Korean J Physiol Pharmacol.  2017 Mar;21(2):241-249. 10.4196/kjpp.2017.21.2.241.

Intracellular calcium-dependent regulation of the sperm-specific calcium-activated potassium channel, hSlo3, by the BK(Ca) activator LDD175

• Wijerathne TD
• Kim J
• Yang D
• Lee KP

• KMID: 2371043
• DOI: http://doi.org/10.4196/kjpp.2017.21.2.241

Abstract

Plasma membrane hyperpolarization associated with activation of Ca²âº-activated K⁺ channels plays an important role in sperm capacitation during fertilization. Although Slo3 (slowpoke homologue 3), together with the auxiliary γ2-subunit, LRRC52 (leucine-rich-repeat-containing 52), is known to mediate the pH-sensitive, 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 BK(Ca) activators, NS1619 and LDD175, on human Slo3, heterologously expressed in HEK293 cells together with its functional interacting γ2 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 Ca²âº 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 Ca²âº. In contrast, elevation of intracellular Ca²âº 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 Ca²âº-dependent activation of hSlo3. Thus, LDD175 potentially activates native KSper and may induce membrane hyperpolarization-associated hyperactivation in human sperm.

Keyword

Ca²⁺-activated potassium channel; hLRRC52; hSlo3; KSper; LDD175; Sperm motility

MeSH Terms

4-Aminopyridine
Animals
Cell Membrane
Fertilization
HEK293 Cells
Humans
Hydrogen-Ion Concentration
Membrane Potentials
Membranes
Mice
Potassium Channels, Calcium-Activated*
Sperm Capacitation
Sperm Motility
Spermatozoa
Tea
4-Aminopyridine
Potassium Channels, Calcium-Activated
Tea

Figure

• 

  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<0.05.

• 

  Fig. 2 Effects of the BKCa activator NS1619 on hSlo3 K+ currents.(A) Left panel: Extracellular application of 50 µM NS1619 inhibits hSLo3+hLRRC52 currents. Inhibition was calculated as the percentage of current remaining after treatment relative to that prior to treatment at 116 mV. Right panel: Representative traces showing I~V relationships before (●) and after (○) treatment. (B) Inhibition of hSlo3+hLRRC52 current by 50 µM NS1619, determined by measuring changes in membrane potential before and after treatment. Left panel: NS1619-induced membrane potential depolarization. Right panel: Representative continuous trace showing changes in membrane potential during the application of 50 µM NS1619. (C) Left panel: Concentration-response curve for NS1619 inhibition of hSlo3+hLRRC52. Right panel: Representative I~V traces showing changes in current with the application of NS1619. The number of cells is indicated in a bar graph. *p<0.05, **p<0.01.

• 

  Fig. 3 LDD175 activates hSLo3+hLRRC52 currents.(A) Left panel: Extracellular application of 30 µM LDD175 enhances hSLo3+hLRRC52 currents. Activation was calculated as the percentage of current remaining after treatment relative to that prior to treatment at 116 mV. Right panel: Representative traces showing I~V relationships before (●) and after (○) treatment. (B) Left panel: Concentration-response curve for LDD175-induced activation of hSlo3+hLRRC52. Right panel: Representative I~V traces showing changes in current with the application of LDD175. (C) Activation of hSlo3+hLRRC52 current by 30 µM LDD175, as evidenced by changes in membrane potential after treatment. Left panel: Treatment with 30 µM LDD175 induces membrane potential hyperpolarization. Right panel: Representative continuous trace showing changes in membrane potential during the application of 30 µM LDD175. The number of cells is indicated in a bar graph. **p<0.01.

• 

  Fig. 4 LDD175 potentiates K+ currents in both rSlo1-transfected and hSlo3+hLRRC52–co-transfected cells.(A) The application of 10 µM LDD175 causes a rightward shift in the rSlo1 steady-state G~V relationship (left panel), significantly decreases Vh values (middle panel), and increases the amplitude of step currents. (B, C) External application of 10 µM LDD175 significantly increases the relative conductivity of hSlo3+hLRRC52 at pH 7.3 (B, left panel) and pH 8.0 (C, left panel), and increases the amplitude and changes the shape of step currents (right panels). The number of cells is indicated in a bar graph. **p<0.01

• 

  Fig. 5 Intracellular-Ca2+–dependent regulation of hSlo3+hLRRC52 currents by LDD175 in co-transfected HEK293 cells.(A) Left panel: Steady-state G~V relationships for hSlo3+hLRRC52 at different [Ca2+]i, and their half-activation voltages. Right panel: Increasing internal free Ca2+ facilitates the effect of LDD175 (○) compared with controls (●). (B) Left panel: Activation time constants calculated in the presence or absence of LDD175. Right panel: Representative currents.

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