involved in regulating the sperm hyperactivation process necessary for motility. Whereas the Ca2+ permeating channel Catsper can be
Within minutes of vaginal deposition, sperm begin to leave the seminal fluid and swim toward the ovum. The journey of sperm towards the oocyte is a guided event that is capacitated by changes in the local environment surrounding the sperm, including alkalinization and chemotactic hormonal and temperature gradients, among others. Ion channels in sperm are responsible for capacitation and capacitation-associated activities
Author contributions: T.D.W., J.H.K., D.K.Y., and K.P.L. designed the research; T.D.W. and J.H.K. performed the experiments; all authors contri buted to the analysis of data and preparation of the manuscript.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License, which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Copyright © Korean J Physiol Pharmacol, pISSN 1226-4512, eISSN 2093-3827
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Korean J Physiol Pharmacol 2017;21(2):241-249
242 activated directly by intracellular alkalinization and progesterone, mSlo3, which mediates a weakly outwardly rectifying K+ conductance in sperm known as KSper, can sense pH or Ca2+ and set the membrane potential in a way that favors Catsper-mediated Ca2+ influx. However, the characteristics of this K+ conductance in human sperm have not been fully elucidated. Electrophysiological studies have characterized the pH-sensitive + K conductance that sets the membrane potential in mouse sperm [1-3]. Subsequent studies have shown that this current is almost certainly mediated by a complex comprising the pore-forming mSlo3 a-subunit and the auxiliary g2-subunit, LRRC52 (leucinerich-repeat–containing 52) [4]. However, the electrophysiological properties of human KSper differ from those of mouse KSper in that KSper can be inhibited by progesterone, exhibits a distinct pharmacological profile, and is more dependent on regulation by Ca2+ [5]. On the basis of these observations, Mannowetz et al. proposed that Slo1, also known as the BKCa (large-conductance Ca 2+-activated potassium) channel (KCNMA1), rather than Slo3, is responsible for human KSper. Brenker et al. subsequently demonstrated that human KSper exhibits hallmarks of human Slo3 (hSlo3), including insensitivity to the BKCa channel inhibitors iberiotoxin (IbTx) and tetraethylammonium (TEA), Ca 2+dependent rather than pH-dependent activation, and inhibition by progesterone. Collectively, these studies suggest that discrepancies in physiology between human and mouse spermatozoa may reflect the involvement of a different regulatory subunit or subfamily in the modulation of KSper, highlighting the importance of understanding the specific pharmacological profile of hSlo3. The fact that mSlo3 is responsible for capacitation in murine sperm has motivated the search for strong pharmacological modulators of hSlo3 that may provide a biological tool for validating the role of hSlo3 in human sperm physiology and help to establish new therapeutic concepts for male infertility. Several recent studies have demonstrated that general BKCa channel blockers inhibit heterologously expressed Slo3 and human native KSper. However, the resulting modulators display limited preference for hSlo3 relative to hSlo1, and thus do not markedly discriminate between the two channels in vivo or in vitro [5,6]. Therefore, we undertook an alternative approach, applying wellknown BKCa activators to heterologously expressed hSlo3 K+ channels and validating their electrophysiological properties. We found that the BKCa activators, LDD175 (which is recognized also as CTBIC) and NS1619, inversely regulated hSlo3, with NS1619 suppressing hSlo3 activity and LDD175 enhancing hSlo3 activity in a Ca 2+-dependent manner, rather than an intracellular pH (pHi)-dependent manner.
Korean J Physiol Pharmacol 2017;21(2):241-249
Wijerathne TD et al
Methods Cell culture and transfection Human embryonic kidney 293 (HEK293) cells were cultured at 37oC and 5% CO2 in Dulbecco’s Modified Eagle Medium (GIBCO) supplemented with 1X antibiotic-antimycotic reagent (Life Technologies) and 10% fetal bovine serum. HEK293 cells were transiently transfected using Lipofectamine 2000 Transfection Reagent (Life Technologies) as recommended by the vendor. In brief, 1 µg DNA and 5 µl transfection reagent were separately and thoroughly mixed in 50 µ l and 45 µ l of Opti-MEM (31985-070; Life Technologies), respectively, and incubated at room temperature for 5 min. Thereafter, the two solutions were mixed and incubated at room temperature for 20 min before adding to 90% confluent HEK293 cells grown in a 12-well plate in antibiotic- and serum-free Opti-MEM media. hSlo3 function was investigated by co-expressing 0.45 µg of an hSlo3 (NP_114016.1) pCDNA3 expression plasmid, 0.45 µg of an hLRRC52 (NP_114016.1) pCDNA3 expression plasmid and 0.1 µg of a GFP-expressing vector. For rat Slo1 (rSlo1)-overexpression experiments, 0.5 µg rSlo1 (NM_001005214.3) in pCDNA3.1 vector was co expressed with 0.5 µg empty pCDNA3.1 vector. Cells were harvested 24 h after transfection, plated onto coverslips, and analyzed by electrophysiology using the wholecell configuration of the patch-clamp technique.
Electrophysiology Patch pipettes were pulled from thin-wall filament glass capillaries GC 150TF-7.5 (Harvard Apparatus) to a resistance of 3~4 M Ω using a vertical pipette puller (PC-10; Narishige Group Products). An inverted microscope (ECLIPSE Ti; Nikon) was used to identify transfected cells based on their green fluorescence upon illumination at 514 nm. Whole-cell voltageclamp experiments were performed at room temperature using an Axopatch 200B patch-clamp amplifier (Axon Instruments) connected to a Digidata-1440A Digitizer (Axon Instruments). A total of 12 step-pulses from –100 mV to +140 mV were applied from a holding potential of –100 mV. Each test pulse was 400ms long, and the gap between pulses was 600 ms. Linear voltageramp protocols from –100 to +140 mV (holding potential -100 mV) were used in tests of drug responses. Recorded currents were compensated for cell capacitance. All experiments were performed at room temperature (21~25oC). Gigaohm seals were formed in a standard extracellular solution (140 mM NaCl, 5 mM KCl, 10 mM HEPES and 0.5 mM EGTA; pH adjusted to 7.4 using NaOH). Activation of hSlo3 by alkalinization was studied using the following pipette solution: 130 mM K-aspartate, 10 mM NaCl, 1 mM EGTA, 5 mM HEPES, 15 mM D-glucose, pH 6.2 or 7.3 (adjusted using KOH). Activation of hSlo3 by Ca2+ was studied using the following pipette solutions: divalent-free solution https://doi.org/10.4196/kjpp.2017.21.2.241
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Pharmacological regulation of KSper by LDD175
(130 mM K-aspartate, 10 mM NaCl, 1 mM EGTA, and 20 mM HEPES, adjusted to pH 7.3 using KOH), intracellular solutions with different free Ca2+ concentrations (130 mM K-aspartate, 10 mM NaCl, 5 mM EGTA, and 20 mM HEPES; adjusted to pH 7.3 using KOH). Different volumes of a calcium stock solution were added to this base solution in order to obtain final free calcium concentrations of 80, 200, and 1,000 mM. Free Ca2+ concentrations were calculated using the MaxChelator tool (maxchelator. stanford.edu/).
Solutions and chemicals
Results Characterization of hSlo3 Prior to measurement of hSlo3 currents, we examined the response of endogenous potassium currents to the test pro tocols. A +116 mV voltage step elicited a 64.63±6.02 pA/pF (n=20) current in non-transfected HEK293 cells (Fig. 1A). Potassium-carrying currents were not significantly increased by overexpression of hSlo3 (p=0.99, n=8) or hLRRC52 (p=0.94, n=8) alone (data not shown). However, overexpression of hSlo3
TEA (tetraethylammonium), NH4Cl, 4-AP (4-aminopyridine), and NS1619 (1,3-dihydro-1-[2-hydroxy-5-(trifluoromethyl) phenyl]-5-(trifluoromethyl)-2H-benzimidazole-2-one) were purchased from Sigma Aldrich (USA). Iberiotoxin (IbTX) was purchased from TOCRIS Biosciences (USA). LDD175 (4-chloro7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid) was kindly provided by Professor Chul-Seung Park (GIST, Gwangju, Korea). TEA and NH4Cl were directly diluted in physiological perfusion solution prior to use. Stock solutions of 4-AP (1 M) and IbTX (100 mM) were prepared by dissolving the appropriate amount of each chemical in distilled water. Stock solutions of LDD175 (100 mM) and NS1619 (50 mM) were prepared in DMSO (Sigma Aldrich, USA) and 99% ethanol, respectively. All stock solutions were stored at –20oC and used freshly reconstituted to appropriate concentrations in perfusion solutions prior to use.
Statistical analysis Statistical significance of differences among three or more groups was calculated using a one-way analysis of variance (ANOVA) with Bonferroni correction using Origin Pro 8.1 software, and differences between two groups were calculated using unpaired or paired Student’s t-test. All data are given as means±standard error. Gating conductance (G)–voltage (V) curves were calculated by fitting the slope of current (I)– voltage (V) curves to the linear relation, Y=mX+c. Independent Boltzmann fitting (y=A2+(A1–A2)/(1+exp((x–x0)/dx)) was used to calculate activation and inactivation kinetics. Maximum conductivity recorded for each cell was used as the maximum conductivity (Gmax) to normalize the conductivity of each cell. A standard exponential fit in Clampfit software 10.6.2.2 (Molecular Devices, USA) was used to calculate the time constant in step pulses.
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Fig. 1. Pharmacological characterization of hSlo3 currents. (A) Representative traces of endogenous currents in HEK293 cells (upper panel; MOCK), currents in cells transfected only with the hLRRC52 subunit (middle panel; hLRRC52), and currents in cells co-transfected with hSlo3 and the hLRRC52 subunit (lower panel; hSlo3+hLRRC52). Currents were recorded using a step-depolarizing pulse from –100 to +140 mV, with a holding potential of –100 mV. (B) Upper panel: Pharmacological experiments designed to verify hSlo3 K + currents, recorded as in (A) in the presence of TEA (20 mM), IbTx (100 nM), NH4Cl (10 mM), or 4-AP (25 mM). Lower panel: Inhibition was calculated as the percentage of current remaining after treatment relative to that prior to treatment at 116 mV. (C) G~V curves shifted towards the left with increasing alkalinization and internal Ca2+. Left panel: hSlo3 G~V curves, tested at various pHi values: pH 6.0 (●), pH 7.3 (○), pH 8.0 (□), pH 9.0 (□). Right panel: hSlo3 G~V curves, tested at various [Ca2+]i: DVF (●), 80 µM Ca (▲), 200 µM Ca (■), 1000 µM Ca (▼). Data are presented as averages of results from five patches. Lines are fits to the Boltzmann equation. Vh values for each Boltzmann component are indicated for each condition. The number of cells is indicated in a bar graph. *p
