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Study questionDoes a man (patient 1) with a previously described deficiency in principle cation channel of sperm (CatSper) function have a mutation in the CatSper-epsilon (CATSPERE) and/or CatSper-zeta (CATSPERZ) gene?Summary answerPatient 1 has a homozygous in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE.What is known alreadyCatSper is the principal calcium channel of mammalian spermatozoa. Spermatozoa from patient 1 had a specific loss of CatSper function and were unable to fertilize at IVF. Loss of CatSper function could not be attributed to genetic abnormalities in coding regions of seven CatSper subunits. Two additional subunits (CatSper-epsilon (CATPSERE) and CatSper-zeta (CATSPERZ)) were recently identified, and are now proposed to contribute to the formation of the mature channel complex.Study design, size, durationThis was a basic medical research study analysing genomic data from a single patient (patient 1) for defects in CATSPERE and CATSPERZ.Participants/materials, setting, methodsThe original exome sequencing data for patient 1 were analysed for mutations in CATSPERE and CATSPERZ. Sanger sequencing was conducted to confirm the presence of a rare variant.Main results and the role of chancePatient 1 is homozygous for an in-frame 6-bp deletion in exon 18 (c.2393_2398delCTATGG, rs761237686) of CATSPERE that is predicted to be highly deleterious.Limitations, reasons for cautionThe nature of the molecular deficit caused by the rs761237686 variant and whether it is exclusively responsible for the loss of CatSper function remain to be elucidated.Wider implications of the findingsPopulation genetics are available for a significant number of predicted deleterious variants of CatSper subunits. The consequence of homozygous and compound heterozygous forms on sperm fertilization potential could be significant. Selective targeting of CatSper subunit expression maybe a feasible strategy for the development of novel contraceptives.Study funding/competing interest(s)This study was funded by project grants from the MRC (MR/K013343/1 and MR/012492/1), Chief Scientist Office/NHS research Scotland. This work was also supported by NIH R01GM111802, Pew Biomedical Scholars Award 00028642 and Packer Wentz Endowment Will to P.V.L. C.L.R.B is the editor-in-chief of Molecular Human Reproduction, has received lecturing fees from Merck and Ferring, and is on the Scientific Advisory Panel for Ohana BioSciences. C.L.R.B was chair of the World Health Organization Expert Synthesis Group on Diagnosis of Male infertility (2012-2016).

To help students develop into confident and effective laboratory scientists it is necessary to facilitate their understanding and ability to participate effectively in all processes involved in experimental design and implementation in an engaging and realistic manner. Our challenge was to develop a 20-credit, ordinary degree (without honours) level, practical skills-based module for Biomedical Science students who are not on a National Health Service placement that encompasses elements of professionalism. We describe the design of a module that utilises Inquiry-based learning (IBL) and Quality by Design (QbD) principles and discuss staff experience and reflections following analysis of six years of anonymised module feedback data which includes quantitative and qualitative data. Key positive themes from the qualitative analysis were satisfaction at being able to make evidenced based decisions for changes in experimental factors and development of personal skills which was perceived as helpful for their preparation for their honours year project. The level of challenge that the format of the module presented was perceived negatively. We conclude that this mode of learning is challenging to implement and monitor for academics and challenges students intellectual and professional development, but that the benefits are worth the investment. [ABSTRACT FROM AUTHOR]

Study questionAre significant abnormalities in outward (K(+)) conductance and resting membrane potential (Vm) present in the spermatozoa of patients undertaking IVF and ICSI and if so, what is their functional effect on fertilization success?Summary answerNegligible outward conductance (≈5% of patients) or an enhanced inward conductance (≈4% of patients), both of which caused depolarization of Vm, were associated with a low rate of fertilization following IVF.What is known alreadySperm-specific potassium channel knockout mice are infertile with defects in sperm function, suggesting that these channels are essential for fertility. These observations suggest that malfunction of K(+) channels in human spermatozoa might contribute significantly to the occurrence of subfertility in men. However, remarkably little is known of the nature of K(+) channels in human spermatozoa or the incidence and functional consequences of K(+) channel defects.Study design, size and durationSpermatozoa were obtained from healthy volunteer research donors and subfertile IVF and ICSI patients attending a hospital assisted reproductive techniques clinic between May 2013 and December 2015. In total, 40 IVF patients, 41 ICSI patients and 26 normozoospermic donors took part in the study.Participants/materials, setting, methodsSamples were examined using electrophysiology (whole-cell patch clamping). Where abnormal electrophysiological characteristics were identified, spermatozoa were further examined for Ca(2+) influx induced by progesterone and penetration into viscous media if sufficient sample was available. Full exome sequencing was performed to specifically evaluate potassium calcium-activated channel subfamily M α 1 (KCNMA1), potassium calcium-activated channel subfamily U member 1 (KCNU1) and leucine-rich repeat containing 52 (LRRC52) genes and others associated with K(+) signalling. In IVF patients, comparison with fertilization rates was done to assess the functional significance of the electrophysiological abnormalities.Main results and the role of chancePatch clamp electrophysiology was used to assess outward (K(+)) conductance and resting membrane potential (Vm) and signalling/motility assays were used to assess functional characteristics of sperm from IVF and ICSI patient samples. The mean Vm and outward membrane conductance in sperm from IVF and ICSI patients were not significantly different from those of control (donor) sperm prepared under the same conditions, but variation between individuals was significantly greater (P< 0.02) with a large number of outliers (>25%). In particular, in ≈10% of patients (7/81), we observed either a negligible outward conductance (4 patients) or an enhanced inward current (3 patients), both of which caused depolarization of Vm. Analysis of clinical data from the IVF patients showed significant association of depolarized Vm (≥0 mV) with low fertilization rate (P= 0.012). Spermatozoa with electrophysiological abnormities (conductance and Vm) responded normally to progesterone with elevation of [Ca(2+)]i and penetration of viscous medium, indicating retention of cation channel of sperm (CatSper) channel function.Limitations, reasons for cautionFor practical, technical, ethical and logistical reasons, we could not obtain sufficient additional semen samples from men with conductance abnormalities to establish the cause of the conductance defects. Full exome sequencing was only available in two men with conductance defects.Wider implications of the findingsThese data add significantly to the understanding of the role of ion channels in human sperm function and its impact on male fertility. Impaired potassium channel conductance (Gm) and/or Vm regulation is both common and complex in human spermatozoa and importantly is associated with impaired fertilization capacity when the Vm of cells is completely depolarized.Study funding/competing interestsThe majority of the data were obtained using funding from MRC project grants (#MR/K013343/1, MR/012492/1). Additional funding was provided by NHS Tayside, TENOVUS, Chief Scientist Office NRS Fellowship and University of Abertay. The authors declare that there is no conflict of interest.Trial registration numberNot applicable.

Study questionAre significant abnormalities of CatSper function present in IVF patients with normal sperm concentration and motility and if so what is their functional significance for fertilization success?Summary answerSperm with a near absence of CatSper current failed to respond to activation of CatSper by progesterone and there was fertilization failure at IVF.What is known alreadyIn human spermatozoa, Ca(2+) influx induced by progesterone is mediated by CatSper, a sperm-specific Ca(2+) channel. A suboptimal Ca(2+) influx is significantly associated with, and more prevalent in, men with abnormal semen parameters, and is associated with reduced fertilizing capacity. However, abnormalities in CatSper current can only be assessed directly using electrophysiology. There is only one report of a CatSper-deficient man who showed no progesterone potentiated CatSper current. A CatSper 2 genetic abnormality was present but there was no information on the [Ca(2+)]i response to CatSper activation by progesterone. Additionally, the semen samples had indicating significant abnormalities (oligoasthenoteratozoospermia) multiple suboptimal functional responses in the spermatozoon. As such it cannot be concluded that impaired CatSper function alone causes infertility or that CatSper blockade is a potential safe target for contraception.Study design, size, durationSpermatozoa were obtained from donors and subfertile IVF patients attending a hospital assisted reproductive techniques clinic between January 2013 and December 2014. In total 134 IVF patients, 28 normozoospermic donors and 10 patients recalled due to a history of failed/low fertilization at IVF took part in the study.Participants/materials, setting, methodsSamples were primarily screened using the Ca(2+) influx induced by progesterone and, if cell number was sufficient, samples were also assessed by hyperactivation and penetration into viscous media. A defective Ca(2+) response to progesterone was defined using the 99% confidence interval from the distribution of response amplitudes in normozoospermic donors. Samples showing a defective Ca(2+) response were further examined in order to characterize the potential CatSper abnormalities. In men where there was a consistent and robust failure of calcium signalling, a direct assessment of CatSper function was performed using electrophysiology (patch clamping), and a blood sample was obtained for genetic analysis.Main results and the role of chanceA total of 101/102 (99%) IVF patients and 22/23 (96%) donors exhibited a normal Ca(2+) response. The mean (± SD) normalized peak response did not differ between donors and IVF patients (2.57 ± 0.68 [n = 34 ejaculates from 23 different donors] versus 2.66 ± 0.68 [n = 102 IVF patients], P = 0.63). In recall patients, 9/10 (90%) showed a normal Ca(2+) response. Three men were initially identified with a defective Ca(2+) influx. However, only one (Patient 1) had a defective response in repeat semen samples. Electrophysiology experiments on sperm from Patient 1 showed a near absence of CatSper current and exon screening demonstrated no mutations in the coding regions of the CatSper complex. There was no increase in penetration of viscous media when the spermatozoa were stimulated with progesterone and importantly there was failed fertilization at IVF.Limitations, reasons for cautionA key limitation relates to working with a specific functional parameter (Ca(2+) influx induced by progesterone) in fresh sperm samples from donors and patients that have limited viability. Therefore, for practical, technical and logistical reasons, some men (∼ 22% of IVF patients) could not be screened. As such the incidence of significant Ca(2+) abnormalities induced by progesterone may be higher than the ∼ 1% observed here. Additionally, we used a strict definition of a defective Ca(2+) influx such that only substantial abnormalities were selected for further study. Furthermore, electrophysiology was only performed on one patient with a robust and repeatable defective calcium response. This man had negligible CatSper current but more subtle abnormalities (e.g. currents present but significantly smaller) may have been present in men with either normal or below normal Ca(2+) influx.Wider implications of the findingsThese data add significantly to the understanding of the role of CatSper in human sperm function and its impact on male fertility. Remarkably, these findings provide the first direct evidence that CatSper is a suitable and specific target for human male contraception.

Analysis of membrane currents recorded from hormone-deprived H441 cells showed that the membrane potential (Vm) in single cells (approximately -80 mV) was unaffected by lowering [[Na.sup.+]][sub.o] or [[Cl.sup.-]][sub.o] indicating that cellular [Na.sup.+] and [Cl.sup.-] conductances ([G.sub.Na] and [G.sub.Cl], respectively) are negligible. Although insulin (20 nM, ~24 h) and dexamethasone (0.2 [micro]M, ~24 h) both depolarized Vm by ~20 mV, the response to insulin reflected a rise in [G.sup.Cl] mediated via phosphatidylinositol 3-kinase (PI3K) whereas dexamethasone acted by inducing a serum- and glucocorticoid-regulated kinase I (SGK1)-dependent rise in [G.sup.Na]. Although insulin stimulation/P13K-P110[alpha] expression did not directly increase [G.sup.Na], these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGKl-induced [G.sup.Na] in single cells discriminated poorly between [Na.sup.+] and [K.sup.+] ([P.sub.Na]/[P.sub.K] ~0.6), was insensitive to amiloride (1 mM), but was partially blocked by La[Cl.sub.3] ([La.sup.3+]; 1 mM, ~80%), pimozide (0.1 mM, ~40%), and dichlorobenzamil (15 [micro]M, ~15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10 [micro]M), selective [G.sup.Na] that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective GNu, (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGK1 and subject to PI3K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of [Na.sup.+] selectivity and amiloride sensitivity. epithelial [Na.sup.+] channel; serum- and glucocorticoid regulated kinase 1; phosphatidylinositol 3-kinase; H441 cells; pulmonary [Na.sup.+] absorption

By analysis of whole cell membrane currents in [Na.sup.+]-absorbing H441 human airway epithelial cells, we have identified a [K.sup.+] conductance ([G.sub.K]) resistant to [Ba.sup.2+] but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocalne, and quinidine inhibit basolateral membrane [K.sup.+] current ([I.sub.BI]) whereas [Ba.sup.2+] has only a weak inhibitory effect. [I.sub.BI] was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in [I.sub.BI], acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive [K.sup.+] channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current ([I.sub.SC]), and basolateral bupivacaine, lidocalne, quinidine, and [Ba.sup.2+] inhibited [I.sub.SC] at concentrations similar to those needed to inhibit [I.sub.BI], suggesting that the [K.sup.+] channels underlying [I.sub.IB] are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain [K.sup.+] (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie [G.sub.K] in unstimulated cells and so maintain the driving force for [Na.sup.+] absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology. airway sodium channel transport; patch clamping; Ussing chamber doi:10.1152/ajplung.00424.2006

G protein-gated inward rectifier (Kir3) channels are inhibited by activation of [G.sub.q/11]-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PI[P.sub.2]). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 cells after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the [G.sub.q/11]-mediated, M3 receptor, inhibition of channel activity. Recovery from [M.sub.3]-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PI[P.sub.2], were still irreversibly inhibited by [M.sub.3] receptor stimulation. When adenosine AI receptors were co-expressed, inclusion of PIP2 rescued the [A.sub.1]-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kit3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-[delta] mediates channel inhibition because recombinant PKC-[delta] inhibited channel activity, [M.sub.3]-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-[delta] constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-[delta] to the plasma membrane on [M.sub.3] receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-[delta] after [M.sub.3] receptor activation in HEK-293 cells. phosphatidylinositol 4,5-bisphosphate; phorbol 12-myristate 13-acetate; receptor for activated C kinase; A kinase anchoring protein; carbachol; 5'-N-ethylcarboxyamidoadenosine

Study Question: How are progesterone (P4)-induced repetitive intracellular Ca2+ concentration ([Ca2+]i) signals (oscillations) in human sperm generated?Summary Answer: P4-induced [Ca2+]i oscillations are generated in the flagellum by membrane potential (Vm)-sensitive Ca2+-influx through CatSper channels.What Is Known Already: A subset of human sperm display [Ca2+]i oscillations that regulate flagellar beating and acrosome reaction. Although pharmacological manipulations indicate involvement of stored Ca2+ in these oscillations, influx of extracellular Ca2+ is also required.Study Design, Size, Duration: This was a laboratory study that used >20 sperm donors and involved more than 100 separate experiments and analysis of more than 1000 individual cells over a period of 2 years.Participants/materials, Setting, Methods: Semen donors and patients were recruited in accordance with local ethics approval from Birmingham University and Tayside ethics committees. [Ca2+]i responses and Vm of individual cells were examined by fluorescence imaging and whole-cell current clamp.Main Results and the Role Of Chance: P4-induced [Ca2+]i oscillations originated in the flagellum, spreading to the neck and head (latency of 1-2 s). K+-ionophore valinomycin (1 µM) was used to investigate the role of membrane potential (Vm). Direct assessment by whole-cell current-clamp confirmed that Vm in valinomycin-exposed cells was determined primarily by K+ equilibrium potential (EK) and was rapidly 'reset' upon manipulation of [K+]o. Pre-treatment of sperm with valinomycin ([K+]o = 5.4 mM) had no effect on the P4-induced [Ca2+] transient (P = 0.95; eight experiments), but application of valinomycin to P4-pretreated sperm suppressed activity in 82% of oscillating cells (n = 257; P = 5 × 10-55 compared to control) and significantly reduced both the amplitude and frequency of persisting oscillations (P = 0.0001). Upon valinomycin washout, oscillations re-started in most cells. When valinomycin was applied in saline with elevated [K+], the inhibitory effect of valinomycin was reduced and was dependent on EK (P = 10-25). Amplitude and frequency of [Ca2+]i oscillations that persisted in the presence of valinomycin showed similar sensitivity to EK (P < 0.01). The CatSper inhibitor RU1968 (4.8 and 11 µM) caused immediate and reversible arrest of activity in 36% and 96% of oscillating cells, respectively (P < 10-10). Quinidine (300 µM) which blocks the sperm K+ current (IKsper) completely, inhibited [Ca2+]i oscillations.Large Scale Data: N/A.Limitations, Reasons For Caution: This was an in-vitro study and caution must be taken when extrapolating these results to in-vivo regulation of sperm.Wider Implications Of the Findings: [Ca2+]i oscillations in human sperm are functionally important and their absence is associated with failed fertilisation at IVF. The data reported here provide new understanding of the mechanisms that underlie the regulation and generation (or failure) of these oscillations.Study Funding/competing Interest(s): E.T.-N. was in receipt of a postgraduate scholarship from the CAPES Foundation (Ministry of Education, Brazil). E.M-M received travel funds from the Programa de Apoyo a los Estudios de Posgrado (Maestria y Doctorado en Ciencias Bioquimicas-Universidad Autonoma de Mexico). SGB and CLRB are recipients of a Chief Scientist Office (NHS Scotland) grant TCS/17/28. The authors have no conflicts of interest. [ABSTRACT FROM AUTHOR]

Study Question: Can pharma drug discovery approaches be utilized to transform investigation into novel therapeutics for male infertility?Summary Answer: High-throughput screening (HTS) is a viable approach to much-needed drug discovery for male factor infertility.What Is Known Already: There is both huge demand and a genuine clinical need for new treatment options for infertile men. However, the time, effort and resources required for drug discovery are currently exorbitant, due to the unique challenges of the cellular, physical and functional properties of human spermatozoa and a lack of appropriate assay platform.Study Design, Size, Duration: Spermatozoa were obtained from healthy volunteer research donors and subfertile patients undergoing IVF/ICSI at a hospital-assisted reproductive techniques clinic between January 2012 and November 2016.Participants/materials, Setting, Methods: A HTS assay was developed and validated using intracellular calcium ([Ca2+]i) as a surrogate for motility in human spermatozoa. Calcium fluorescence was detected using a Flexstation microplate reader (384-well platform) and compared with responses evoked by progesterone, a compound known to modify a number of biologically relevant behaviours in human spermatozoa. Hit compounds identified following single point drug screen (10 μM) of an ion channel-focussed library assembled by the University of Dundee Drug Discovery Unit were rescreened to ensure potency using standard 10 point half-logarithm concentration curves, and tested for purity and integrity using liquid chromatography and mass spectrometry. Hit compounds were grouped by structure activity relationships and five representative compounds then further investigated for direct effects on spermatozoa, using computer-assisted sperm assessment, sperm penetration assay and whole-cell patch clamping.Main Results and the Role Of Chance: Of the 3242 ion channel library ligands screened, 384 compounds (11.8%) elicited a statistically significant increase in calcium fluorescence, with greater than 3× median absolute deviation above the baseline. Seventy-four compounds eliciting ≥50% increase in fluorescence in the primary screen were rescreened and evaluated further, resulting in 48 hit compounds that produced a concentration-dependent increase in [Ca2+]i. Sperm penetration studies confirmed in vitro exposure to two hit compounds (A and B) resulted in significant improvement in functional motility in spermatozoa from healthy volunteer donors (A: 1 cm penetration index 2.54, 2 cm penetration index 2.49; P < 0.005 and B: 1 cm penetration index 2.1, 2 cm penetration index 2.6; P < 0.005), but crucially, also in patient samples from those undergoing fertility treatment (A: 1 cm penetration index 2.4; P = 0.009, 2 cm penetration index 3.6; P = 0.02 and B: 1 cm penetration index 2.2; P = 0.0004, 2 cm penetration index 3.6; P = 0.002). This was primarily as a result of direct or indirect CatSper channel action, supported by evidence from electrophysiology studies of individual sperm.Limitations, Reasons For Caution: Increase and fluxes in [Ca2+]i are fundamental to the regulation of sperm motility and function, including acrosome reaction. The use of calcium signalling as a surrogate for sperm motility is acknowledged as a potential limitation in this study.Wider Implications Of the Findings: We conclude that HTS can robustly, efficiently, identify novel compounds that increase [Ca2+]i in human spermatozoa and functionally modify motility, and propose its use as a cornerstone to build and transform much-needed drug discovery for male infertility.Study Funding/competing Interest(s): The majority of the data were obtained using funding from TENOVUS Scotland and Chief Scientist Office NRS Fellowship. Additional funding was provided by NHS Tayside, MRC project grants (MR/K013343/1, MR/012492/1) and University of Abertay. The authors declare that there is no conflict of interest.Trail Registration Number: N/A. [ABSTRACT FROM AUTHOR]

Progesterone released from the cumulus cells of the oocyte causes a number of physiological responses in human sperm cells including hyperactivation, acrosome reaction and chemotaxis. We employed a validated sperm mobility assay, which involves measuring the ability of sperm to penetrate an inert cell separation solution over time, to assess the ability of progesterone to enhance the mobility of boar spermatozoa. Cells maximally penetrate the solution over 50 minutes. 100nM progesterone significantly (P = 0.01) increased the mobility of non-capacitated sperm cells causing a doubling in the rate at which the cells penetrated through the cell separation solution (control half maximal penetration rate [Km] = 18.0±2.2; +100nM progesterone Km = 8.8±0.8min). Similarly, capacitated cells penetrated at a rate (Km = 19.2±3.0 min) not significantly different from non-capacitated cells and 100nM progesterone also significantly increased the rate of penetration of capacitated cells (Km = 9.5±1.0 min, P<0.05). The T-type voltage gated calcium channel blocker mibefradil (30mM) significantly inhibited both the control and progesterone enhanced mobility in non-capacitated and capacitated sperm. Only capacitated cells showed a significant increase in the acrosome reaction in response to 100nM progesterone (control non-reacted = 75±4%, +100nM progesterone non-reacted = 47±10%). Western blot analysis confirmed that there was an increase in the total protein tyrosine phosphorylation levels in capacitated cells. In conclusion, we have demonstrated that 100nM progesterone accelerates the mobility of boar sperm cells through an inert cell separation solution in an extracellular calcium dependent manner. [ABSTRACT FROM AUTHOR]

Analysis of membrane currents recorded from hormone-deprived H44 I cells showed that the membrane potential (Vm) in single cells (approximately -80 mV) was unaffected by lowering [Na+ ] or [Cl- ]0, indicating that cellular N+ and C1 conductances (GNa and Gc1, respectively) are negligible. Although insulin (20 nM, -24 h) and dexamethasone (0.2 μM, -24 h) both depolarized Vm by --20 mV, the response to insulin reflected a rise in Gc1 mediated via phosphatidylinositol 3-kinase (P13K) whereas dexamethasone acted by inducing a serum and glucocorticoid-regulated kinase 1 (SGKI) dependent rise in GNa. Although insulin stimulationfPl3K-Pl lOct expression did not directly increase GNa, these maneuvers augmented the dexamethasone-induced conductance. The glucocorticoid/SGKl-induced GNa in single cells discriminated poorly between Na+ and K+ (PNaIPK --0.6), was insensitive to amiloride (1 mM), but was partially blocked by LaCI3 (La3+ 1 mM, -80%), pimozide(0.I mM, --40%), and dichlorobenzamil (15 riM, -.15%). Cells growing as small groups, on the other hand, expressed an amiloride-sensitive (10μM), selective GNNa that displayed the same pattern of hormonal regulation as the nonselective conductance in single cells. These data therefore 1) confirm that H441 cells can express selective or nonselective GNa (14, 48), 2) show that these conductances are both induced by glucocorticoids/SGKI and subject to P13K-dependent regulation, and 3) establish that cell-cell contact is vitally important to the development of Na+ selectivity and amiloride sensitivity. [ABSTRACT FROM AUTHOR]

By analysis of whole cell membrane currents in Nat-absorbing H441 human airway epithelial cells, we have identified a K+ conductance (GK) resistant to Ba2+ but sensitive to bupivacaine or extracellular acidification. In polarized H441 monolayers, we have demonstrated that bupivacaine, lidocaine, and quinidine inhibit basolateral membrane K+ current (IB1) whereas Ba2+ has only a weak inhibitory effect. IB1 was also inhibited by basolateral acidification, and, although subsequent addition of bupivacaine caused a further fall in IB1 acidification had no effect after bupivacaine, demonstrating that cells grown under these conditions express at least two different bupivacaine-sensitive K+ channels, only one of which is acid sensitive. Basolateral acidification also inhibited short-circuit current (ISC), and basolateral bupivacaine, lidocaine, quinidine, and Ba2+ inhibited ISC at concentrations similar to those needed to inhibit IB1, suggesting that the K+ channels underlying IB1 are part of the absorptive mechanism. Analyses using RT-PCR showed that mRNA encoding several two-pore domain K+ (K2P) channels was detected in cells grown under standard conditions (TWIK-1, TREK-1, TASK-2, TWIK-2, KCNK-7, TASK-3, TREK-2, THIK-1, and TALK-2). We therefore suggest that K2P channels underlie GK in unstimulated cells and so maintain the driving force for Na+ absorption. Since this ion transport process is vital to lung function, K2P channels thus play an important but previously undocumented role in pulmonary physiology. [ABSTRACT FROM AUTHOR]

G protein-gated inward rectifier (Kit3) channels are inhibited by activation of Gq/11-coupled receptors and this has been postulated to involve the signaling molecules protein kinase C (PKC) and/or phosphatidylinositol 4,5-bisphosphate (PIP2). Their precise roles in mediating the inhibition of this family of channels remain controversial. We examine here their relative roles in causing inhibition of Kir3.1/3.2 channels stably expressed in human embryonic kidney (HEK)-293 ceils after muscarinic M3 receptor activation. In perforated patch mode, staurosporine prevented the Gq/11-mediated, M3 receptor, inhibition of channel activity. Recovery from M3-mediated inhibition was wortmannin sensitive. Whole cell currents, where the patch pipette was supplemented with PIP2, were still irreversibly inhibited by M3 receptor stimulation. When adenosine A1 receptors were co-expressed, inclusion of PIP2 rescued the A1-mediated response. Recordings from inside-out patches showed that catalytically active PKC applied directly to the intracellular membrane face inhibited the channels: a reversible effect modulated by okadaic acid. Generation of mutant heteromeric channel Kir3.1S185A/Kir3.2C-S178A, still left the channel susceptible to receptor, pharmacological, and direct kinase-mediated inhibition. Biochemically, labeled phosphate is incorporated into the channel. We suggest that PKC-δ mediates channel inhibition because recombinant PKC-δ inhibited channel activity, M3-mediated inhibition of the channel, was counteracted by overexpression of two types of dominant negative PKC-δ constructs, and, by using confocal microscopy, we have demonstrated translocation of green fluorescent protein-tagged PKC-δ to the plasma membrane on M3 receptor stimulation. Thus Kir3.1/3.2 channels are sensitive to changes in membrane phospholipid levels but this is contingent on the activity of PKC-δ after M3 receptor activation in HEK-293 cells. [ABSTRACT FROM AUTHOR]

Despite recent advances in male reproductive health research, there remain many elements of male infertility where our understanding is incomplete. Consequently, diagnostic tools and treatments for men with sperm dysfunction, other than medically assisted reproduction, are limited. On the other hand, the gaps in our knowledge of the mechanisms which underpin sperm function have hampered the development of male non-hormonal contraceptives. The study of mature spermatozoa is inherently difficult. They are a unique and highly specialised cell type which does not actively transcribe or translate proteins and cannot be cultured for long periods of time or matured in vitro. One large-scale approach to both increasing the understanding of sperm function and the discovery and development of compounds that can modulate sperm function is to directly observe responses to compounds with phenotypic screening techniques. These target agnostic approaches can be developed into high-throughput screening platforms with the potential to drastically increase advances in the field. Here, we discuss the rationale and development of high-throughput phenotypic screening platforms for mature human spermatozoa and the multiple potential applications these present, as well as the current limitations and leaps in our understanding and the capabilities needed to overcome them. Further development and use of these technologies could lead to the identification of compounds which positively or negatively affect sperm cell motility or function or novel platforms for toxicology or environmental chemical testing among other applications. Ultimately, each of these potential applications is also likely to increase the understanding within the field of sperm biology.

[Table: see text] Seven PPADS ( P yridoxal-5'- P hosphate 6- A zophenyl 2',4'- D i S ulfonate) analogs were investigated at Group 1 P2X receptors expressed in Xenopus oocytes. All seven analogs potently inhibited P2X 1 (IC 50 range, 5-32 nM) and P2X 3 (IC 50 range, 22-345 nM), the two Group I P2X receptor subtypes. Analogs showed greater inhibitory activity where the pyridoxal moiety of PPADS contained a 5'-phosphonate group, rather than a 5'-phosphate group. Analogs also showed greater potency where disulfonate groups were removed from, or substituted at, the azophenyl moiety. The most active analog was MRS 2257 (pyridoxal-5'-phosphonate 6-azophenyl 3',5'-bismethylenephosphonate) at P2X 1 (IC 50 , 5 nM) and P2X 3 (IC 50 , 22 nM) receptors, being 14-fold and 10-fold more potent than PPADS itself. MRS 2257 produced a nonsurmountable inhibition when tested against a range of ATP concentrations, although blockade was reversed by about 85% after 20 minutes of washout. TNP-ATP and Ip 5 I were equipotent with MRS 2257 at P2X 1 receptors, whereas TNP-ATP was 64-fold more potent than MRS 2257 at P2X 3 receptors. In conclusion, the PPADS template can be altered at the pyridoxal and phenyl moieties to produce P2X 1 and P2X 3 receptor antagonists showing higher potency and greater degree of reversibility than the parent compound at these Group I P2X receptors.