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Niflumic acid blocks native and recombinant T-type channels
Enrique Balderas1, Rogelio Ateaga-Tlecuitl, Manuel Rivera
1Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 2001, Col. Chamilpa, Cuernavaca Mor., Mexico.
Abstract:
Voltage-dependent calcium channels are widely distributed in animal cells, including spermatozoa. Calcium is fundamental in many sperm functions such as: motility, capacitation, and the acrosome reaction (AR), all essential for fertilization. Pharmacological evidence has suggested T-type calcium channels participate in the AR. Niflumic acid (NA), a non-steroidal anti-inflammatory drug commonly used as chloride channel blocker, blocks T-currents in mouse spermatogenic cells and Cl(-) channels in testicular sperm. Here we examine the mechanism of NA blockade and explore if it can be used to separate the contribution of different Ca(V)3 members previously detected in these cells. Electrophysiological patch-clamp recordings were performed in isolated mouse spermatogenic cells and in HEK cells heterologously expressing Ca(V)3 channels. NA blocks mouse spermatogenic cell T-type currents with an IC(50) of 73.5 µM, without major voltage-dependent effects. The NA blockade is more potent in the open and in the inactivated state than in the closed state of the T-type channels. Interestingly, we found that heterologously expressed Ca(V)3.1 and Ca(V)3.3 channels were more sensitive to NA than Ca(V)3.2 channels, and this drug substantially slowed the recovery from inactivation of the three isoforms. Molecular docking modeling of drug-channel binding predicts that NA binds preferentially to the extracellular face of Ca(V)3.1 channels. The biophysical characteristics of mouse spermatogenic cell T-type currents more closely resemble those from heterologously expressed Ca(V)3.1 channels, including their sensitivity to NA. As Ca(V)3.1 null mice maintain their spermatogenic cell T-currents, it is likely that a novel Ca(V)3.2 isoform is responsible for them.
Insights
Niflumic acid blocks T-type calcium channels in mouse sperm, aiding research into calcium channel roles in fertilization. This study identifies Ca(V)3.1 channels as key players in sperm function.
Area of Science:
- Spermatogenesis and Fertilization Biology
- Molecular and Cellular Physiology
- Pharmacology of Ion Channels
Background:
- Voltage-dependent calcium channels (CaV) are crucial for sperm functions like motility and acrosome reaction (AR).
- T-type calcium channels (CaV3) are implicated in the AR, but their specific roles and isoforms in sperm remain unclear.
- Niflumic acid (NA), an anti-inflammatory drug, is known to block T-currents and chloride channels in sperm.
Purpose of the Study:
- To investigate the mechanism of niflumic acid (NA) blockade on T-type calcium channels in mouse spermatogenic cells.
- To determine if NA can differentiate between CaV3 channel isoforms (CaV3.1, CaV3.2, CaV3.3) in sperm.
- To elucidate the specific CaV3 isoform responsible for T-type currents in mouse spermatogenic cells.
Main Methods:
- Electrophysiological patch-clamp recordings in isolated mouse spermatogenic cells and HEK cells expressing CaV3 isoforms.
- Pharmacological analysis of niflumic acid (NA) blockade kinetics and voltage-dependence.
- Molecular docking simulations to predict NA binding sites on CaV3 channels.
Main Results:
- Niflumic acid (NA) effectively blocks T-type currents in mouse spermatogenic cells with an IC50 of 73.5 µM.
- NA blockade is state-dependent, being more potent on open and inactivated channels.
- Heterologously expressed CaV3.1 and CaV3.3 channels show higher sensitivity to NA than CaV3.2, and NA slows recovery from inactivation for all three isoforms.
- Molecular docking suggests NA binds to the extracellular side of CaV3.1.
- Spermatogenic cell T-type currents resemble CaV3.1 in biophysical properties and NA sensitivity.
Conclusions:
- Niflumic acid (NA) is a valuable tool for studying T-type calcium channels in sperm.
- Mouse spermatogenic cell T-type currents share characteristics with CaV3.1 channels.
- Despite CaV3.1 null mice retaining T-currents, the data suggest CaV3.1's significant role, potentially with compensatory mechanisms or involvement of other isoforms like CaV3.2.
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