Sperm capacitation is a organic and indispensable physiological process that spermatozoa must undergo in order to acquire fertilization capability. of the potential K+ channels were involved in this hyperpolarization we used different K+ channel inhibitors including charybdotoxin slotoxin and iberiotoxin (which target Slo1) and clofilium (a more specific blocker for Slo3). All these K+ channel antagonists inhibited membrane hyperpolarization to a similar extent suggesting that both users of the Slo family may potentially participate. Two very recent papers recorded K+ currents in human sperm electrophysiologically with some contradictory results. In the present work we show through immunoblotting that Slo3 channels are present in the human sperm membrane. In addition we found that human Slo3 channels expressed in CHO cells were sensitive to clofilium (50 μM). Considered altogether our data show that Slo1 and Slo3 could share the preponderant role in the capacitation-associated hyperpolarization of human sperm in contrast to what has been previously reported for mouse sperm where Slo3 channels are the main contributors to the hyperpolarization event. (2013) recently showed that Slo3 is the principal channel responsible for mouse hyperpolarization during capacitation and that its K+ permeability increases three times during this process while Na+ and Cl? permeabilities remain practically the same. Despite the different functions that membrane hyperpolarization may play during capacitation this Em switch has not been demonstrated in human MF1 sperm. You will find few reports regarding this subject. Linares-Hernandez (1998) reported that this Emr of non-capacitated human sperm is around ?40 mV whereas Patrat (2002) reported that capacitated sperm exhibit an Emr of about ?58 mV. An exploratory study using cell sorting and an Em sensitive dye reported that capacitated human sperm undergo a BRD9757 hyperpolarization (Brewis I and III sites. A III site was inserted into the hSlo3 cDNA sequence using oligonucleotide primers 5′ GGG AAG CTT GAG TCT AGA Take action AGT ATA GTG GCT 3′ and 5′ CGC ATT TAA CCC BRD9757 TCA CTA AAG 3′ synthesized by IBT/UNAM (Cuernavaca BRD9757 Mexico). PCR was performed using high-fidelity Phusion? DNA polymerase (New England BioLabs Ipswich MA USA) starting with a denaturation step at 98°C for 30 s followed by 30 cycles under the following conditions: 98°C for 10 s 45 for 20 s and 72°C for 70 s. The final elongation step was performed at 72°C for 10 min. The presence of a single product (～3500 bp) was confirmed in a 1% agarose gel and it was purified using a quick gel extraction protocol by column (Fermentas). The amplified fragment was digested with appropriate restriction enzymes and ligated into the pcDNA3.1(?) vector using standard protocols. Recombinant clones were subsequently sequenced on both strands to confirm the identity of the construct. Mouse Slo3 channel cDNA (Santi I and I restriction sites. Cell culture CHO cells were maintained in culture using Advanced Dulbecco’s altered Eagle’s medium (Gibco Invitrogen) supplemented with 1% antibiotics and 10% BSA (Gibco Life Technologies Paisley UK). Cells were grown in plastic Petri dishes incubated in a humidity-controlled incubator at 37°C under 5% CO2 (VWR Scientific 2100). Transient expression of hSlo3 channels in CHO cells Fifty percent of confluent CHO cells were transfected with 1 μg of the hSlo3 construct mixed with Lipofectamine (Invitrogen) according to the manufacturer’s protocol. Transfected CHO cells were managed in advanced DMEM medium and were used in the studies explained >48 h post-transfection. BRD9757 Control experiments include cells transfected with vacant pcDNA3.1(?) vector. Design and synthesis of anti-hSlo3 antibodies Two chicken anti-Slo3 channel antibodies (U1493 and U1504) were commissioned from New England Peptides using the antigen peptide Ac-CELKNPSNIHFIE-amide corresponding to either amino acid residues 864-875 of human (NCBI Gene BRD9757 ID: 157855) or 869-880 of mouse (NCBI Gene ID: 16532) Slo3 channels. SDS-PAGE and western blot analysis Human sperm after swim-up separation (～30 × 106 cells/lane) were washed once (600for 20 min the supernatants were collected and 20 μg of total protein was mixed with loading buffer (500 mM Tris 8 mM EDTA; 1 μg bromophenol blue/mL 10 v/v SDS 50 v/v glycerol and 5% v/v 2-mercaptoethanol)..