Potassium Equilibrium Potential Research Articles

Single infrared light pulses induce excitatory and inhibitory neuromodulation.

The excitatory and inhibitory effects of single and brief infrared (IR) light pulses (2µm) with millisecond durations and various power levels are investigated with a custom-built fiber amplification system. Intracellular recordings from motor axons of the crayfish opener neuromuscular junction are performed ex vivo. Single IR light pulses induce a membrane depolarization during the light pulses, which is followed by a hyperpolarization that can last up to 100 ms. The depolarization amplitude is dependent on the optical pulse duration, total energy deposition and membrane potential, but is insensitive to tetrodotoxin. The hyperpolarization reverses its polarity near the potassium equilibrium potential and is barium-sensitive. The membrane depolarization activates an action potential (AP) when the axon is near firing threshold, while the hyperpolarization reversibly inhibits rhythmically firing APs. In summary, we demonstrate for the first time that single and brief IR light pulses can evoke initial depolarization followed by hyperpolarization on individual motor axons. The corresponding mechanisms and functional outcomes of the dual effects are investigated.

Modeling of sustained spontaneous network oscillations of a sexually dimorphic brainstem nucleus: the role of potassium equilibrium potential.

Intrinsic oscillators in the central nervous system play a preeminent role in the neural control of rhythmic behaviors, yet little is known about how the ionic milieu regulates their output patterns. A powerful system to address this question is the pacemaker nucleus of the weakly electric fish Apteronotus leptorhynchus. A neural network comprised of an average of 87 pacemaker cells and 20 relay cells produces tonic oscillations, with higher frequencies in males compared to females. Previous empirical studies have suggested that this sexual dimorphism develops and is maintained through modulation of buffering of extracellular K+ by a massive meshwork of astrocytes enveloping the pacemaker and relay cells. Here, we constructed a model of this neural network that can generate sustained spontaneous oscillations. Sensitivity analysis revealed the potassium equilibrium potential, EK (as a proxy of extracellular K+ concentration), and corresponding somatic channel conductances as critical determinants of oscillation frequency and amplitude. In models of both the pacemaker nucleus network and isolated pacemaker and relay cells, the frequency increased almost linearly with EK, whereas the amplitude decreased nonlinearly with increasing EK. Our simulations predict that this frequency increase is largely caused by a shift in the minimum K+ conductance over one oscillation period. This minimum is close to zero at more negative EK, converging to the corresponding maximum at less negative EK. This brings the resting membrane potential closer to the threshold potential at which voltage-gated Na+ channels become active, increasing the excitability, and thus the frequency, of pacemaker and relay cells.

Serotonin hyperpolarizes the dorsal raphe nucleus neurons of mice by activating G protein-coupled inward rectifier potassium channels.

Serotonin (5-HT) has an important role in the pathophysiology of the mood disorders like major depression and anxiety disorders in central nervous system. On the one hand, dorsal raphe nucleus (DRN) neurons send serotonergic projections to almost all brain regions. On the other hand, they affect themselves through 5-HT1A autoreceptors. Many electrophysiological studies have investigated the ionic mechanism of the 5-HTs effect on the DRN neurons of the rat. However, there is no study characterizing the current that mediates the 5-HTs effect on mouse DRN neurons. In the present electrophysiological study, the whole-cell patch-clamp technique was used in the neurons of the DRN from one-month-old Balb/c mice to investigate the effect of 5-HT on the DRN neurons of mice and its ionic mechanism of action. The application of 5-HT resulted in a 14.3 ± 3.1 mV hyperpolarization (n = 9, P < 0.01) of resting membrane potential and 25.7 ± 3.5 pA outward current (n = 7) in the DRN neurons. The reversal potential (E5-HT) of the current induced by 5-HT was close to the potassium equilibrium potential (EK). This current had an inward rectification feature and was blocked by quinine pretreatment (n = 5, P < 0.05). In conclusion, 5-HT inhibits the DRN neurons of mice by inducing a current that is carried by potassium ions through G-protein-coupled inward rectifier potassium channels.

Development of a sexual dimorphism in a central pattern generator driving a rhythmic behavior: The role of glia-mediated potassium buffering in the pacemaker nucleus of the weakly electric fish Apteronotus leptorhynchus.

Central pattern generators play a critical role in the neural control of rhythmic behaviors. One of their characteristic features is the ability to modulate the oscillatory output. An important yet little-studied type of modulation involves the generation of oscillations that are sexually dimorphic in frequency. In the weakly electric fish Apteronotus leptorhynchus, the pacemaker nucleus serves as a central pattern generator that drives the electric organ discharge of the fish in a one-to-one fashion. Males discharge at higher frequencies than females-a sexual dimorphism that develops under the influence of steroid hormones. The two principal neurons that constitute the oscillatory network of the pacemaker nucleus are the pacemaker and relay cells. Whereas the number and size of the pacemaker and relay cells are sexually monomorphic, pronounced sex-dependent differences exist in the morphology, and subcellular properties of astrocytes, which form a syncytium closely associated with these neurons. In females, compared to males, the astrocytic syncytium covers a larger area surrounding the pacemaker and relay cells and exhibits higher levels of expression of connexin-43 expression. The latter indicates a strong gap-junction coupling of the individual cells within the syncytium. It is hypothesized that these sex-specific differences result in an increased capacity for buffering of extracellular potassium ions, thereby lowering the potassium equilibrium potential, which, in turn, leads to a decrease in the oscillation frequency. This hypothesis has received strong support from simulations based on computational models of individual neurons and the whole neural network of the pacemaker nucleus.

Glia-mediated modulation of extracellular potassium concentration determines the sexually dimorphic output frequency of a model brainstem oscillator

Sexual dimorphism in behavior is widespread among animals, but the cellular mechanisms underlying neural control of this phenomenon are largely unknown. One behavior that has provided some important clues about how such sex differences might develop is the electric organ discharge of Apteronotus leptorhynchus. In this weakly electric fish, the mean discharge frequencies of males and females are 880 Hz and 740 Hz, respectively, with little overlap of the two frequency bands. The discharges are controlled, in a one-to-one fashion, by the neural oscillations of the pacemaker nucleus in the medulla oblongata. Experimental evidence has shown that the astrocytic syncytium associated with the neural network that generates these oscillations is significantly larger, and stronger coupled via gap junctions, in females than in males. In the present study, modeling of this network was performed to test the hypotheses that the sex-dependent differences in the structure and properties of the astrocytic syncytium mediate better buffering of extracellular potassium in females than in males, which in turn causes, via a lowering of the potassium equilibrium potential, a decrease in the oscillation frequency. Simulations of the neural activity of the pacemaker nucleus and its individual components demonstrated that under both spontaneous and induced conditions the oscillation frequency and the potassium equilibrium potential are strongly positively correlated. These simulations predict that sufficient separation of the electric organ discharge frequencies for establishment of the sexual dimorphism can be achieved by rather minor alterations in the concentration of the extracellular potassium concentration in the pacemaker nucleus.

Direct evidence of impaired neuronal Na/K-ATPase pump function in alternating hemiplegia of childhood

Mutations in ATP1A3 encoding the catalytic subunit of the Na/K-ATPase expressed in mammalian neurons cause alternating hemiplegia of childhood (AHC) as well as an expanding spectrum of other neurodevelopmental syndromes and neurological phenotypes. Most AHC cases are explained by de novo heterozygous ATP1A3 mutations, but the fundamental molecular and cellular consequences of these mutations in human neurons are not known. In this study, we investigated the electrophysiological properties of neurons generated from AHC patient-specific induced pluripotent stem cells (iPSCs) to ascertain functional disturbances underlying this neurological disease. Fibroblasts derived from two subjects with AHC, a male and a female, both heterozygous for the common ATP1A3 mutation G947R, were reprogrammed to iPSCs. Neuronal differentiation of iPSCs was initiated by neurogenin-2 (NGN2) induction followed by co-culture with mouse glial cells to promote maturation of cortical excitatory neurons. Whole-cell current clamp recording demonstrated that, compared with control iPSC-derived neurons, neurons differentiated from AHC iPSCs exhibited a significantly lower level of ouabain-sensitive outward current (‘pump current’). This finding correlated with significantly depolarized potassium equilibrium potential and depolarized resting membrane potential in AHC neurons compared with control neurons. In this cellular model, we also observed a lower evoked action potential firing frequency when neurons were held at their resting potential. However, evoked action potential firing frequencies were not different between AHC and control neurons when the membrane potential was clamped to −80 mV. Impaired neuronal excitability could be explained by lower voltage-gated sodium channel availability at the depolarized membrane potential observed in AHC neurons. Our findings provide direct evidence of impaired neuronal Na/K-ATPase ion transport activity in human AHC neurons and demonstrate the potential impact of this genetic defect on cellular excitability.

Postsynaptic GABA(B) Receptors Contribute to the Termination of Giant Depolarizing Potentials in CA3 Neonatal Rat Hippocampus.

During development, hippocampal CA3 network generates recurrent population bursts, so-called Giant Depolarizing Potentials (GDPs). GDPs are characterized by synchronous depolarization and firing of CA3 pyramidal cells followed by afterhyperpolarization (GDP-AHP). Here, we explored the properties of GDP-AHP in CA3 pyramidal cells using gramicidin perforated patch clamp recordings from neonatal rat hippocampal slices. We found that GDP-AHP occurs independently of whether CA3 pyramidal cells fire action potentials (APs) or remain silent during GDPs. However, the amplitude of GDP-AHP increased with the number of APs the cells fired during GDPs. The reversal potential of the GDP-AHP was close to the potassium equilibrium potential. During voltage-clamp recordings, current-voltage relationships of the postsynaptic currents activated during GDP-AHP were characterized by reversal near the potassium equilibrium potential and inward rectification, similar to the responses evoked by the GABA(B) receptor agonists. Finally, the GABA(B) receptor antagonist CGP55845 strongly reduced GDP-AHP and prolonged GDPs, eventually transforming them to the interictal and ictal-like discharges. Together, our findings suggest that the GDP-AHP involves two mechanisms: (i) postsynaptic GABA(B) receptor activated potassium currents, which are activated independently on whether the cell fires or not during GDPs; and (ii) activity-dependent, likely calcium activated potassium currents, whose contribution to the GDP-AHP is dependent on the amount of firing during GDPs. We propose that these two complementary inhibitory postsynaptic mechanisms cooperate in the termination of GDP.

Three types of ion channels in the cell membrane of mouse fibroblasts.

Patch clamp recordings carried out in the inside-out configuration revealed activity of three kinds of channels: nonselective cation channels, small-conductance K(+) channels, and large-conductance anion channels. The nonselective cation channels did not distinguish between Na(+) and K(+). The unitary conductance of these channels reached 28 pS in a symmetrical concentration of 200 mM NaCl. A lower value of this parameter was recorded for the small-conductance K(+) channels and in a 50-fold gradient of K(+) (200 mM/4 mM) it reached 8 pS. The high selectivity of these channels to potassium was confirmed by the reversal potential (-97 mV), whose value was close to the equilibrium potential for potassium (-100 mV). One of the features of the largeconductance anion channels was high conductance amounting to 493 pS in a symmetrical concentration of 200 mM NaCl. The channels exhibited three subconductance levels. Moreover, an increase in the open probability of the channels at voltages close to zero was observed. The anion selectivity of the channels was low, because the channels were permeable to both Cl(-) and gluconate - a large anion. Research on the calcium dependence revealed that internal calcium activates nonselective cation channels and small-conductance K(+) channels, but not largeconductance anion channels.

Effects of Propranolol and Verapamil on Changes in TQ and ST Segment Potentials During Graded Coronary Flow Reduction in a Porcine Myocardial Ischemia Model.

Acute myocardial ischemia causes TQ depression and ST elevation. However, the effects of cardioprotective drugs such as β-blockers and Ca++-antagonists on the extent of TQ depression, ST elevation, and myocardial ischemic injury are not fully understood.We created a carotid-coronary shunt in 30 pigs, and extracellular K+ ([K+]e), TQ, and ST segments were recorded simultaneously with K+-selective plunge electrodes placed in the left anterior descending artery (LAD) distribution during graded LAD flow reduction before and after administration of propranolol or verapamil. Unipolar DC-coupled electrograms were recorded from the reference pole of the K+-selective plunge electrodes. The microvolt readings from the K+-selective electrodes were converted to [K+]e and then to the changes in potassium equilibrium potential (ΔEK). The shunted LAD flow was reduced in a stepwise fashion at 5-minute intervals.segment depression at the similar ΔEK was not affected by propranolol or verapamil. However, ST segment elevation was reduced by propranolol but exacerbated by verapamil at the similar ΔEK.TQ-ST changes recorded by AC coupled ECG are not a reliable index of ischemia and therefore cannot be used to evaluate the effects of drugs that might affect the electrophysiologic properties of ischemic myocardium.

μ opioid receptor activation hyperpolarizes respiratory-controlling Kölliker-Fuse neurons and suppresses post-inspiratory drive.

In addition to reductions in respiratory rate, opioids also cause aspiration and difficulty swallowing, indicating impairment of the upper airways. The Kölliker-Fuse (KF) maintains upper airway patency and a normal respiratory pattern. In this study, activation of μ opioid receptors in the KF reduced respiratory frequency and tidal volume in anaesthetized rats. Nerve recordings in an in situ preparation showed that activation of μ opioid receptors in the KF eliminated the post-inspiration phase of the respiratory cycle. In brain slices, μ opioid agonists hyperpolarized a distinct population (61%) of KF neurons by activation of an inwardly rectifying potassium conductance. These results suggest that KF neurons that are hyperpolarized by opioids could contribute to opioid-induced respiratory disturbances, particularly the impairment of upper airways. Opioid-induced respiratory effects include aspiration and difficulty swallowing, suggesting impairment of the upper airways. The pontine Kölliker-Fuse nucleus (KF) controls upper airway patency and regulates respiration, in particular the inspiratory/expiratory phase transition. Given the importance of the KF in coordinating respiratory pattern, the mechanisms of μ opioid receptor activation in this nucleus were investigated at the systems and cellular level. In anaesthetized, vagi-intact rats, injection of opioid agonists DAMGO or [Met(5) ]enkephalin (ME) into the KF reduced respiratory frequency and amplitude. The μ opioid agonist DAMGO applied directly into the KF of the in situ arterially perfused working heart-brainstem preparation of rat resulted in robust apneusis (lengthened low amplitude inspiration due to loss of post-inspiratory drive) that was rapidly reversed by the opioid antagonist naloxone. In brain slice preparations, activation of μ opioid receptors on KF neurons hyperpolarized a distinct population (61%) of neurons. As expected, the opioid-induced hyperpolarization reduced the excitability of the neuron in response to either current injection or local application of glutamate. In voltage-clamp recordings the outward current produced by the opioid agonist ME was concentration dependent, reversed at the potassium equilibrium potential and was blocked by BaCl2 , characteristics of a G protein-coupled inwardly rectifying potassium (GIRK) conductance. The clinically used drug morphine produced an outward current in KF neurons with similar potency to morphine-mediated currents in locus coeruleus brain slice preparations. Thus, the population of KF neurons that are hyperpolarized by μ opioid agonists are likely mediators of the opioid-induced loss of post-inspiration and induction of apneusis.

Mu opioid receptors hyperpolarize respiratory‐controlling Kölliker‐Fuse neurons (712.1)

Respiratory depression is the primary cause of death from opioid overdose, yet little is known about the cellular mechanisms of opioids on respiratory‐controlling neurons. The pontine Kölliker‐Fuse (KF) regulates respiration, especially the inspiratory/expiratory phase transition. In the in situ arterially perfused working heart‐brainstem preparation of rat, injection of the mu opioid agonist DAMGO into the KF caused robust apneusis that was similar to complete silencing of the KF. The apneusis was rapidly reversed by perfusion of the opioid antagonist naloxone. In whole‐cell recordings from KF neurons contained in rat brain slices, activation of mu opioid receptors hyperpolarized a majority (75%) of KF neurons. The hyperpolarizing current produced by [Met5]enkephalin (ME) was concentration‐dependent, reversed at the potassium equilibrium potential and was blocked by BaCl2, characteristics of a GIRK conductance. Unexpectedly, the partial agonist morphine produced the same amplitude current as full agonists DAMGO or ME, indicating a large receptor reserve. The presence of many spare receptors could reduce the degree of desensitization induced by prolonged application of ME. Indeed, only minimal acute desensitization of the ME‐mediated GIRK current was observed. This lack of desensitization correlates with the lack of tolerance to the respiratory depressant effects of opioids.Grant Funding Source: Supported by DA08163 (JTW) and DA33036 (ESL)

A selective 5-HT1areceptor agonist improves respiration in a mouse model of Rett syndrome

Rett syndrome is a neurological disorder caused by loss of function mutations in the gene that encodes the DNA binding protein methyl-CpG-binding protein 2 (Mecp2). A prominent feature of the syndrome is disturbances in respiration characterized by frequent apnea and an irregular interbreath cycle. 8-Hydroxy-2-dipropylaminotetralin has been shown to positively modulate these disturbances (Abdala AP, Dutschmann M, Bissonnette JM, Paton JF, Proc Natl Acad Sci U S A 107: 18208-18213, 2010), but the mode of action is not understood. Here we show that the selective 5-HT1a biased agonist 3-chloro-4-fluorophenyl-(4-fluoro-4-{[(5-methylpyrimidin-2-ylmethyl)-amino]-methyl}-piperidin-1-yl)-methanone (F15599) decreases apnea and corrects irregularity in both heterozygous Mecp2-deficient female and in Mecp2 null male mice. In whole cell voltage-clamp recordings from dorsal raphe neurons, F15599 potently induced an outward current, which was blocked by barium, reversed at the potassium equilibrium potential, and was antagonized by the 5-HT1a antagonist WAY100135. This is consistent with somatodendritic 5-HT1a receptor-mediated activation of G protein-coupled inwardly rectifying potassium channels (GIRK). In contrast, F15599 did not activate 5-HT1b/d receptors that mediate inhibition of glutamate release from terminals in the nucleus accumbens by a presynaptic mechanism. Thus F15599 activated somatodendritic 5-HT1a autoreceptors, but not axonal 5-HT1b/d receptors. In unanesthetized Mecp2-deficient heterozygous female mice, F15599 reduced apnea in a dose-dependent manner with maximal effect of 74.5 ± 6.9% at 0.1 mg/kg and improved breath irrregularity. Similarly, in Mecp2 null male mice, apnea was reduced by 62 ± 6.6% at 0.25 mg/kg, and breathing became regular. The results indicate respiration is improved with a 5-HT1a agonist that activates GIRK channels without affecting neurotransmitter release.

Teaching and learning the Hodgkin-Huxley model based on software developed in NEURON's programming language hoc.

BackgroundWe present a software tool called SENB, which allows the geometric and biophysical neuronal properties in a simple computational model of a Hodgkin-Huxley (HH) axon to be changed. The aim of this work is to develop a didactic and easy-to-use computational tool in the NEURON simulation environment, which allows graphical visualization of both the passive and active conduction parameters and the geometric characteristics of a cylindrical axon with HH properties.ResultsThe SENB software offers several advantages for teaching and learning electrophysiology. First, SENB offers ease and flexibility in determining the number of stimuli. Second, SENB allows immediate and simultaneous visualization, in the same window and time frame, of the evolution of the electrophysiological variables. Third, SENB calculates parameters such as time and space constants, stimuli frequency, cellular area and volume, sodium and potassium equilibrium potentials, and propagation velocity of the action potentials. Furthermore, it allows the user to see all this information immediately in the main window. Finally, with just one click SENB can save an image of the main window as evidence.ConclusionsThe SENB software is didactic and versatile, and can be used to improve and facilitate the teaching and learning of the underlying mechanisms in the electrical activity of an axon using the biophysical properties of the squid giant axon.

Effects of Stresscopin on Rat Hypothalamic Paraventricular Nucleus Neurons In Vitro

The effects of stresscopin (SCP) on rat paraventricular nucleus (PVN) neurons were examined using whole-cell patch-clamp recordings and single-cell reverse-transcription multiplex polymerase chain reaction (SC-RT-mPCR) techniques. Under current-clamp conditions, bath application of SCP (100 nM) induced inhibition in 35.2% (37/105) of putative magnocellular neurons and 24.7% (20/81) of putative parvocellular neurons, and excitation in 5.7% (6/105) of putative magnocellular neurons and 18.5% (15/81) of putative parvocellular neurons. SCP-induced inhibition persisted in the presence of a mixture of TTX, a voltage-gated Na+ channel blocker, CNQX, an AMPA/kainate receptor antagonist and bicuculline, a GABAA receptor antagonist, whereas SCP-induced excitation of PVN neurons was reversed by the mixture. The SCP-induced inhibition of PVN neurons was abolished by bath application of antisauvagine-30, a selective CRF receptor 2 (CRF-R2) antagonist. Under voltage-clamp conditions, SCP evoked outward currents at the holding potential (−60 mV), which reversed near the potassium equilibrium potential. The SCP-evoked membrane currents were completely blocked by bath application of tertiapin-Q, a selective blocker of G protein-activated inwardly rectifying potassium (GIRK) channels. SC-RT-mPCR analysis indicated that all the SCP-sensitive PVN neurons (57 SCP-inhibited neurons, 21 SCP-excited neurons) expressed CRF-R1 and CRF-R2 mRNAs. Among SCP-hyperpolarized PVN neurons, oxytocin (OT) mRNA was detected in 91.8% of putative magnocellular neurons and 45.0% of putative parvocellular neurons. OT mRNA was also detected in 26.6% of SCP-depolarized parvocellular neurons, but not in SCP-depolarized magnocellular neurons. These results indicate that SCP inhibits a subpopulation of PVN neurons, especially OTergic magnocellular neurons, by enhancing the activity of GIRK channels via CRF-R2.

Modulation of action potential and calcium signaling by levetiracetam in rat sensory neurons

Levetiracetam (LEV), a new anticonvulsant agent primarily used to treat epilepsy, has been used in pain treatment but the cellular mechanism of this action remains unclear. This study aimed to investigate effects of LEV on the excitability and membrane depolarization-induced calcium signaling in isolated rat sensory neurons using the whole-cell patch clamp and fura 2–based ratiometric Ca2+-imaging techniques. Dorsal root ganglia (DRG) were excised from neonatal rats, and cultured following enzymatic and mechanical dissociation. Under current clamp conditions, acute application of LEV (30 µM, 100 µM and 300 µM) significantly increased input resistance and caused the membrane to hyperpolarize from resting membrane potential in a dose-dependent manner. Reversal potentials of action potential (AP) after hyperpolarising amplitudes were shifted to more negative, toward to potassium equilibrium potentials, after application of LEV. It also caused a decrease in number of APs in neurons fired multiple APs in response to prolonged depolarization. Fura-2 fluorescence Ca2+ imaging protocols revealed that HiK+ (30 mM)-induced intracellular free Ca2+ ([Ca2+]i) was inhibited to 97.8 ± 4.6% (n = 17), 92.6 ± 4.8% (n = 17, p < 0.01) and 89.1 ± 5.1% (n = 18, p < 0.01) after application of 30 µM, 100 µM and 300 µM LEV (respectively), without any significant effect on basal levels of [Ca2+]i. This is the first evidence for the effect of LEV on the excitability of rat sensory neurons through an effect which might involve activation of potassium channels and inhibition of entry of Ca2+, providing new insights for cellular mechanism(s) of LEV in pain treatment modalities.

GIRK channel-mediated inhibition of melanin-concentrating hormone neurons by nociceptin/orphanin FQ

Targeting the melanin-concentrating hormone (MCH) system has been suggested as a potential treatment for obesity, anxiety disorders, as well as addiction. Despite the therapeutic potential of MCH agonists and antagonists, the endogenous factors regulating MCH activity, in particular those implicated in anxiety and reward, are ill-defined. The present study investigated the cellular effects of nociceptin/orphanin FQ (N/OFQ), an endogenous opioid with anxiolytic and antireward properties, on MCH neurons. We found that N/OFQ induced a concentration-dependent reversible outward current in MCH neurons (EC(50) = 50.7 nM), an effect that was blocked by the competitive antagonist of the nociceptin opioid peptide (NOP) receptor UFP-101. N/OFQ-induced outward currents persisted in TTX, reversed near the potassium equilibrium potential, and displayed inward rectification, suggesting direct postsynaptic potassium channel activation. Tertiapin-Q completely abolished the N/OFQ effect, whereas glibenclamide did not, implicating protein G-dependent inwardly rectifying potassium (GIRK) and not ATP-sensitive potassium (K(ATP)) channels as the effector ion channel. The N/OFQ-induced outward current desensitized during repeated applications and occluded the inhibitory effect of dynorphin, suggesting that dynorphin and N/OFQ activate the same pathway. N/OFQ also reversibly inhibited voltage-gated calcium currents in MCH neurons. In conclusion, our study indicates N/OFQ as a robust endogenous regulator of MCH neurons, which may play a role in anxiety and drug addiction.

Two pore domain potassium channels in cerebral ischemia: a focus on K2P9.1 (TASK3, KCNK9)

BackgroundRecently, members of the two-pore domain potassium channel family (K2P channels) could be shown to be involved in mechanisms contributing to neuronal damage after cerebral ischemia. K2P3.1-/- animals showed larger infarct volumes and a worse functional outcome following experimentally induced ischemic stroke. Here, we question the role of the closely related K2P channel K2P9.1.MethodsWe combine electrophysiological recordings in brain-slice preparations of wildtype and K2P9.1-/- mice with an in vivo model of cerebral ischemia (transient middle cerebral artery occlusion (tMCAO)) to depict a functional impact of K2P9.1 in stroke formation.ResultsPatch-clamp recordings reveal that currents mediated through K2P9.1 can be obtained in slice preparations of the dorsal lateral geniculate nucleus (dLGN) as a model of central nervous relay neurons. Current characteristics are indicative of K2P9.1 as they display an increase upon removal of extracellular divalent cations, an outward rectification and a reversal potential close to the potassium equilibrium potential. Lowering extracellular pH values from 7.35 to 6.0 showed comparable current reductions in neurons from wildtype and K2P9.1-/- mice (68.31 ± 9.80% and 69.92 ± 11.65%, respectively). These results could be translated in an in vivo model of cerebral ischemia where infarct volumes and functional outcomes showed a none significant tendency towards smaller infarct volumes in K2P9.1-/- animals compared to wildtype mice 24 hours after 60 min of tMCAO induction (60.50 ± 17.31 mm3 and 47.10 ± 19.26 mm3, respectively).ConclusionsTogether with findings from earlier studies on K2P2.1-/- and K2P3.1-/- mice, the results of the present study on K2P9.1-/- mice indicate a differential contribution of K2P channel subtypes to the diverse and complex in vivo effects in rodent models of cerebral ischemia.

Barbiturates Require the N Terminus and First Transmembrane Domain of the δ Subunit for Enhancement of α1β3δ GABAA Receptor Currents

GABA(A) receptors are composed predominantly of alphabetagamma receptors, which mediate primarily synaptic inhibition, and alphabetadelta receptors, which mediate primarily extrasynaptic inhibition. At saturating GABA concentrations, the barbiturate pentobarbital substantially increased the amplitude and desensitization of the alpha1beta3delta receptor but not the alpha1beta3gamma2L receptor currents. To explore the structural domains of the delta subunit that are involved in pentobarbital potentiation and increased desensitization of alpha1beta3delta currents, chimeric cDNAs were constructed by progressive replacement of gamma2L subunit sequence with a delta subunit sequence or a delta subunit sequence with a gamma2L subunit sequence, and HEK293T cells were co-transfected with alpha1 and beta3 subunits or alpha1 and beta3 subunits and a gamma2L, delta, or chimeric subunit. Currents evoked by a saturating concentration of GABA or by co-application of GABA and pentobarbital were recorded using the patch clamp technique. By comparing the extent of enhancement and changes in kinetic properties produced by pentobarbital among chimeric and wild type receptors, we concluded that although potentiation of alpha1beta3delta currents by pentobarbital required the delta subunit sequence from the N terminus to proline 241 in the first transmembrane domain (M1), increasing desensitization of alpha1beta3delta currents required a delta subunit sequence from the N terminus to isoleucine 235 in M1. These findings suggest that the delta subunit N terminus and N-terminal portion of the M1 domain are, at least in part, involved in transduction of the allosteric effect of pentobarbital to enhance alpha1beta3delta currents and that this effect involves a distinct but overlapping structural domain from that involved in altering desensitization.

Activation of α1 and α2 noradrenergic receptors exert opposing effects on excitability of main olfactory bulb granule cells

The mammalian main olfactory bulb (MOB) receives a dense noradrenergic innervation from the pontine nucleus locus coeruleus that is important for neonatal odor preference learning and odor processing in mature animals. Modulation of GABAergic granule cells (GCs) is thought to play a key role in the net functional impact of norepinephrine (NE) release in the MOB, yet there are few direct studies of the influence of NE on these cells. In the present study we investigated noradrenergic modulation of GC excitability using electrophysiological approaches in rat MOB slices. A moderate concentration of NE (10 μM) and the α1 receptor agonist phenylephrine (10 μM) depolarized and increased spontaneous or current injection-evoked spiking in GCs. By contrast, low NE concentrations (0.1–1.0 μM) or the α2 receptor agonist clonidine (Clon, 10 μM) hyperpolarized and decreased the discharge of GCs. The effects of NE (10 μM) were blocked by antagonism of α1 and α2 receptors. Inhibitory effects of low NE concentrations were blocked or converted to excitatory responses by α2 receptor blockade, whereas excitatory effects of the moderate NE concentration were converted to inhibitory responses after α1 receptor blockade. NE (10 μM) and phenylephrine elicited inward currents that reversed near the potassium equilibrium potential. The effects of NE and phenylephrine were associated with increased membrane input resistance. Clonidine elicited an outward current associated with decreased membrane input resistance that reversed near the potassium equilibrium potential. These results indicate that α1 and α2 receptor activation exert opposing effects on GC excitability. Low concentrations of NE acting via α2 receptors suppress GC excitability, while higher concentrations of NE acting at α1 receptors increase GC excitability. These findings are consistent with recent findings that α1 and α2 receptor activation increase and decrease, respectively, GABAergic inhibition of mitral cells. The differential affinities of α1 and α2 noradrenergic receptor subtypes may allow for differential modulation of GABA release and olfactory processing as a function of the level of NE release, which in turn, is regulated by behavioral state.

Characterization of a stretch‐activated potassium channel in chondrocytes

Chondrocytes possess the capacity to transduce load-induced mechanical stimuli into electrochemical signals. The aim of this study was to functionally characterize an ion channel activated in response to membrane stretch in isolated primary equine chondrocytes. We used patch-clamp electrophysiology to functionally characterize this channel and immunohistochemistry to examine its distribution in articular cartilage. In cell-attached patch experiments, the application of negative pressures to the patch pipette (in the range of 20–200 mmHg) activated ion channel currents in six of seven patches. The mean activated current was 45.9 ± 1.1 pA (n = 4) at a membrane potential of 33 mV (cell surface area approximately 240 µm2). The mean slope conductance of the principal single channels resolved within the total stretch-activated current was 118 ± 19 pS (n = 6), and reversed near the theoretical potassium equilibrium potential, EK+, suggesting it was a high-conductance potassium channel. Activation of these high-conductance potassium channels was inhibited by extracellular TEA (Kd approx. 900 µM) and iberiotoxin (Kd approx. 40 nM). This suggests that the current was largely carried by BK-like potassium (MaxiK) channels. To further characterize these BK-like channels, we used inside-out patches of chondrocyte membrane: we found these channels to be activated by elevation in bath calcium concentration. Immunohistochemical staining of equine cartilage samples with polyclonal antibodies to the α1- and β1-subunits of the BK channel revealed positive immunoreactivity for both subunits in superficial zone chondrocytes. These experiments support the hypothesis that functional BK channels are present in chondrocytes and may be involved in mechanotransduction and chemotransduction.