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Results for “patch clamp”

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PubMed1992

Alternate pathways for chloride conductance activation in normal and cystic fibrosis airway epithelial cells.

Using whole cell patch-clamp and perforated patch recording techniques on human cystic fibrosis (CF) and non-CF airway epithelial cells, we sought to determine whether a single Cl- conductance (GCl) could be modulated via different regulatory pathways or whether multiple conductances could be identified. Cl- current in both CF and non-CF cells was activated by cellular swelling as well as by an elevation in intracellular calcium ([Ca2+]i). While the adenosine 3',5'-cyclic monophosphate (cAMP)-activated GCl was absent in CF cells, its activation in non-CF cells was only observed in the perforated patch configuration at lower temperatures (24 degrees C) or infrequently in the whole cell configuration at elevated temperatures (33 degrees C). Currents activated by all three regulatory pathways were sensitive to the Cl- channel blocker 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). Further increases in current activation could be produced by cellular swelling after maximal Ca2+ or cAMP-induced current activation. Intracellular application of a peptide inhibitor of Ca(2+)-calmodulin-dependent protein kinase selectively blocked the Ca(2+)-dependent current activation while leaving the swelling-induced current increase intact. These results are consistent with the presence of multiple anion conductances in both CF and non-CF airway cells. The heterogeneity of the responses to the three regulatory stimuli, however, prevented the correlation of a specific anion conductance with a separate modulatory pathway based on characteristic voltage-dependent kinetics and conductance.

Calcium↗

PubMed1999

Intracellular H+ regulates the alpha-subunit of ENaC, the epithelial Na+ channel.

Protons regulate electrogenic sodium absorption in a variety of epithelia, including the cortical collecting duct, frog skin, and urinary bladder. Recently, three subunits (alpha, beta, gamma) coding for the epithelial sodium channel (ENaC) were cloned. However, it is not known whether pH regulates Na+ channels directly by interacting with one of the three ENaC subunits or indirectly by interacting with a regulatory protein. As a first step to identifying the molecular mechanisms of proton-mediated regulation of apical membrane Na+ permeability in epithelia, we examined the effect of pH on the biophysical properties of ENaC. To this end, we expressed various combinations of alpha-, beta-, and gamma-subunits of ENaC in Xenopus oocytes and studied ENaC currents by the two-electrode voltage-clamp and patch-clamp techniques. In addition, the effect of pH on the alpha-ENaC subunit was examined in planar lipid bilayers. We report that alpha,beta,gamma-ENaC currents were regulated by changes in intracellular pH (pHi) but not by changes in extracellular pH (pHo). Acidification reduced and alkalization increased channel activity by a voltage-independent mechanism. Moreover, a reduction of pHi reduced single-channel open probability, reduced single-channel open time, and increased single-channel closed time without altering single-channel conductance. Acidification of the cytoplasmic solution also inhibited alpha, beta-ENaC, alpha,gamma-ENaC, and alpha-ENaC currents. We conclude that pHi but not pHo regulates ENaC and that the alpha-ENaC subunit is regulated directly by pHi.

Acids↗

PubMed1990

Long-lasting openings of single slow (L-type) Ca2+ channels in chick embryonic heart cells.

Single-channel currents were recorded in cultured embryonic chick (3-day-old) cardiomyocytes in cell-attached patch-clamp experiments. The patch electrode contained 50 mM Ba2+. The cell was bathed in an external solution containing 150 mM K+ (pH 7.4) at room temperature. Depolarizing pulses above -30 mV, from a holding potential of -80 mV, elicited inward unitary currents. The conductance of the channel for this unitary current was 26 pS. The activity of these channels was completely blocked by nifedipine (3 microM). These results indicate that the channel is a slow (L-type) Ca2+ channel. The channels exhibited long-lasting openings, in addition to conventional brief openings. These long openings resembled the long openings produced by the dihydropyridine Ca2+ agonist BAY K 8644 and resultant mode 2 behavior (Hess et al., Nature Lond. 311: 538-544, 1984). The long-lasting openings were observed in 25 patches out of a total of 29 patches in which single-channel activity was present. High open-state probability (Po) sweeps (with Po greater than 0.65), which mainly contain long-lasting openings, accounted for 20.7% of all sweeps. The open-time histogram for the Ca2+ channels was fitted by two exponential components. The time constants of the two components were 0.45 ms (fast) and 6.30 ms (slow). These kinetic properties were similar to those of the previous reports using BAY K 8644. Thus the slow (L-type) Ca2+ channels in young embryonic chick heart cells naturally produce many long-lasting openings in the absence of any added dihydropyridine Ca2+ agonist.

Animals↗

PubMed1993

Transient enhancement of low-threshold calcium current in thalamic relay neurons after corticectomy.

1. The alterations of voltage-sensitive calcium currents produced in thalamic cells by injury were investigated under voltage clamp using patch-clamp recordings in the whole-cell configuration. 2. One day after unilateral cortical ablation in immature rats (postnatal day 7), low-threshold transient calcium (T) currents in acutely isolated thalamic relay neurons (RNs) were increased by 68% compared with contralateral controls (P < 0.001). Three days after the operation, T currents in injured neurons were at 44% of control levels (P < 0.001). On the other hand, high-threshold (L) calcium currents in RNs did not change over the same interval. 3. To investigate the mechanism for the increase of T current, both kinetics and voltage dependency of activation and inactivation were examined. At a test voltage of -40 mV, the activation time constant decreased from 4.1 to 3.2 ms (P < 0.05); however, this small change was insufficient to explain the large increase in T current. Time constants for both fast and slow inactivation did not change significantly, nor did voltage dependence of activation or inactivation of thalamic T currents. 4. Methyl-phenyl-succinimide (MPS, 1 mM), a compound known to block T currents, was used to examine possible alterations in the pharmacological properties of T channels after injury. MPS was more effective in reducing T currents in normal versus injured RNs (24 and 20% reductions, respectively; P < 0.05), suggesting that pharmacological properties of T channels in the injured RNs may be different from those of the normal RNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Afferent Pathways↗

PubMed1992

Adrenergic stimulation of bovine non-pigmented ciliary epithelial cells raises cAMP but has no effect on K+ or Cl- currents.

Freshly isolated bovine nonpigmented ciliary epithelial cells were studied using the whole-cell voltage-clamp and permeabilized patch techniques. In a study of 148 cells three classes of cell were found; those containing inward currents alone, 31%; those containing outward currents alone, 37% and those containing both inward and outward currents, 32%. The outward currents exhibited slow, delayed activation, were blocked by tetraethylammonium (TEA+) but were not effected by changes in [Cai] suggesting they are K(+)-currents similar to IK(V), the delayed rectifier. The inward currents were reduced by TEA+ and blocked by Cs+ suggesting they are inward rectifier K(+)-currents. Intracellular cAMP levels were increased by isoprenaline with a Ka of 20 nM. However, the resting membrane potential recorded from whole ciliary processes in intracellular microelectrode studies was not effected by adrenergic stimulation, neither were the leak current, the outward current nor the sustained inward current significantly effected by isoprenaline, noradrenaline or dibutyryl-cAMP in whole-cell and permeabilized patch clamp studies.

Adrenergic Agonists↗

PubMed1999

Differential sensitivity of voltage-gated potassium channels Kv1.5 and Kv1.2 to acidic pH and molecular identification of pH sensor.

Kv1.2 and Kv1.5 are two subtypes of voltage-gated potassium channels expressed in heart that are thought to contribute to phase 1 (ITO) and phase 3 (IK) components of cardiac action potential repolarization. Although the effect of decreased pH in prolonging cardiac action potentials is well documented, the molecular target of acidification has not previously been determined. We expressed Kv1.2 and Kv1.5 in Xenopus oocytes to study the effect of acidic and alkaline extracellular pH on channel function. Using two-electrode voltage clamp and cellattached patch clamp, we demonstrate that Kv1. 5 channels show enhanced C-type inactivation at acidic pH that is relevant to pathophysiological conditions. In contrast, homologous Kv1.2 channels are resistant to acidic pH. Both channel types are insensitive to alkaline pH. A histidine residue in the third extracellular loop of Kv1.5 (H452) accounts for the difference in pH sensitivity between the Kv1.5 and Kv1.2 channels. Mutation of histidine H452 to a glutamine residue in Kv1.5 yields a channel that no longer shows enhanced inactivation with acidification. These data provide insight into mechanisms subserving known pH effects on cellular signaling functions. Our results demonstrate that H452 in the third extracellular loop of Kv1.5 plays a role in C-type inactivation, thus expanding the known complement of protein regions that contribute to the slow K+ channel inactivation mechanism.

Amino Acid Sequence↗

PubMed1988

[Apamin--a highly specific and effective blockader of calcium-dependent potassium conductance].

Apamin is a toxic polypeptide extracted from bee venom. It has been considered as a neurotoxin with central action, but its low concentrations (10(-8)-10(-7) M) were shown to reversibly block the nonadrenergic inhibition and effects of externally applied ATP, noradrenaline and caffeine in smooth muscles of the gastrointestinal tract. All these processes are related to the activation of Ca-dependent potassium permeability. Current-clamp, voltage-clamp and patch-clamp experiments have also shown that apamin blocks specifically some types of these conductances in other tissues: skeletal muscles, mammalian neurons and neuroblastoma, hepatocytes. Nowadays apamin is the most specific but not a universal blocker of the Ca-activated potassium conductance.

Animals↗

PubMed1999

Activation of a Ca2+-permeable cation channel by two different inducers of apoptosis in a human prostatic cancer cell line.

1. We have combined patch clamp recording with simultaneous [Ca2+]i measurements in single LNCaP cells (a human prostate cancer cell line), to study the activation of Ca2+-permeable channels by two different inducers of apoptosis, ionomycin and serum deprivation. 2. In perforated patch recording, LNCaP cells had a membrane potential of -40 mV and a resting [Ca2+]i of 90 nM. Application of ionomycin at levels that induced apoptosis in these cells (10 microM) produced a biphasic increase in [Ca2+]i. The first rise in [Ca2+]i was due to release of Ca2+ from internal stores and it was associated with a membrane hyperpolarization to -77 mV. The latter was probably due to the activation of high conductance, Ca2+- and voltage-dependent K+ channels (maxi-K). Conversely, the second rise in [Ca2+]i was always preceded by and strictly associated with membrane depolarization and required external Ca2+. Serum deprivation, another inducer of apoptosis, unmasked a voltage-independent Ca2+ permeability as well. 3. A lower concentration of ionomycin (1 microM) did not induce apoptosis, and neither depolarized LNCaP cells nor produced the biphasic increase in [Ca2+]i. However, the first increment in [Ca2+]i due to release from internal Ca2+ stores was evident at this concentration of ionomycin. 4. Simultaneous recordings of [Ca2+]i and ion channel activity in the cell attached configuration of patch clamp revealed a Ca2+-permeable, Ca2+-independent, non-selective cation channel of 23 pS conductance. This channel was activated only during the second increment in [Ca2+]i induced by ionomycin. The absence of serum activated the 23 pS channel as well, albeit at a lower frequency than with ionomycin. 5. Thus, the 23 pS channel can be activated by two unrelated inducers of apoptosis and it could be another Ca2+ influx mechanism in programmed cell death of LNCaP cells.

Apoptosis↗

PubMed1996

Endotoxin-induced L-arginine pathway produces nitric oxide and modulates the Ca2+-activated K+ channel in cultured human dermal papilla cells.

Endotoxin includes an enzyme that synthesizes nitric oxide (NO) from l-arginine (NO synthase) in vascular smooth muscles cells, macrophages, and fibroblasts, leading to the release of NO. We evaluated the release of NO and its intracellular action on the Ca2+-activated K+ channel (KCa channel) in cultured human dermal papilla cells by use of the electron paramagnetic response (EPR) spin trapping method and the patch clamp technique. In dermal papilla cells pretreated for 24 h with endotoxin (1 microgram/microliter), application of 1 microM L-arginine generated NO, although no measurable release of NO was observed in cells without endotoxin pretreatment, as determined by the EPR spin trapping method. With the patch clamp technique, we found that the KCa channel of dermal papilla cells had high conductance and was voltage dependent. In addition, after endotoxin pretreatment, the extracellular application of 100 microM l-arginine modulated the KCa channel in the cell-attached patch configurations. In inside-out patch configuration, however, NO produced by L-arginine itself did not modulate the Kca channel. The modulation of the KCa channel was suppressed by pretreatment with 100 microM N omega-nitro-L arginine methyl ester, and inhibitor of inducible and constitutive NO synthases. Methylene blue, a blocker of guanylate cyclase, inhibited the L-arginine-induced activation of the Kca channel. Theses results indicate that the endotoxin-induced L-arginine pathway cell generates No, which consequently modulated the KCa channel in cultured human dermal papilla cells by increasing of cyclic GMP-dependent phosphorylation.

Arginine↗

PubMed2005

The human Ca2+-activated K+ channel, IK, can be blocked by the tricyclic antihistamine promethazine.

Phenothiazines can be used as psychopharmaceutical agents and are known to cause many side effects during treatment since they interfere with many different cellular systems. Recently, phenothiazines were reported to block Ca(2+)-activated potassium channels of the SK type. Therefore we investigated their effect on the functionally related class of Ca(2+)-activated potassium channels of the IK type. The representative phenothiazine derivative promethazine (PTZ) blocked IK channels almost independently from the extracellular pH(o) with an IC(50) of 49 +/- 0.2 microM (pH(o) 7.4, n = 5) and 32 +/- 0.2 microM (pH(o) 6.2, n = 5) in whole cell experiments. The extracellularly applied membrane impermeable PTZ analogue methyl-promethazine (M-PTZ) had a strongly reduced blocking potency compared to PTZ. In contrast, intracellularly applied PTZ and M-PTZ had the same blocking potency on IK channels in excised inside out patch clamp experiments (K(d) = 9.3 +/- 0.5 microM for PTZ, n = 7 and 6.7 +/- 0.4 microM for M-PTZ, n = 5). The voltage dependency of the PTZ and M-PTZ block was investigated in excised inside out patch clamp experiments at a concentration of 100 microM. For both compounds the block was more pronounced at positive membrane potentials. The steepness of the voltage dependency was found to be 70 +/- 10 mV (for PTZ) and 61 +/- 6 mV (for M-PTZ) indicating that both compounds sensed approximately 40% of the entire membrane spanning electrical field from the inside. We conclude that PTZ and M-PTZ bind to a side in IK channels, which is located within the electrical field and is accessible from the intracellular side.

Calcium↗

PubMed1995

A transient effect of estrogen on calcium currents and electrophysiological responses to gonadotropin-releasing hormone in ovine gonadotropes.

Episodic release of luteinizing hormone (LH) by the pituitary gland is controlled by hypothalamic gonadotropin-releasing hormone (GnRH). In the period leading up to the preovulatory surge of LH, estrogen increases the number of pituitary receptors for GnRH and sensitises the gonadotropes to GnRH. The postreceptor events that are responsible for the increase in responsiveness to GnRH are not clearly delineated, but LH release is known to be Ca2+ dependent. The present study addressed the question as to whether or not estrogen may act to modify voltage-dependent Ca2+ entry in normal gonadotropes. Primary cultures enriched in gonadotropes or somatotropes were produced from anestrous female sheep. Conventional whole-cell patch-clamp recording was used to measure inward membrane current in the absence of GnRH treatment, with and without 10 nM estradiol-17beta (E2) treatment for 0 to 36 h. Nystatin-perforated whole-cell patch-clamp recording was used to record membrane voltage responses to GnRH. Ca2+ current density (ICa, pA/pF) began to increase after 2 h exposure to E2, and reached peak values of about 200% of control by 16-20 h (p < 0.005), then declined. If E2 was withdrawn at 24 h, ICa returned towards control values by 36 h. If E2 treatment was continued beyond 24 h, however, ICa fell to about 75% of control by 36 h (p < 0.005). Actinomycin D prevented the enhancement of ICa. E2 was without effect on Na+ current density in gonadotropes, or on ICa in somatotropes. The proportion of ICa carried by L-type and N-type channels in gondadotropes was not changed by E2. Ovine gonadotropes respond to GnRH with membrane potential fluctuations driven by periodic activation of Ca2+-dependent K+ channels, and synchronised action potential generation. This response was found to be sensitive to E2. Responses were categorised according to the pattern of activity evoked by 10 nM GnRH. Without E2 treatment, 11/14 cells responded with oscillations and 3/14 cells responded with spiking (hyperpolarizations following single action potentials). After 20 h 10 nM E2, just 1/14 cells responded with hyperpolarizing oscillations while 13/14 cells showed spiking activity. The predominance of the spiking pattern in E2-treated cells is consistent with the increased Ca2+ flux, and with enhanced LH release. We conclude that E2 has a transient effect on gonadotropes to enhance voltage-gated Ca2+ channel function. The time-course and biphasic nature of the influence of E2 on ICa may be physiologically appropriate to the preovulatory LH surge. Enhanced Ca2+ influx may participate in increased Ca2+-dependent hormone release, while the delayed inhibitory action of E2 on ICa may serve to limit the duration of the surge.

Animals↗

PubMed2005

Cyclopiazonic acid activates a Ca2+-permeable, nonselective cation conductance in porcine and bovine tracheal smooth muscle.

Capacitative Ca2+ entry has been examined in several tissues and, in some, appears to be mediated by nonselective cation channels collectively referred to as "store-operated" cation channels; however, relatively little is known about the electrophysiological properties of these channels in airway smooth muscle. Consequently we examined the electrophysiological characteristics and changes in intracellular Ca2+ concentration associated with a cyclopiazonic acid (CPA)-evoked current in porcine and bovine airway smooth muscle using patch-clamp and Ca2+-fluorescence techniques. In bovine tracheal myocytes, CPA induced an elevation of intracellular Ca2+ that was dependent on extracellular Ca2+ and was insensitive to nifedipine (an L-type voltage-gated Ca2+ channel inhibitor). Using patch-clamp techniques and conditions that block both K+ and Cl- currents, we found that CPA rapidly activated a membrane conductance (I(CPA)) in porcine and bovine tracheal myocytes that exhibits a linear current-voltage relationship with a reversal potential around 0 mV. Replacement of extracellular Na+ resulted in a marked reduction of I(CPA) at physiological membrane potentials (i.e., -60 mV) that was accompanied by a shift in the reversal potential for I(CPA) toward more negative membrane potentials. In addition, I(CPA) was markedly inhibited by 10 microM Gd3+ and La3+ but was largely insensitive to 1 microM nifedipine. We conclude that CPA induces capacitative Ca2+ entry in porcine and bovine tracheal smooth muscle via a Gd3+- and La3+-sensitive, nonselective cation conductance.

Animals↗

PubMed2000

Inhibition of 5-HT3 receptors by propofol: equilibrium and kinetic measurements.

Patch-clamp/rapid solution exchange experiments as well as tracer ([14C]-guanidinium) influx measurements were applied to investigate effects of propofol on 5-HT3 receptor channels and compare the results with those obtained with pentobarbital. Currents induced by 30 microM 5-HT were recorded in outside-out patches from N1E-115 cells. Application of propofol 45 s before and during 5-HT application inhibited peak-currents and integrated current responses in a concentration-dependent manner (IC50 values=14.5 and 10.5 microM; Hill coefficients -1.5 and -1.3, respectively). The inhibitory effect of propofol in the current measurements was similar to the propofol-induced inhibition in tracer influx experiments in whole N1E-115 cells (Barann et al., 1993. Naunyn-Schmiedeberg's Archives of Pharmacology 347, 125-132). Pentobarbital-induced inhibition of 5-HT3 receptors in both patch-clamp (Barann et al., 1997. Neuropharmacology 36, 655-664) and tracer influx measurements indicated a lower potency and lower slope (IC50 values=130 and 55 microM; Hill coefficients -0.8 and -0.7, respectively) compared to propofol. Propofol, in contrast to pentobarbital, showed nearly the full potency when applied to the patches exclusively 45 s before 5-HT. Propofol was least effective when administered exclusively during 5-HT. The onset of inhibition of 5-HT-induced peak currents by propofol had a time constant of 220 ms, similar to the kinetics of 5-HT-induced desensitization.

Anesthetics, General↗

PubMed2006

Early evaluation of compound QT prolongation effects: a predictive 384-well fluorescence polarization binding assay for measuring hERG blockade.

INTRODUCTION: A large number of drugs from a variety of pharmacological classes have been demonstrated to cause adverse effects on cardiac rhythm, including the life-threatening arrhythmia Torsades de Pointes. These side effects are often associated with prolongation of the QT interval and are mediated via blockade of the human ether-a-go-go related gene (hERG) encoded potassium channel. In order to manage this risk in the pharmaceutical industry it is desirable to evaluate QT prolongation as early as possible in the drug discovery process. METHODS: Here we describe the development of a 384-well fluorescence polarization (FP) binding assay compatible with high-throughput assessment of compound blockade of the hERG channel during the lead optimisation process. To characterise the fluorescent ligand that was developed, competition binding studies, kinetic studies and electrophysiology studies were performed. Furthermore, to validate the assay as a key screening method a series of competition binding studies were performed and correlated with functional data obtained via patch-clamp. RESULTS: Evaluation of the assay indicates that high quality data is obtained (Z'>0.6), that the K(i) values determined are equivalent to more traditional radiometric methods and that it is predictive for functional hERG blockade as assessed by patch clamp. DISCUSSION: Whilst FP assays, utilizing a variety of fluors, have become well established for the evaluation of G-protein-coupled receptor (GPCRs) and kinase ligand interactions, this technique has not been applied widely to the study of ion channels. Therefore, this represents a novel assay format that is amenable to the evaluation of thousands of compounds per day. Whilst other assay formats have proven predictive or high throughput, this assay represents one of few that combines both attributes, moreover it represents the most cost effective assay, making it truly amenable to early assessment of hERG blockade.

Binding, Competitive↗

PubMed2000

Dihydropyridine-sensitive Ca(2+) channels in human glomerular mesangial cells.

In mesangial cells (MC), the response of intracellular Ca(2+) concentration ([Ca(2+)](i)) to a contractile agonist is biphasic with a large, transient increase in [Ca(2+)](i) followed by a smaller but sustained elevation as Ca(2+) flows into the cell from the extracellular fluid. It has been postulated that membrane depolarization precedes opening of Ca(2+) channels in the plasmalemmal membrane. However, a role for voltage-gated Ca(2+) channels (VGCC) in human MC has been controversial, and their existence has not been verified with single-channel analysis. We used fura 2 fluorescence and patch-clamp techniques to determine the properties of the Ca(2+) entry pathway responsible for the sustained response of [Ca(2+)](i) in human MC. We found that ANG II at 10 nM, 100 nM, and 1 microM increased [Ca(2+)](i) to sustained levels of 22%, 35%, and 49%, respectively, above baseline. The sustained response to 1 microM ANG II was attenuated by diltiazem and was reduced to a value less than baseline in the absence of external Ca(2+). None of the peak responses (due to release of intracellular stores of Ca(2+)) were affected by removal of external Ca(2+) or addition of diltiazem. Upon elevating the extracellular [K(+)] from 5 mM to 75 mM, [Ca(2+)](i) reached a sustained level of 48% greater than baseline. This effect of high K(+) was attenuated by either Ca(2+) removal or addition of diltiazem. In the presence of 75 or 140 mM K(+), the dihydropyridine agonist BAY K 8644 (1 microM and 10 microM) initiated sustained [Ca(2+)](i) responses averaging 18% and 25%, respectively, greater than baseline. With <10 nM Ca(2+) in the external solution, BAY K 8644 did not significantly affect [Ca(2+)](i). In separate patch-clamp experiments, barium-selective channels were found in cell-attached patches with 90 mM BaCl(2) and 10 microM BAY K 8644 in the pipette solution. The single-channel conductance was 11.2 pS, and the open probability increased steeply at membrane potentials between -30 mV and 0 mV. It is concluded that human glomerular MC contain dihydropyridine-sensitive Ca(2+) channels responsible for the voltage-regulated entry of Ca(2+) into the cell during an agonist-induced contraction.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMedDate not supplied

IgE-mediated degranulation of mast cells does not require opening of ion channels.

Rat peritoneal mast cells respond to antigenic stimulation by releasing histamine through exocytosis. The dynamics of exocytosis can be investigated by dialysing single cells with patch pipettes using the whole-cell recording configuration of the patch-clamp technique. However, dialysed cells fail to respond to external stimuli such as compound 48/80 or antigens, suggesting that essential cytoplasmic components have been washed out. We have developed a new patch-clamp configuration in which the patch under the pipette tip is not disrupted but instead permeabilized, preventing the diffusion of large molecules out of the cell. In this configuration the cell responds to external stimulation, and the capacitance as well as the conductance of the cell membrane can be recorded during degranulation. On antigenic stimulation, the cell capacitance (proportional to plasma membrane area), after an initial delay, increases by a factor of about 3. This increase in capacitance is often preceded by a transient increase in conductance. Agents that block Ca-activated channels inhibit this conductance change without affecting the amplitude and time course of degranulation. We therefore conclude that, in contrast to excitable secretory cells such as chromaffin cells, mast cells do not use ion channels in stimulus-secretion coupling.

Animals↗

PubMed2005

Functional properties of endogenous receptor- and store-operated calcium influx channels in HEK293 cells.

Activation of phospholipase C (PLC)-mediated signaling pathways in non-excitable cells causes the release of calcium (Ca2+) from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores and activation of Ca2+ influx via plasma membrane Ca2+ channels. The properties and molecular identity of plasma membrane Ca2+ influx channels in non-excitable cells is a focus of intense investigation. In the previous studies we used patch clamp electrophysiology to describe the properties of Ca2+ influx channels in human carcinoma A431 cell lines. Now we extend our studies to human embryonic kidney HEK293 cells. By using a combination of Ca2+ imaging and whole cell and single channel patch clamp recordings we discovered that: 1) HEK293 cells contain four types of plasma membrane Ca2+ influx channels: I(CRAC), Imin, Imax, and I(NS); 2) I(CRAC) channels are highly Ca2+-selective (P(Ca/Cs)>1000) and I(CRAC) single channel conductance is too small for single channel analysis; 3) Imin channels in HEK293 cells display functional properties identical to Imin channels in A431 cells, with single channel conductance of 1.2 pS for divalent cations, 10 pS for monovalent cations, and divalent cation selectivity P(Ba/K)=20; 4) Imin channels in HEK293 cells are activated by InsP3 and inhibited by phosphatidylinositol 4,5-bisphosphate, but store-independent; 5) when compared with Imin, Imax channels have higher conductance for divalent (17 pS) and monovalent (33 pS) cations, but less selective for divalent cations (P(Ba/K)=4), 6) Imax channels in HEK293 cells can be activated by InsP3 or by Ca2+ store depletion; 7) I(NS) channels are non-selective (P(Ba/K)=0.4) and display a single channel conductance of 5 pS; and 8) I(NS) channels are not gated by InsP3 but activated by depletion of intracellular Ca2+ stores. Our findings provide novel information about endogenous Ca2+ channels supporting receptor-operated and store-operated Ca2+ influx pathways in HEK293 cells.

Calcium↗

PubMedDate not supplied

Physiological and proteomic approaches to studying prefertilization events in the human.

This research aims firstly to understand, in cellular and molecular terms, how a mature human spermatozoon is prepared for fertilization, and secondly, to identify what factors are involved in the initial signalling interactions between the egg and spermatozoon. In order to achieve these objectives, a combination of approaches is being used, including single-cell imaging, patch clamping and proteomics. Single-cell imaging reveals hidden complexity and heterogeneity in signalling responses in spermatozoa. Characterization of cell physiology at the single-cell level must be a future aim, including the study of ion channel expression and function by patch clamping. Proteomic experiments are aimed at identifying defects in protein expression in specific subgroups of men, e.g. those with globozoospermia. A better understanding of prefertilization events will allow the development of non-assisted reproductive therapy, drug-based treatments for male infertility.

Amino Acid Sequence↗