title: Niflumic acid blocks native and recombinant T-type channels.
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.
authors: Departamento de Genética del Desarrollo y Fisiología Molecular, Instituto de Biotecnología, Universidad Nacional Autónoma de México (UNAM), Avenida Universidad 2001, Col. Chamilpa, Cuernavaca Mor., Mexico.
authors: Enrique
authors: E
authors: Balderas
authors: Rogelio
authors: R
authors: Ateaga-Tlecuitl
authors: Manuel
authors: M
authors: Rivera
authors: Juan C
authors: JC
authors: Gomora
authors: Alberto
authors: A
authors: Darszon
mesh_terms: D000818:Animals; D001665:Binding Sites; D002121:Calcium Channel Blockers / Q000737:chemistry / Q000494:pharmacology*; D020747:Calcium Channels, T-Type / Q000737:chemistry / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004558:Electric Stimulation; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D008297:Male; D008564:Membrane Potentials; D051379:Mice; D008958:Models, Molecular; D015394:Molecular Structure; D009544:Niflumic Acid / Q000737:chemistry / Q000494:pharmacology*; D018408:Patch-Clamp Techniques; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000187:drug effects / Q000378:metabolism; D013091:Spermatogenesis / Q000187:drug effects*; D013094:Spermatozoa / Q000187:drug effects* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection
chemical_list: C121855:CACNA1G protein, human; C479952:CACNA1H protein, human; C469757:CACNA1I protein, human; D002121:Calcium Channel Blockers; D020747:Calcium Channels, T-Type; D011994:Recombinant Proteins; D009544:Niflumic Acid