Reversal Potential Of Current Research Articles

Conductance properties of the α9α10 nicotinic acetylcholine receptor of neonatal mouse inner and outer hair cells

In the developing cochlea, just before the onset of hearing on postnatal day 12, the medial olivocochlear efferent axons in synaptic contact with the inner hair cells (IHCs) start withdrawing and new efferent synaptic connections are formed on the outer hair cells (OHCs), thereby progressing towards the adult pattern of medial olivocochlear efferent innervation. The synapses are inhibitory, calcium influx through the α9α10 nicotinic acetylcholine receptors (nAChRs) driving opening of calcium-dependent potassium channels. The nAChRs appear to function similarly in IHCs and OHCs, although with probable kinetic differences. Our aim was to assess their functional similarity in the neonatal mouse cochlea by making whole-cell recordings from both hair cell types between postnatal day 7 and 10 when nAChRs are expressed. ACh was applied to voltage-clamped hair cells by pressure-ejection from a pipette. The cells were dialysed with a Cs+-based solution designed to eliminate calcium-dependent potassium currents. There were differences in amplitude, voltage-sensitivity and reversal potential of the nAChR currents between IHCs and OHCs. There was also some indication that IHC nAChRs have slower activation and desensitization kinetics, although the relatively slow ACh application limited interpretation of this result. These differences, particularly concerning the reversal potential, might indicate the presence of different auxiliary protein subunits of the α9α10 receptor in neonatal IHCs and OHCs.

Tonic excitation by astrocytic GABA causes neuropathic pain by augmenting neuronal activity and glucose metabolism

Neuropathic pain is a debilitating condition caused by the hyperexcitability of spinal dorsal horn neurons and is often characterized by allodynia. Although neuron-independent mechanisms of hyperexcitability have been investigated, the contribution of astrocyte-neuron interactions remains unclear. Here, we show evidence of reactive astrocytes and their excessive GABA release in the spinal dorsal horn, which paradoxically leads to the tonic excitation of neighboring neurons in a neuropathic pain model. Using multiple electrophysiological methods, we demonstrated that neuronal hyperexcitability is attributed to both increased astrocytic GABA synthesis via monoamine oxidase B (MAOB) and the depolarized reversal potential of GABA-mediated currents (EGABA) via the downregulation of the neuronal K+/Cl− cotransporter KCC2. Furthermore, longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET imaging demonstrated increased regional glucose metabolism in the ipsilateral dorsal horn, reflecting neuronal hyperexcitability. Importantly, inhibiting MAOB restored the entire astrocytic GABA-mediated cascade and abrogated the increased glucose metabolism and mechanical allodynia. Overall, astrocytic GABA-mediated tonic excitation is critical for neuronal hyperexcitability, leading to mechanical allodynia and neuropathic pain.

Activation of Kir4.1/Kir5.1 contributes to the cyclosporin A-induced stimulation of the renal NaCl cotransporter and hyperkalemic hypertension.

Cyclosporin A (CsA) is a widely used immunosuppressive drug that causes hypertension and hyperkalemia. Moreover, CsA-induced stimulation of the thiazide-sensitive NaCl cotransporter (NCC) in the kidney has been shown to be responsible for the development of hyperkalemic hypertension. In this study, we tested whether CsA induces the activation of NCC by stimulating the basolateral Kir4.1/Kir5.1 channel in the distal convoluted tubule (DCT). Electrophysiology, immunoblotting, metabolic cages, and radio-telemetry methods were used to examine the effects of CsA on Kir4.1/Kir5.1 activity in the DCT, NCC function, and blood pressure in wild-type (WT) and kidney-specific Kir4.1 knockout (KS-Kir4.1 KO) mice. The single-channel patch clamp experiment demonstrated that CsA stimulated the basolateral 40 pS K+ channel in the DCT. Whole-cell recording showed that short-term CsA administration (2h) not only increased DCT K+ currents but also shifted the K+ current (IK ) reversal potential to the negative range (hyperpolarization). Furthermore, CsA administration increased phosphorylated NCC (pNCC) levels and inhibited renal Na+ and K+ excretions in WT mice but not in KS-Kir4.1 KO mice, suggesting that Kir4.1 is required to mediate CsA effects on NCC function. Finally, long-term CsA infusion (14 days) increased blood pressure, plasma K+ concentration, and total NCC or pNCC abundance in WT mice, but these effects were blunted in KS-Kir4.1 KO mice. We conclude that CsA stimulates basolateral K+ channel activity in the DCT and that Kir4.1 is essential for CsA-induced NCC activation and hyperkalemic hypertension.

Consequences of somatic mutations of GIRK1 detected in primary malign tumors on expression and function of G-protein activated, inwardly rectifying, K+ channels.

A search in the GDC Data Portal revealed 304 documented somatic mutations of the KCNJ3 gene in primary tumors (out of 10.202 cases). Most affected tumor types were carcinomas from uterus, skin and lung, while breast cancer exerted the lowest number of somatic mutations. We focused our research on 15 missense mutations within the region between TM1 and TM2, comprising the pore helix and ion selectivity signature. Expression was measured by confocal laser scan microscopy of eGFP tagged GIRK1 subunits, expressed with and without GIRK4 in oocytes of Xenopus laevis. GIRK ion currents were activated via coexpressed m2Rs and measured by the Two Electrode Voltage Clamp technique. Magnitude of the total GIRK current, as well as the fraction of current inducible by the agonist, were measured. Ion selectivity was gauged by assessment of the PNa+/PK+ ratio, calculated by the GIRK current reversal potential in extracellular media at different Na+ and K+ concentrations. None of the tested mutations was able to form functional GIRK1 homooligomeric ion channels. One of the mutations, G145A, which locates directly to the ion selectivity signature, exerted an increased PNa+/PK+ ratio. Generally, the missense mutations studied can be categorized into three groups: (i) normal/reduced expression accompanied by reduced/absent function (S132Y, F136L, E139K, G145A, R149Q, R149P, G178D, S185Y, Q186R), (ii) normal/increased expression as well as increased function (E140M, A142T, M184I) and (iii) miniscule expression but increased function relative to expression levels (I151N, G158S). We conclude, that gain of function mutations, identical or similar to categories (ii) and (iii), may potentially be involved in genesis and progression of malignancies in tissues that exert a high rate of occurrence of somatic mutations of KCNJ3.

Certain calcium channel inhibitors exhibit a number of secondary effects on the physiological properties in Nitellopsis obtusa: a voltage clamp approach.

An unsolved problem of contemporary plant electrophysiology is the identity of Ca2+ channels responsible for the initiation of the action potential. We took a pharmacological approach and applied several Ca2+ channel blockers (verapamil, tetrandrine, and NED-19) on a Characean (Nitellopsis obtusa ) algae model system. The impact of the selected pharmaceuticals on the parameters of excitation transients of a single cell was analysed employing the two-electrode voltage clamp technique. It was revealed that tetrandrine exerted no effect, while both verapamil and NED-19 prolonged activation and inactivation durations of the excitatory Cl- current. NED-19 also significantly depolarised the excitation threshold membrane potential and shifted Ca2+ current reversal potential. Thus, NED-19 most specifically targeted Ca2+ channels. A viability assay paired with observations of cytoplasmic streaming revealed that verapamil affected not only Ca2+ channels but also exhibited non-specific effects, which eventually lead to cell death. Since many potential Ca2+ channel blockers exert additional undesirable non-specific effects, our study underlines the necessity to search for new more specific modulators of plant Ca2+ transport systems.

Deletion of renal Nedd4-2 abolishes the effect of high K+ intake on Kir4.1/Kir5.1 and NCC activity in the distal convoluted tubule.

High-dietary K+ (HK) intake inhibits basolateral Kir4.1/Kir5.1 activity in the distal convoluted tubule (DCT), and HK-induced inhibition of Kir4.1/Kir5.1 is essential for HK-induced inhibition of NaCl cotransporter (NCC). Here, we examined whether neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) deletion compromises the effect of HK on basolateral Kir4.1/Kir5.1 and NCC in the DCT. Single-channel recording and whole cell recording showed that neither HK decreased nor low-dietary K+ (LK) increased basolateral Kir4.1/Kir5.1 activity of the DCT in kidney tubule-specific Nedd4-2 knockout (Ks-Nedd4-2 KO) mice. In contrast, HK inhibited and LK increased Kir4.1/Kir5.1 activity in control mice [neural precursor cell expressed developmentally downregulated 4-like (Nedd4l)flox/flox]. Also, HK intake decreased the negativity of K+ current reversal potential in the DCT (depolarization) only in control mice but not in Ks-Nedd4-2 KO mice. Renal clearance experiments showed that HK intake decreased, whereas LK intake increased, hydrochlorothiazide-induced renal Na+ excretion only in control mice, but this effect was absent in Ks-Nedd4-2 KO mice. Western blot analysis also demonstrated that HK-induced inhibition of phosphorylated NCC (Thr53) and total NCC was observed only in control mice but not in Ks-Nedd4-2 KO mice. Furthermore, expression of all three subunits of the epithelial Na+ channel in Ks-Nedd4-2 KO mice on HK was higher than in control mice. Thus, plasma K+ concentrations were similar between Nedd4lflox/flox and Ks-Nedd4-2 KO mice on HK for 7 days despite high NCC expression. We conclude that Nedd4-2 plays a role in regulating HK-induced inhibition of Kir4.1/Kir5.1 and NCC in the DCT.NEW & NOTEWORTHY Basolateral Kir4.1/Kir5.1 in the distal convoluted tubule plays an important role as a "K+ sensor" in the regulation of renal K+ excretion after high K+ intake. We found that neural precursor cell expressed developmentally downregulated 4-2 (Nedd4-2) a role in mediating the effect of K+ diet on Kir4.1/Kir5.1 and NaCl cotransporter because high K+ intake failed to inhibit basolateral Kir4.1/Kir5.1 and NaCl cotransporter in kidney tubule-specific Nedd4-2 knockout mice.

Variants inCHRNB2andCHRNA4Identified in Patients with Insular Epilepsy

Our purpose was to determine the role of CHRNA4 and CHRNB2 in insular epilepsy. We identified two patients with drug-resistant predominantly sleep-related hypermotor seizures, one harboring a heterozygous missense variant (c.77C>T; p. Thr26Met) in the CHRNB2 gene and the other a heterozygous missense variant (c.1079G>A; p. Arg360Gln) in the CHRNA4 gene. The patients underwent electrophysiological and neuroimaging studies, and we performed functional characterization of the p. Thr26Met (c.77C>T) in the CHRNB2 gene. We localized the epileptic foci to the left insula in the first case (now seizure-free following epilepsy surgery) and to both insulae in the second case. Based on tools predicting the possible impact of amino acid substitutions on the structure and function of proteins (sorting intolerant from tolerant and PolyPhen-2), variants identified in this report could be deleterious. Functional expression in human cell lines of α4β2 (wild-type), α4β2-Thr26Met (homozygote), and α4β2/β2-Thr26Met (heterozygote) nicotinic acetylcholine receptors revealed that the mutant subunit led to significantly higher whole-cell nicotinic currents. This feature was observed in both homo- and heterozygous conditions and was not accompanied by major alterations of the current reversal potential or the shape of the concentration-response relation. This study suggests that variants in CHRNB2 and CHRNA4, initially linked to autosomal dominant nocturnal frontal lobe epilepsy, are also found in patients with predominantly sleep-related insular epilepsy. Although the reported variants should be considered of unknown clinical significance for the moment, identification of additional similar cases and further functional studies could eventually strengthen this association.

A Cellular Mechanism of Learning-Induced Enhancement of Synaptic Inhibition: PKC-Dependent Upregulation of KCC2 Activation

Long-term memory of complex olfactory learning is expressed by wide spread enhancement in excitatory and inhibitory synaptic transmission onto piriform cortex pyramidal neurons. A particularly interesting modification in synaptic inhibition is the hyperpolarization of the reversal potential of the fast post synaptic inhibitory potential (fIPSP). Here we study the mechanism underlying the maintenance of such a shift in the fIPSP. Blocking of the neuronal specific K+-Cl− co-transporter (KCC2) in neurons of trained rats significantly depolarized the averaged fIPSP reversal potential of the spontaneous miniature inhibitory post synaptic currents (mIPSCs), to the averaged pre-training level. A similar effect was obtained by blocking PKC, which was previously shown to upregulate KCC2. Accordingly, the level of PKC-dependent phosphorylation of KCC2, at the serine 940 site, was significantly increased after learning. In contrast, blocking two other key second messenger systems CaMKII and PKA, which have no phosphorylation sites on KCC2, had no effect on the fIPSP reversal potential. Importantly, the PKC inhibitor also reduced the averaged amplitude of the spontaneous miniature excitatory synaptic currents (mEPSCs) in neurons of trained rats only, to the pre-training level. We conclude that learning-induced hyper-polarization of the fIPSP reversal potential is mediated by PKC-dependent increase of KCC2 phosphorylation.

Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

Polymorphic variants of the gene encoding for metabotropic glutamate receptor 3 (mGlu3) are linked to schizophrenia. Because abnormalities of cortical GABAergic interneurons lie at the core of the pathophysiology of schizophrenia, we examined whether mGlu3 receptors influence the developmental trajectory of cortical GABAergic transmission in the postnatal life. mGlu3-/- mice showed robust changes in the expression of interneuron-related genes in the prefrontal cortex (PFC), including large reductions in the expression of parvalbumin (PV) and the GluN1 subunit of NMDA receptors. The number of cortical cells enwrapped by perineuronal nets was increased in mGlu3-/- mice, suggesting that mGlu3 receptors shape the temporal window of plasticity of PV+ interneurons. Electrophysiological measurements of GABAA receptor-mediated responses revealed a more depolarized reversal potential of GABA currents in the somata of PFC pyramidal neurons in mGlu3-/- mice at postnatal d 9 associated with a reduced expression of the K+/Cl- symporter. Finally, adult mGlu3-/- mice showed lower power in electroencephalographic rhythms at 1-45 Hz in quiet wakefulness as compared with their wild-type counterparts. These findings suggest that mGlu3 receptors have a strong impact on the development of cortical GABAergic transmission and cortical neural synchronization mechanisms corroborating the concept that genetic variants of mGlu3 receptors may predispose to psychiatric disorders.-Imbriglio, T., Verhaeghe, R., Martinello, K., Pascarelli, M. T., Chece, G., Bucci, D., Notartomaso, S., Quattromani, M., Mascio, G., Scalabrì, F., Simeone, A., Maccari, S., Del Percio, C., Wieloch, T., Fucile, S., Babiloni, C., Battaglia, G., Limatola, C., Nicoletti, F., Cannella, M. Developmental abnormalities in cortical GABAergic system in mice lacking mGlu3 metabotropic glutamate receptors.

CHRNA2 and Nocturnal Frontal Lobe Epilepsy: Identification and Characterization of a Novel Loss of Function Mutation.

Mutations in genes coding for subunits of the neuronal nicotinic acetylcholine receptor (nAChR) have been involved in familial sleep-related hypermotor epilepsy (also named autosomal dominant nocturnal frontal lobe epilepsy, ADNFLE). Most of these mutations reside in CHRNA4 and CHRNB2 genes, coding for the α4 and β2 nAChR subunits, respectively. Two mutations with contrasting functional effects were also identified in the CHRNA2 gene coding for the α2 subunit. Here, we report the third mutation in the CHRNA2, found in a patient showing ADNFLE. The patient was examined by scalp EEG, contrast-enhanced brain magnetic resonance imaging (MRI), and nocturnal video-polysomnographic recording. All exons and the exon-intron boundaries of CHRNA2, CHRNA4, CHRNB2, CRH, KCNT1 were amplified and Sanger sequenced. In the proband, we found a c.754T>C (p.Tyr252His) missense mutation located in the N-terminal ligand-binding domain and inherited from the mother. Functional studies were performed by transient co-expression of α2 and α2Tyr252His, with either β2 or β4, in human embryonic kidney (HEK293) cells. Equimolar amounts of subunits expression were obtained by using F2A-based multi-cistronic constructs encoding for the genes relative to the nAChR subunits of interest and for the enhanced green fluorescent protein. The mutation reduced the maximal currents by approximately 80% in response to saturating concentrations of nicotine in homo- and heterozygous form, in both the α2β4 and α2β2 nAChR subtypes. The effect was accompanied by a strong right-shift of the concentration-response to nicotine. Similar effects were observed using ACh. Negligible effects were produced by α2Tyr252His on the current reversal potential. Moreover, binding of (±)-[3H]Epibatidine revealed an approximately 10-fold decrease of both Kd and Bmax (bound ligand in saturating conditions), in cells expressing α2Tyr252His. The reduced Bmax and whole-cell currents were not caused by a decrease in mutant receptor expression, as minor effects were produced by α2Tyr252His on the level of transcripts and the membrane expression of α2β4 nAChR. Overall, these results suggest that α2Tyr252His strongly reduced the number of channels bound to the agonist, without significantly altering the overall channel expression. We conclude that mutations in CHRNA2 are more commonly linked to ADNFLE than previously thought, and may cause a loss-of-function phenotype.

Norepinephrine-Induced Stimulation of Kir4.1/Kir5.1 Is Required for the Activation of NaCl Transporter in Distal Convoluted Tubule.

The stimulation of β-adrenergic receptor increases thiazide-sensitive NaCl cotransporter (NCC), an effect contributing to salt-sensitive hypertension by sympathetic stimulation. We now test whether the stimulation of β-adrenergic receptor-induced activation of NCC is achieved through activating basolateral Kir4.1 in the distal convoluted tubule (DCT). Application of norepinephrine increased the basolateral 40 pS K+ channel (Kir4.1/Kir5.1 heterotetramer) in the DCT. The stimulatory effect of norepinephrine on the K+ channel was mimicked by cAMP analogue but abolished by inhibiting PKA (protein kinase A). Also, the effect of norepinephrine on the K+ channel in the DCT was recapitulated by isoproterenol but not by α-adrenergic agonist and blocked by propranolol, suggesting that norepinephrine effect on the K+ channel was mediated by β-adrenergic receptor. The whole-cell recording shows that norepinephrine and isoproterenol increased DCT K+ currents and shifted the K+ current ( IK) reversal potential to negative range (hyperpolarization). Continuous norepinephrine perfusion (7 days) increased DCT K+ currents, hyperpolarized IK reversal potential, and increased the expression of total NCC/phosphorylated NCC, but it had no significant effect on the expression of NKCC2 (type 2 Na-Cl-K cotransporter) and ENaC-α (epithelial Na channel-α subunit). Renal clearance study demonstrated that norepinephrine perfusion augmented thiazide-induced urinary Na+ excretion only in wild-type but not in kidney-specific Kir4.1 knockout mice, suggesting that Kir4.1 is required for mediating the effect of norepinephrine on NCC. However, norepinephrine perfusion did not affect urinary K+ excretion. We conclude that the stimulation of β-adrenergic receptor activates the basolateral Kir4.1 in the DCT and that the activation of Kir4.1 is required for norepinephrine-induced stimulation of NCC.

Propensity for Bistability of Bursting and Silence in the Leech Heart Interneuron.

The coexistence of neuronal activity regimes has been reported under normal and pathological conditions. Such multistability could enhance the flexibility of the nervous system and has many implications for motor control, memory, and decision making. Multistability is commonly promoted by neuromodulation targeting specific membrane ionic currents. Here, we investigated how modulation of different ionic currents could affect the neuronal propensity for bistability. We considered a leech heart interneuron model. It exhibits bistability of bursting and silence in a narrow range of the leak current parameters, conductance (gleak) and reversal potential (Eleak). We assessed the propensity for bistability of the model by using bifurcation diagrams. On the diagram (gleak, Eleak), we mapped bursting and silent regimes. For the canonical value of Eleak we determined the range of gleak which supported the bistability. We use this range as an index of propensity for bistability. We investigated how this index was affected by alterations of ionic currents. We systematically changed their conductances, one at a time, and built corresponding bifurcation diagrams in parameter planes of the maximal conductance of a given current and the leak conductance. We found that conductance of only one current substantially affected the index of propensity; the increase of the maximal conductance of the hyperpolarization-activated cationic current increased the propensity index. The second conductance with the strongest effect was the conductance of the low-threshold fast Ca2+ current; its reduction increased the propensity index although the effect was about two times smaller in magnitude. Analyzing the model with both changes applied simultaneously, we found that the diagram (gleak, Eleak) showed a progressively expanded area of bistability of bursting and silence.

Potassium Currents Activated by Depolarization in Odontoblasts.

Increased intracellular free Ca2+ concentrations elicit plasma membrane depolarization, which leads to the activation of K+ currents. However, the precise properties of K+ currents activated by depolarization in odontoblasts remain to be elucidated. The present study identified biophysical and pharmacological characteristics of time-dependent and voltage-activated K+ currents in freshly dissociated rat odontoblasts using patch-clamp recordings in a whole-cell configuration. Using a holding potential of −70 mV, outwardly rectifying time- and voltage-dependent currents were activated by depolarizing voltage. To record pure K+ conductance, we substituted Cl− in both the extracellular and intracellular solutions with gluconate−. Under these conditions, observation of K+ concentration changes in the extracellular solution showed that reversal potentials of tail currents shifted according to the K+ equilibrium potential. The activation kinetics of outward K+ currents were relatively slow and depended on the membrane potential. Kinetics of steady-state inactivation were fitted by a Boltzmann function. The half-maximal inactivation potential was −38 mV. Tetraethylammonium chloride, 4-aminopyridine, and α-dendrotoxin inhibited outward currents in odontoblasts in a concentration-dependent manner, suggesting that rat odontoblasts express the α-subunit of the time- and voltage-dependent K+ channel (Kv) subtypes Kv1.1, 1.2, and/or 1.6. We further examined the effects of Kv activity on mineralization by alizarin red and von Kossa staining. Continuous application of tetraethylammonium chloride to human odontoblasts grown in a mineralization medium over a 21-day period exhibited a dose-dependent decrease in mineralization efficiency compared to cells without tetraethylammonium chloride. This suggests that odontoblasts functionally express voltage-dependent K+ channels that play important roles in dentin formation.

Computational model of interictal discharges triggered by interneurons.

Interictal discharges (IIDs) are abnormal waveforms registered in the periods before or between seizures. IIDs that are initiated by GABAergic interneurons have not been mathematically modeled yet. In the present study, a mathematical model that describes the mechanisms of these discharges is proposed. The model is based on the experimental recordings of IIDs in pyramidal neurons of the rat entorhinal cortex and estimations of synaptic conductances during IIDs. IIDs were induced in cortico-hippocampal slices by applying an extracellular solution with 4-aminopyridine, high potassium, and low magnesium concentrations. Two different types of IIDs initiated by interneurons were observed. The first type of IID (IID1) was pure GABAergic. The second type of IID (IID2) was induced by GABAergic excitation and maintained by recurrent interactions of both GABA- and glutamatergic neuronal populations. The model employed the conductance-based refractory density (CBRD) approach, which accurately approximates the firing rate of a population of similar Hodgkin-Huxley-like neurons. The model of coupled excitatory and inhibitory populations includes AMPA, NMDA, and GABA-receptor-mediated synapses and gap junctions. These neurons receive both arbitrary deterministic input and individual colored Gaussian noise. Both types of IIDs were successfully reproduced in the model by setting two different depolarized levels for GABA-mediated current reversal potential. It was revealed that short-term synaptic depression is a crucial factor in ceasing each of the discharges, and it also determines their durations and frequencies.

Complex CatSper-dependent and independent [Ca2+]i signalling in human spermatozoa induced by follicular fluid.

STUDY QUESTIONDoes progesterone in human follicular fluid (hFF) activate CatSper and do other components of hFF modulate this effect and/or contribute separately to hFF-induced Ca2+ signaling?SUMMARY ANSWERhFF potently stimulates CatSper and increases [Ca2+]i, primarily due to high concentrations of progesterone, however, other components of hFF also contribute to [Ca2+]i signaling, including modulation of CatSper channel activity and inhibition of [Ca2+]i oscillations.WHAT IS KNOWN ALREADYCatSper, the principal Ca2+ channel in spermatozoa, is progesterone-sensitive and essential for fertility. Both hFF and progesterone, which is present in hFF, influence sperm function and increase their [Ca2+]i.STUDY DESIGN, SIZE, DURATIONThis basic medical research study used semen samples from >40 donors and hFF from >50 patients who were undergoing surgical oocyte retrieval for IVF/ICSI.PARTICIPANTS/MATERIALS, SETTING, METHODSSemen donors and patients were recruited in accordance with local ethics approval (13/ES/0091) from the East of Scotland Research Ethics Service REC1. Activities of CatSper and KSper were assessed by patch clamp electrophysiology. Sperm [Ca2+]i responses were examined in sperm populations and single cells. Computer-assisted sperm analysis (CASA) parameters and penetration into viscous media were used to assess functional effects.MAIN RESULTS AND THE ROLE OF CHANCEhFF and progesterone significantly potentiated CatSper currents. Under quasi-physiological conditions, hFF (up to 50%) failed to alter membrane K+ conductance or current reversal potential. hFF and progesterone (at an equivalent concentration) stimulated similar biphasic [Ca2+]i signals both in sperm populations and single cells. At a high hFF concentration (10%), the sustained (plateau) component of the [Ca2+]i signal was consistently greater than that induced by progesterone alone. In single cell recordings, 1% hFF-induced [Ca2+]i oscillations similarly to progesterone but with 10% hFF generation of [Ca2+]i oscillations was suppressed. After treatment to ‘strip’ lipid-derived mediators, hFF failed to significantly stimulate CatSper currents but induced small [Ca2+]i responses that were greater than those induced by the equivalent concentration of progesterone after stripping. Similar [Ca2+]i responses were observed when sperm pretreated with 3 μM progesterone (to desensitize progesterone responses) were stimulated with hFF or stripped hFF. hFF stimulated viscous media penetration and was more effective than the equivalent does of progesterone.LARGE SCALE DATAN/A.LIMITATIONS, REASONS FOR CAUTIONThis was an in vitro study. Caution must be taken when extrapolating these results in vivo.WIDER IMPLICATIONS OF THE FINDINGSThis study directly demonstrates that hFF activates CatSper and establishes that the biologically important effects of hFF reflect, at least in part, action on this channel, primarily via progesterone. However, these experiments also demonstrate that other components of hFF both contribute to the [Ca2+]i signal and modulate the activation of CatSper. Simple in vitro experiments performed out of the context of the complex in vivo environment need to be interpreted with caution.STUDY FUNDING/COMPETING INTEREST(S)Funding was provided by MRC (MR/K013343/1, MR/012492/1) (S.G.B., S.J.P., C.L.R.B.) and University of Abertay (sabbatical for S.G.B.). Additional funding was provided by TENOVUS SCOTLAND (S.M.D.S.), Chief Scientist Office/NHS Research Scotland (S.M.D.S). C.L.R.B. is EIC of MHR and Chair of the WHO ESG on Diagnosis of Male infertility. The remaining authors have no conlicts of interest.

An increased extrasynaptic NMDA tone inhibits A-type K+ current and increases excitability of hypothalamic neurosecretory neurons in hypertensive rats.

A functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K+ current (IA ) influences homeostatic firing responses of magnocellular neurosecretory cells (MNCs) to a physiological challenge. However, whether an altered eNMDAR-IA coupling also contributes to exacerbated MNC activity and neurohumoral activation during disease states is unknown. We show that activation of eNMDARs by exogenously applied NMDA inhibited IA in MNCs obtained from sham, but not in MNCs from renovascular hypertensive (RVH) rats. Neither the magnitude of the exogenously evoked NMDA current nor the expression of NMDAR subunits were altered in RVH rats. Conversely, we found that a larger endogenous glutamate tone, which was not due to blunted glutamate transport activity, led to the sustained activation of eNMDARs that tonically inhibited IA , contributing in turn to higher firing activity in RVH rats. Our studies show that exacerbated activation of eNMDARs by endogenous glutamate contributes to tonic inhibition of IA and enhanced MNC excitability in RVH rats. We recently showed that a functional coupling between extrasynaptic NMDA receptors (eNMDARs) and the A-type K+ current (IA ) influences the firing activity of hypothalamic magnocellular neurosecretory neurons (MNCs), as well as homeostatic adaptive responses to a physiological challenge. Here, we aimed to determine whether changes in the eNMDAR-IA coupling also contributed to exacerbated MNC activity during disease states. We used a combination of patch-clamp electrophysiology and real-time PCR in MNCs in sham and renovascular hypertensive (RVH) rats. Activation of eNMDARs by exogenously applied NMDA inhibited IA in sham rats, but this effect was largely blunted in RVH rats. The blunted response was not due to changes in eNMDAR expression and/or function, since neither NMDA current magnitude or reversal potential, nor the levels of NR1-NR2A-D subunit expression were altered in RVH rats. Conversely, we found a larger endogenous glutamate tone, resulting in the sustained activation of eNMDARs that tonically inhibited IA and contributed also to higher ongoing firing activity in RVH rats. The enhanced endogenous glutamate tone in RVH rats was not due to blunted glutamate transporter activity. Rather, a higher transporter activity was observed, which possibly acted as a compensatory mechanism in the face of the elevated endogenous tone. In summary, our studies indicate that an elevated endogenous glutamate tone results in an exacerbated activation of eNMDARs, which in turn contributes to diminished IA magnitude and increased firing activity of MNCs from hypertensive rats.

Extracellular potassium concentration defines neuronal bursting properties

Many neurons, or populations of neurons, in the brain are capable of producing rhythmic bursting activity. This ability is putatively responsible for rhythmogenic functions like breathing and locomotion. In vivo, rhythms are generated by synaptically interconnected neuronal networks, whereas rhythmic bursting behavior is often induced in vitro by elevating the extracellular potassium concentration (Kout ) [1]. It is known that increasing Kout raises the reversal potentials of potassium and leak currents [2]. However, the complete nature of how these effects underlie bursting activity has yet to be uncovered. A mathematical modeling study was performed to elucidate the interplay between these factors and their roles in a neuron's transition from quiescence to rhythmic bursting. A conductance-based model of a neuron from the pre-Botzinger Complex (pre-BotC) was used as a basis [3]. A potassium ion component was incorporated into the leak current, and model behaviors were investigated at varying concentrations of Kout , taking into account its effect on delayed rectifier potassium current responsible for after-spike hyperpolarization. The primary aim of this modeling study was to evaluate the contribution of extracellular potassium ions in the leak and delayed rectifier potassium current, and the subsequent effect of these altered currents on the bursting properties of neurons. Furthermore, the initial model was modified to replicate experimental results and test for conditions of low Kout as seen in vivo. The analysis of our model shows that: (i) in vitro bursting behavior with elevated Kout may occur due to attenuation of the delayed rectifier potassium current and (ii) no oscillations are generated at physiological levels of extracellular potassium. These results indicate that, according to the commonly-accepted models used in our study, neurons that naturally burst in in vitro preparations may not be able to burst in vivo under any circumstances. Accordingly, rhythmic activity in vivo should rely on other mechanisms. For example, Jasinski et al. [4] have shown that the recurrent synaptic excitation in combination with the sodium-potassium exchanger (pump) can result in the robust rhythmic network activity even with all intrinsic bursting mechanisms blocked.

Measurement of relative Ca²⁺ permeability during sustained activation of TRPV1 receptors.

Some cation permeable ligand-gated ion channels, including the capsaicin-sensitive TRPV1, have been reported to exhibit a time-dependent increase in permeability to large inorganic cations during sustained activation, a phenomenon termed "pore dilation." TRPV1 conducts substantial Ca(2+) entry, and it has been suggested that this channel undergoes a time-dependent change in Ca(2+) permeability relative to Na(+) (P Ca/P Na) that parallels pore dilation. However, our experiments employing whole cell patch clamp photometry and single channel recordings to directly measure relative Ca(2+) current in TRPV1 expressing HEK293 cells show that relative Ca(2+) influx remains constant for the duration of capsaicin-evoked channel activation. Further, we present evidence from patch clamp photometry experiments suggesting that sustained activation of Ca(2+) permeable ion channels in the voltage-clamp configuration leads to rapid saturation of the pipette Ca(2+) chelator, and that subsequent observed shifts in the current reversal potentials in the presence of extracellular Ca(2+) are likely due to intracellular accumulation of this ion and a movement of the Ca(2+) equilibrium potential (E Ca) towards zero. Finally, using an adapted reversal potential-based protocol in which cells are only exposed to Ca(2+) after sustained capsaicin exposure in the absence of added extracellular Ca(2+), we demonstrate that the calculated P Ca/P Na is unaffected by duration of TRPV1 activation. In conclusion, we find no evidence in support of a time-dependent change in P Ca/P Na for TRPV1. Our data further urges caution in estimating relative Ca(2+) permeability using reversal potentials, as there is a limited time window in which the cytosolic Ca(2+) chelator included in the patch pipette can prevent localised elevations in cytosolic free Ca(2+) and thus allow for an accurate estimate of this important channel permeability parameter.

What Currents Underlie Pulmonary Vein Automaticity?

What Currents Underlie Pulmonary Vein Automaticity?

Modification of glial response in hibernation: a patch-clamp study on glial cells acutely isolated from hibernating land snail.

Hibernation is a dormant state of some animal species that enables them to survive harsh environmental conditions during the winter seasons. In the hibernating state, preservation of neuronal rhythmic activity at a low level is necessary for maintenance of suspended forms of behavior. As glial cells support rhythmic activity of neurons, preservation of brain function in the hibernating state implies accompanying modification of glial activity. A supportive role of glia in regulating neuronal activity is reflected through the activity of inwardly rectifying K+ channels (Kir). Therefore, we examined electrophysiological response, particularly Kir current response, of glial cells in mixture with neurons acutely isolated from active and hibernating land snail Helix pomatia. Our data show that hibernated glia have significantly lower inward current density, specific membrane conductance, and conductance density compared with active glia. The observed reduction could be attributed to the Kir currents, since the Ba2+-sensitive Kir current density was significantly lower in hibernated glia. Accordingly, a significant positive shift of the current reversal potential indicated a more depolarized state of hibernated glia. Data obtained show that modification of glial current response could be regulated by serotonin (5-HT) through an increase of cGMP as a secondary messenger, since extracellular addition of 5-HT or intracellular administration of cGMP to active glia induced a significant reduction of inward current density and thus mimicked the reduced response of hibernated glia. Lower Kir current density of hibernated glia accompanied the lower electrical activity of hibernated neurons, as revealed by a decrease in neuronal fast inward Na+ current density. Our findings reveal that glial response is reduced in the hibernating state and suggest seasonal modulation of glial activity. Maintenance of low glial activity in hibernation could be important for preservation of brain rhythmic activity and survival of the animal.