Ion channels in small cells and subcellular structures can be studied with a smart patch-clamp system

We have developed a scanning patch-clamp technique that facilitates single-channel recording from small cells and submicron cellular structures that are inaccessible by conventional methods. The scanning patch-clamp technique combines scanning ion conductance microscopy and patch-clamp recording through a single glass nanopipette probe. In this method the nanopipette is first scanned over a cell surface, using current feedback, to obtain a high-resolution topographic image. This same pipette is then used to make the patch-clamp recording. Because image information is obtained via the patch electrode it can be used to position the pipette onto a cell with nanometer precision. The utility of this technique is demonstrated by obtaining ion channel recordings from the top of epithelial microvilli and openings of cardiomyocyte T-tubules. Furthermore, for the first time we have demonstrated that it is possible to record ion channels from very small cells, such as sperm cells, under physiological conditions as well as record from cellular microstructures such as submicron neuronal processes.     

Comparison of two techniques for the morphometry study on gilthead seabream (Sparus aurata) spermatozoa and evaluation of changes induced by cryopreservation

The development of powerful software has made possible spermatozoa morphology studies. However, some problems have emerged in relation to protocol standardization to compare results from different laboratories. This study was carried out to compare two techniques commonly used (staining vs phase contrast technique) for the morphometry study of gilthead sea bream spermatozoa using an integrated sperm analysis system (ISAS). Spermatozoa morphometry values were significantly affected by the technique used, and phase contrast technique was found to be the more accurate method, showing lower coefficients of variation on spermatozoa morphometry parameters measurements. Moreover, it has been shown that cryopreservation process produces damage in gilthead sea bream spermatozoa, causing negative effects in sperm parameters as spermatozoa morphometry (a decrease in cell volume), motility (from 95 to 68% motile cells) and viability (from 95 to 87% of live cells), being the addition of freezing medium containing cryoprotectant (DMSO) an important factor that caused the morphometry changes.     

Inhibition of TRPC4 channel activity in colonic myocytes by tricyclic antidepressants disrupts colonic motility causing constipation

Tricyclic antidepressants (TCAs) have been used to treat depression and were recently approved for treating irritable bowel syndrome (IBS) patients with severe or refractory IBS symptoms. However, the molecular mechanism of TCA action in the gastrointestinal (GI) tract remains poorly understood. Transient receptor potential channel canonical type 4 (TRPC4), which is a Ca²⁺‐permeable nonselective cation channel, is a critical regulator of GI excitability. Herein, we investigated whether TCA modulates TRPC4 channel activity and which mechanism in colonic myocytes consequently causes constipation. To prove the clinical benefit in patients with diarrhoea caused by TCA treatment, we performed mechanical tension recording of repetitive motor pattern (RMP) in segment, electric field stimulation (EFS)‐induced and spontaneous contractions in isolated muscle strips. From these recordings, we observed that all TCA compounds significantly inhibited contractions of colonic motility in human. To determine the contribution of TRPC4 to colonic motility, we measured the electrical activity of heterologous or endogenous TRPC4 by TCAs using the patch clamp technique in HEK293 cells and murine colonic myocytes. In TRPC4‐overexpressed HEK cells, we observed TCA‐evoked direct inhibition of TRPC4. Compared with TRPC4‐knockout mice, we identified that muscarinic cationic current (mI _cat) was suppressed through TRPC4 inhibition by TCA in isolated murine colonic myocytes. Collectively, we suggest that TCA action is responsible for the inhibition of TRPC4 channels in colonic myocytes, ultimately causing constipation. These findings provide clinical insights into abnormal intestinal motility and medical interventions aimed at IBS therapy.     

Evidence for phosphorylation in the MSP cytoskeletal filaments of amoeboid spermatozoa

Nematode spermatozoa are highly specialized cells that lack flagella and, instead, extend a pseudopod to initiate motility. Crawling spermatozoa display classic features of amoeboid motility (e.g. protrusion of a pseudopod that attaches to the substrate and the assembly and disassembly of cytoskeletal filaments involved in cell traction and locomotion), however, cytoskeletal dynamics in these cells are powered exclusively by Major Sperm Protein (MSP) rather than actin and no other molecular motors have been identified. Thus, MSP-based motility is regarded as a simple locomotion machinery suitable for the study of plasma membrane protrusion and cell motility in general. This recent focus on MSP dynamics has increased the necessity of a standardized methodology to obtain C. elegans sperm extract that can be used in biochemical assays and proteomic analysis for comparative studies. In the present work we have modified a method to reproducibly obtain relative high amounts of proteins from C. elegans sperm extract. We show that these extracts share some of the properties observed in sperm extracts from the parasitic nematode Ascaris including Major Sperm Protein (MSP) precipitation and MSP fiber elongation. Using this method coupled to immunoblot detection, Mass Spectrometry identification, in silico prediction of functional domains and biochemical assays, our results indicate the presence of phosphorylation sites in MSP of Caenorhabditis elegans spermatozoa.     

Effects of phenylalanine substitutions in gramicidin A on the kinetics of channel formation in vesicles and channel structure in SDS micelles

The common occurrence of Trp residues at the aqueous-lipid interface region of transmembrane channels is thought to be indicative of its importance for insertion and stabilization of the channel in membranes. To further investigate the effects of Trp-->Phe substitution on the structure and function of the gramicidin channel, four analogs of gramicidin A have been synthesized in which the tryptophan residues at positions 9, 11, 13, and 15 are sequentially replaced with phenylalanine. The three-dimensional structure of each viable analog has been determined using a combination of two-dimensional NMR techniques and distance geometry-simulated annealing structure calculations. These phenylalanine analogs adopt a homodimer motif, consisting of two beta6.3 helices joined by six hydrogen bonds at their NH2-termini. The replacement of the tryptophan residues does not have a significant effect on the backbone structure of the channels when compared to native gramicidin A, and only small effects are seen on side-chain conformations. Single-channel conductance measurements have shown that the conductance and lifetime of the channels are significantly affected by the replacement of the tryptophan residues (Wallace, 2000; Becker et al., 1991). The variation in conductance appears to be caused by the sequential removal of a tryptophan dipole, thereby removing the ion-dipole interaction at the channel entrance and at the ion binding site. Channel lifetime variations appear to be related to changing side chain-lipid interactions. This is supported by data relating to transport and incorporation kinetics.     

Single-channel properties of N- and L-subtypes of acetylcholine receptor in Ascaris suum

We are interested in the properties of the target site of cholinergic anti-nematodal drugs for therapeutic reasons. The target receptors are ligand-gated ion channels that have different subtypes, and each subtype may have a different pharmacology. In a contraction assay using the parasitic nematode Ascaris suum, our laboratory has identified several subtypes, including an N-subtype, preferentially activated by nicotine, and an L-subtype, preferentially activated by levamisole. Here we use patch-clamp recordings to test the hypothesis that the single-channel selectivities of nicotine and levamisole are different. Unitary currents evoked by nicotine in this preparation were characterised for the first time. In some patches, both nicotine and levamisole activated small- and large-conductance channels. In other patches, the agonists activated just one channel amplitude. Discriminant analysis allowed classification of the one-conductance patch channels into the small or large categories, based on sets defined by the two-conductance patch data. The small channels had a conductance of 26.1+/-1.5 pS, n=18 (mean+/-SEM); the large conductance channels had a conductance of 38.8+/-1.2 pS, n=23 (mean+/-SEM). Analysis of amplitude histograms of the two-conductance patches showed that nicotine preferentially activated the small-conductance channels and levamisole preferentially activated the large-conductance channels. Our observations suggest that the N-subtype receptor channel has a conductance of 26 pS channel and the L-subtype receptor channel has a conductance of 39 pS.     

Patch-clamp 'mapping' of ion channel activity in human sperm reveals regionalisation and co-localisation into mixed clusters

Ion channels are pivotal to many aspects of sperm physiology and function. We have used the patch clamp technique to investigate the distribution of ion channels in the plasma membrane of the head of human spermatozoa. We report that three types of activity are common in the equatorial and acrosomal regions of the sperm head. Two of these (a chloride-permeable anion channel showing long stable openings and a second channel which flickered between open and closed states and was dependent upon cytoplasmic factors for activity) were localised primarily to the equatorial segment. A third type, closely resembling the flickering activity but with different voltage sensitivity of P(open), was more widely distributed but was not detectable over the anterior acrosome. In the anterior acrosomal area channels were present but showed very low levels of spontaneous activity. A unique feature of channel activity in the sperm equatorial region was co-localisation into mixed clusters, most patches were devoid of activity but 'active' patches typically contained two or more types of activity (in a single 200-300 nM diameter patch). We conclude that ion channels in the sperm membrane show regionalisation of type and activity and that the channels are clustered into functional groups, possibly interacting through local effects on membrane potential.     

Ion channels that control fertility in mammalian spermatozoa

Whole-cell voltage clamp of mammalian spermatozoa was first achieved in 2006. This technical advance, combined with genetic deletion strategies, makes unambiguous identification of sperm ion channel currents possible. This review summarizes the ion channel currents that have been directly measured in mammalian sperm, and their physiological roles in fertilization. The predominant currents are a Ca2+-selective current requiring expression of the 4 mCatSper genes, and a rectifying K+ current with properties most similar to mSlo3. Intracellular alkalinization activates both channels and induces hyperactivated motility.     

Noise analysis to study unitary properties of transporter-associated ion channels

Excitatory amino acid transporters (EAATs) do not only mediate secondary-active glutamate uptake but also function as anion channels. We recently used macroscopic current recordings and noise analysis to determine unitary current amplitudes of anion channels associated with a neuronal EAAT isoform, EAAT4. We found that, at symmetrical NO(3)(-), EAAT4 anion channels exhibit a single channel conductance of ~1 pS in the absence as well as in the presence of glutamate. These results indicate that glutamate increases EAAT4 anion currents by modifying exclusively open probabilities, however, leaves unitary current amplitudes unaffected. Noise analysis has been developed for ion channels with a single conductance state and limitations might ensue when using this approach for transporter-associated ion channels. We here performed stochastic simulations of EAAT transporter-associated anion channels and noise analysis of simulated currents to assess the reliability and possible limitations of this technique in studying this special class of ion channels.     

Characterization of mechanosensitive channels in Escherichia coli cytoplasmic membrane by whole-cell patch clamp recording

Whole-cell patch clamp recordings were done on giant protoplasts of Escherichia coli. The pressure sensitivity of the protoplasts was studied. Two different unit conductance mechanosensitive channels, 1100 ± 25 pS and 350 ± 14 pS in 400 mm symmetric KCl solution, were observed upon either applying positive pressure to the interior of the cells or down shocking the cells osmotically. The 1100 pS conductance channel discriminated poorly among the monovalent ions tested and it was permeable to Ca²⁺ and glutamate^?. Both of the two channels were sensitive to the osmotic gradient across the membrane; the unit conductances of the channels remained constant while the mean current of the cell was increased by increasing the osmotic gradient. Both of the channels were voltage sensitive. Voltage-ramp results showed that the pressure sensitivity of protoplasts was voltage dependent: there were more channels active upon depolarization than hyperpolarization. The mech anosensitive channels were reversibly blocked by gadolinium ion. Also they could reversibly be inhibited by protons. Mutations in two of the potassium efflux systems, KefB and KefC, did not affect the channel activity, while a null mutation in the gene for KefA changed the channel activity significantly. This indicates a potential modulation of these channels by KefA.     

Permeation properties of inward-rectifier potassium channels and their molecular determinants

The structural domains contributing to ion permeation and selectivity in K channels were examined in inward-rectifier K(+) channels ROMK2 (Kir1.1b), IRK1 (Kir2.1), and their chimeras using heterologous expression in Xenopus oocytes. Patch-clamp recordings of single channels were obtained in the cell-attached mode with different permeant cations in the pipette. For inward K(+) conduction, replacing the extracellular loop of ROMK2 with that of IRK1 increased single-channel conductance by 25 pS (from 39 to 63 pS), whereas replacing the COOH terminus of ROMK2 with that of IRK1 decreased conductance by 16 pS (from 39 to 22 pS). These effects were additive and independent of the origin of the NH(2) terminus or transmembrane domains, suggesting that the two domains form two resistors in series. The larger conductance of the extracellular loop of IRK1 was attributable to a single amino acid difference (Thr versus Val) at the 3P position, three residues in front of the GYG motif. Permeability sequences for the conducted ions were similar for the two channels: Tl(+) > K(+) > Rb(+) > NH(4)(+). The ion selectivity sequence for ROMK2 based on conductance ratios was NH(4)(+) (1.6) > K(+) (1) > Tl(+) (0.5) > Rb(+) (0.4). For IRK1, the sequence was K(+) (1) > Tl(+) (0.8) > NH(4)(+) (0.6) > Rb(+) (0.1). The difference in the NH(4)(+)/ K(+) conductance (1.6) and permeability (0.09) ratios can be explained if NH(4)(+) binds with lower affinity than K(+) to sites within the pore. The relatively low conductances of NH(4)(+) and Rb(+) through IRK1 were again attributable to the 3P position within the P region. Site-directed mutagenesis showed that the IRK1 selectivity pattern required either Thr or Ser at this position. In contrast, the COOH-terminal domain conferred the relatively high Tl(+) conductance in IRK1. We propose that the P-region and the COOH terminus contribute independently to the conductance and selectivity properties of the pore.     

Sperm treatment affects capacitation parameters and penetration ability of ejaculated and epididymal boar spermatozoa

This work was designed to study how this ability is affected by different sperm treatments routinely used for in vitro fertilization (IVF) assay. In this study, boar sperm samples from epididymal or ejaculated origin were processed by three different methods: left unwashed (NW group), washed in Dulbecco's phosphate-buffered saline supplemented with 0.1% BSA (BSA group), and washed on a Percoll^® gradient (PERCOLL group). After preparation of semen samples, changes in motility patterns were studied by CASA, calcium uptake by spectrofluorimetry, and ROS generation, spontaneous acrosome reaction, and lipid disorder by means of flow cytometry. Finally IVF assays were also performed with the different semen samples and penetrability results evaluated at 2 and 4 h post insemination (hpi). Independently of the sperm treatment, epididymal spermatozoa showed higher values of progressive motility, percentage of live cells with low lipid disorder, and penetration ability at 4 hpi than the corresponding ejaculated spermatozoa. Ejaculated spermatozoa showed higher levels of calcium uptake, ROS generation and percentage of spontaneous acrosome reaction than epididymal sperm. Regarding sperm treatments, PERCOLL group showed the highest values for some motility parameters (linearity of the curvilinear trajectory, straightness, and average path velocity/curvilinear velocity), ROS generation and penetration ability at 2 and 4 hpi; however this same group showed the lowest values for sperm curvilinear velocity and lateral head displacement. From all experimental groups, ejaculated-PERCOLL-treated spermatozoa showed the highest fertilization ability after 2 hpi. Results suggest that capacitation pathways can be regulated by suitable treatments making the ejaculated sperm able to reach capacitation and fertilize oocytes in similar levels than epididymal spermatozoa, although most of the studied capacitation-associated changes do not correlate with this ability.     

Sperm motility in fishes. (II) Effects of ions and osmolality: a review

The spermatozoa of most fish species are immotile in the testis and seminal plasma. Therefore, motility is induced after the spermatozoa are released into the aqueous environment during natural reproduction or into the diluent during artificial reproduction. There are clear relationships between seminal plasma composition and osmolality and the duration of fish sperm motility. Various parameters such as ion concentrations (K+, Na+, and Ca2+), osmotic pressure, pH, temperature and dilution rate affect motility. In the present paper, we review the roles of these ions on sperm motility in Salmonidae, Cyprinidae, Acipenseridae and marine fishes, and their relationship with seminal plasma composition. Results in the literature show that: 1. K+ is a key ion controlling sperm motility in Salmonidae and Acipenseridae in combination with osmotic pressure; this control is more simple in other fish species: sperm motility is prevented when the osmotic pressure is high (Cyprinidae) or low (marine fishes) compared to that of the seminal fluid. 2. Cations (mostly divalent, such as Ca2+) are antagonistic with the inhibitory effect of K+ on sperm motility. 3. In many species, Ca2+ influx and K+ or Na+ efflux through specific ionic channels change the membrane potential and eventually lead to an increase in cAMP concentration in the cell, which constitutes the initiation signal for sperm motility in Salmonidae. 4. Media that are hyper- and hypo-osmotic relative to seminal fluid trigger sperm motility in marine and freshwater fishes, respectively. 5. The motility of fish spermatozoa is controlled through their sensitivity to osmolality and ion concentrations. This phenomenon is related to ionic channel activities in the membrane and governs the motility mechanisms of axonemes.     

Effects of osmolality on sperm morphology, motility and flagellar wave parameters in Northern pike (Esox lucius L.)

Northern pike (Esox lucius L.) spermatozoa are uniflagellated cells differentiated into a head without acrosome, a midpiece and a flagellar tail region flanked by a fin structure. Total, flagellar, head and midpiece lengths of spermatozoa were measured and show mean values of 34.5, 32.0, 1.32, 1.17 μm, respectively, with anterior and posterior widths of the midpiece measuring 0.8 and 0.6 μm, respectively. The osmolality of seminal plasma ranged from 228 to 350 mOsmol kg⁻¹ (average: 283.88 ± 33.05). After triggering of sperm motility in very low osmolality medium (distilled water), blebs appeared along the flagellum. At later periods in the motility phase, the tip of the flagellum became curled into a loop shape which resulted in a shortening of the flagellum and a restriction of wave development to the proximal part (close to head). Spermatozoa velocity and percentage of motile spermatozoa decreased rapidly as a function of time postactivation and depended on the osmolality of activation media (P < 0.05). In general, the greatest percentage of motile spermatozoa and highest spermatozoa velocity were observed between 125 and 235 mOsmol kg⁻¹. Osmolality above 375 mOsmol kg⁻¹ inhibited the motility of spermatozoa. After triggering of sperm motility in activation media, beating waves propagated along the full length of flagella, while waves appeared dampened during later periods in the motility phase, and were absent at the end of the motility phase. By increasing osmolality, the velocity of spermatozoa reached the highest value while wave length, amplitude, number of waves and curvatures also were at their highest values. This study showed that sperm morphology can be used for fish classification. Sperm morphology, in particular, the flagellar part showed several changes during activation in distilled water. Sperm motility of pike is inhibited due to high osmolality in the seminal plasma. Osmolality of activation medium affects the percentage of motile sperm and spermatozoa velocity due to changes in flagellar wave parameters.     

Specific cytogenetic labeling of bovine spermatozoa bearing X or Y chromosomes using fluorescent in situ hybridization (FISH)

X and Y specific probes were identified in order to apply the fluorescent in situ hybridization (FISH) technique to bovine spermatozoa. For Y chromosome detection, the BRY4a repetitive probe, covering three quarters of the chromosome, was used. For X chromosome detection, a goat Bacterial Artificial Chromosome (BAC) specific to the X chromosome of bovine and goats and giving a strong FISH signal was used. Each probe labeled roughly 45% of sperm cells. The hybridization method will be useful for evaluating the ratio of X- and Y- bearing spermatozoa in a sperm sample and consequently can be used to evaluate the efficiency of sperm sorting by different techniques such as flow cytometry.     

The Xenopus Oocyte Cut-open Vaseline Gap Voltage-clamp Technique With Fluorometry

The cut-open oocyte Vaseline gap (COVG) voltage clamp technique allows for analysis of electrophysiological and kinetic properties of heterologous ion channels in oocytes. Recordings from the cut-open setup are particularly useful for resolving low magnitude gating currents, rapid ionic current activation, and deactivation. The main benefits over the two-electrode voltage clamp (TEVC) technique include increased clamp speed, improved signal-to-noise ratio, and the ability to modulate the intracellular and extracellular milieu.Here, we employ the human cardiac sodium channel (hNa_V1.5), expressed in Xenopus oocytes, to demonstrate the cut-open setup and protocol as well as modifications that are required to add voltage clamp fluorometry capability.The properties of fast activating ion channels, such as hNa_V1.5, cannot be fully resolved near room temperature using TEVC, in which the entirety of the oocyte membrane is clamped, making voltage control difficult. However, in the cut-open technique, isolation of only a small portion of the cell membrane allows for the rapid clamping required to accurately record fast kinetics while preventing channel run-down associated with patch clamp techniques.In conjunction with the COVG technique, ion channel kinetics and electrophysiological properties can be further assayed by using voltage clamp fluorometry, where protein motion is tracked via cysteine conjugation of extracellularly applied fluorophores, insertion of genetically encoded fluorescent proteins, or the incorporation of unnatural amino acids into the region of interest¹. This additional data yields kinetic information about voltage-dependent conformational rearrangements of the protein via changes in the microenvironment surrounding the fluorescent molecule.     

Effects of porcine pre-ovulatory oviductal fluid on boar sperm function

Sperm storage within the oviductal isthmus prior to ovulation typically involves binding to oviductal epithelial cells, which are thought to modulate sperm functions including internal calcium concentration, membrane fluidity, and motility. Around the time of ovulation the spermatozoa are gradually released so that they eventually encounter the oocytes within the oviductal ampulla. Previous studies have shown that the oviductal epithelial cells selectively sequester high quality spermatozoa, but the role of oviductal fluid as a selective modulator of sperm function has been investigated to a lesser extent. Here we address the hypothesis that oviductal fluid is also likely to modulate sperm function. Using samples of porcine oviductal fluid collected in the follicular phase of the estrus cycle, we show that short exposure (20 min to 50 μg/mL of oviductal fluid proteins) to either of two separate proteins fractions (> or < 100 kDa) promotes boar sperm viability and acrosomal integrity, decreases sperm plasma membrane fluidity (measured using merocyanine S540), and increases zona binding and polyspermy during in vitro fertilization. Exposure to the lower molecular fraction significantly inhibited, but did not abolish, the bicarbonate-induced stimulation of motility. The results show that subpopulations of spermatozoa respond differentially to oviductal fluid, and suggest that exposure to oviductal fluid in vivo could exert a further level of functional sperm selection.     

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

M₂, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M₂ baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t _Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ～ Na > Li. The pH activation, amantadine block and ion selectivity of the M₂ protein ion channel in bilayers are consistent with those observed on expression of the M₂ protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M₂ protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

Interleukin-4 activates ion channels in B lymphocytes.

The lymphokine interleukin-4 (IL-4) has been shown to induce dramatic changes in the physiology of resting B cells. We have applied the patch clamp technique in the cell attached and inside/out configurations to resting and IL-4-treated B cells to determine whether specific ion conductances result as a consequence of IL-4 action. We report here that two distinct ion channel events occur in B lymphocytes after treatment with IL-4, (i) induction of an inward rectifying K+ channel that is not observed in untreated cells, and (ii) activation of a large conductance anion channel that is normally silent in non-treated cells in the cell attached patch configuration. These data present the first evidence of a direct effect by IL-4 on ion channels and we suggest roles for these two ionic conductances in IL-4-induced B cell activation.