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Journal of Physiology
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Activity and distribution of intracellular carbonic anhydrase II and their effects on the transport activity of anion exchanger AE1/SLC4A1
We have investigated the previously published ‘metabolon hypothesis' postulating that a close association of the anion exchanger 1 (AE1) and cytosolic carbonic anhydrase II (CAII) exists that greatly increases the transport activity of AE1. We study whether there is a physical association of and direct functional interaction between CAII and AE1 in the native human red cell and in tsA201 cells coexpressing heterologous fluorescent fusion proteins CAII-CyPet and YPet-AE1. In these doubly transfected tsA201 cells, YPet-AE1 is clearly associated with the cell membrane, whereas CAII-CyPet is homogeneously distributed throughout the cell in a cytoplasmic pattern. Förster resonance energy transfer measurements fail to detect close proximity of YPet-AE1 and CAII-CyPet. The absence of an association of AE1 and CAII is supported by immunoprecipitation experiments using Flag-antibody against Flag-tagged AE1 expressed in tsA201 cells, which does not co-precipitate native CAII but co-precipitates coexpressed ankyrin. Both the CAII and the AE1 fusion proteins are fully functional in tsA201 cells as judged by CA activity and by cellular HCO3– permeability ( ) sensitive to inhibition by 4,4-Diisothiocyano-2,2-stilbenedisulfonic acid. Expression of the non-catalytic CAII mutant V143Y leads to a drastic reduction of endogenous CAII and to a corresponding reduction of total intracellular CA activity. Overexpression of an N-terminally truncated CAII lacking the proposed site of interaction with the C-terminal cytoplasmic tail of AE1 substantially increases intracellular CA activity, as does overexpression of wild-type CAII. These variously co-transfected tsA201 cells exhibit a positive correlation between cellular and intracellular CA activity. The relationship reflects that expected from changes in cytoplasmic CA activity improving substrate supply to or removal from AE1, without requirement for a CAII–AE1 metabolon involving physical interaction. A functional contribution of the hypothesized CAII–AE1 metabolon to erythroid AE1-mediated HCO3– transport was further tested in normal red cells and red cells from CAII-deficient patients that retain substantial CA activity associated with the erythroid CAI protein lacking the proposed AE1-binding sequence. Erythroid was indistinguishable in these two cell types, providing no support for the proposed functional importance of the physical interaction of CAII and AE1. A theoretical model predicts that homogeneous cytoplasmic distribution of CAII is more favourable for cellular transport of HCO3– and CO2 than is association of CAII with the cytoplasmic surface of the plasma membrane. This is due to the fact that the relatively slow intracellular transport of H+ makes it most efficient to place the CA in the vicinity of the haemoglobin molecules, which are homogeneously distributed over the cytoplasm.
Effects of optional structural elements, including two alternative amino termini and a new splicing cassette IV, on the function of the sodium-bicarbonate cotransporter NBCn1 (SLC4A7)
The SLC4A7 gene encodes the electroneutral sodium/HCO3 cotransporter NBCn1, which plays important physiological and pathophysiological roles in many cell types. Previous work identified six NBCn1 variants differing in the sequence of the extreme N terminus – MEAD in rat only, MERF in human only – as well as in the optional inclusion of cassettes I, II, and III. Earlier work also left open the question of whether optional structural elements (OSEs) affect surface abundance or intrinsic (per-molecule) transport activity. Here, we demonstrate for the first time that SLC4A7 from one species can express both MEAD- and MERF-NBCn1. We also identify a novel cassette IV of 20 aa, and extend by 10 the number of full-length NBCn1 variants. The alternative N termini and four cassettes could theoretically produce 32 major variants. Moreover, we identify a group of cDNAs predicted to encode just the cytosolic N-terminal domain (Nt) of NBCn1. A combination of electrophysiology and biotinylation shows that the OSEs can affect surface abundance and intrinsic HCO3– transport activity of NBCn1, as expressed in Xenopus oocytes. Specifically, MEAD tends to increase whereas novel cassette IV reduces surface abundance. Cassettes II, III and novel cassette IV all appear to increase the intrinsic activity of NBCn1.
Phosphatidylethanolamine binding protein 1 in vacular endothelial cell autophagy and atherosclerosis
We previously found that phosphatidylcholine-specific phospholipase C (PC-PLC) was a key inducing element of atherosclerosis, and might negatively regulate human umbilical vein endothelial cell (HUVEC) autophagy. To further investigate the mechanism of PC-PLC action, we initially identified phosphatidylethanolamine binding protein 1 (PEBP1) as a binding partner of PC-PLC by using mass spectrometry (MS, MALDI-TOF/TOF). We found that PEBP1 positively regulated PC-PLC activity in HUVECs, and inhibition of PC-PLC by its inhibitor D609 suppressed PEBP1 expression dramatically. Moreover, both PC-PLC and PEBP1 negatively regulated HUVEC autophagy independently of mammalian target of rapamycin (mTOR). Furthermore, the PEBP1 level was elevated during the development of atherosclerosis, while D609 significantly decreased the upregulated PEBP1 level in apoE–/– mice.
We test a novel, stochastic model of osmotic water transport in aquaporins. A solute molecule present at the pore mouth can either be reflected or permeate the pore. We assume that only reflected solute molecules induce osmotic transport of water through the pore, while permeating solute molecules give rise to no water transport. Accordingly, the rate of water transport is proportional to the reflection coefficient , while the solute permeability, PS, is proportional to 1 – . The model was tested in aquaporins heterologously expressed in Xenopus oocytes. A variety of aquaporin channel sizes and geometries were obtained with the two aquaporins AQP1 and AQP9 and mutant versions of these. Osmotic water transport was generated by adding 20 mm of a range of different-sized osmolytes to the outer solution. The osmotic water permeability and the reflection coefficient were measured optically at high resolution and compared to the solute permeability obtained from short-term uptake of radio-labelled solute under isotonic conditions. For each type of aquaporin there was a linear relationship between solute permeability and reflection coefficient, in accordance with the model. We found no evidence for coupling between water and solute fluxes in the pore. In confirmation of molecular dynamic simulations, we conclude that the magnitude of the osmotic water permeability and the reflection coefficient are determined by processes at the arginine selectivity filter located at the outward-facing end of the pore.
Two-photon calcium imaging in awake, head-fixed animals enables the measurement of neuronal activity during behaviour. Often, licking for the retrieval of water reward is used as a measurable report of the animal's decision during reward-driven behaviour. However, licking behaviour can induce severe motion artifacts that interfere with two-photon imaging of cellular activity. Here, we describe a simple method for the online correction of licking-induced focus shifts for two-photon calcium imaging of neocortical neurons in the head-fixed mouse. We found that licking causes a stereotyped drop of neocortical tissue, shifting neurons up to 20 m out of focus. Based on the measurement of licking with a piezo film sensor, we developed a feedback model, which provides a corrective signal for fast optical focus adjustments with an electrically tunable lens. Using online correction with this feedback model, we demonstrate a reduction of licking-related focus changes below 3 m, minimizing motion artifact contamination of cellular calcium signals. Focus correction with a tunable lens is a simple and effective method to improve the ability to monitor neuronal activity during reward-based behaviour.
Persistent changes in excitatory and inhibitory synaptic strengths to the ventral tegmental area (VTA) dopamine (DA) neurons in response to addictive drugs may underlie the transition from casual to compulsive drug use. While an enormous amount of work has been done in the area of glutamatergic plasticity of the VTA, little is known regarding the learning rules governing GABAergic plasticity in the VTA. Spike timing-dependent plasticity, STDP, has attracted considerable attention primarily due to its potential roles in processing and storage of information in the brain and there is emerging evidence for the existence of STDP at inhibitory synapses. We therefore used whole-cell recordings in rat midbrain slices to investigate whether near-coincident pre- and postsynaptic firing induces a lasting change in synaptic efficacy of VTA GABAergic synapses. We found that a Hebbian form of STDP including long-term potentiation (LTP) and long-term depression (LTD) can be induced at GABAergic synapses onto VTA DA neurons and relies on the precise temporal order of pre- and postsynaptic spiking. Importantly, GABAergic STDP is heterosynaptic (NMDA receptor dependent): triggered by correlated activities of the presynaptic glutamatergic input and postsynaptic DA cells. GABAergic STDP is postsynaptic and has an associative component since pre- or postsynaptic spiking per se did not induce STDP. STDP of GABAergic synapses in the VTA provides physiologically relevant forms of inhibitory plasticity that may underlie natural reinforcement of reward-related behaviours. Moreover, this form of inhibitory plasticity may mediate some of the reinforcing, aversive and addictive properties of drugs of abuse.
The ionotropic glutamate receptors are primary mediators of fast excitatory neurotransmission, and their properties are determined both by their subunit composition and their association with auxiliary subunits. The neuropilin and tolloid-like 1 and 2 proteins (Neto1 and Neto2) have been recently identified as auxiliary subunits for kainate-type glutamate receptors. Heteromeric kainate receptors (KARs) can be assembled from varying combinations of low-affinity (GluK1–GluK3) and high-affinity (GluK4–GluK5) subunits. To better understand the functional impact of auxiliary subunits on KARs, we examined the effect of Neto1 on the responses of recombinant homomeric and heteromeric KARs to varying concentrations of glutamate. We found that co-expression of Neto1 with homomeric GluK2 receptors had a small effect on sensitivity of the receptors to glutamate, but decreased the onset of desensitization while speeding recovery from desensitization. In the absence of Neto1, addition of GluK5 subunits to form GluK2/GluK5 heteromeric receptors slowed the onset of desensitization at low glutamate concentrations, compared with GluK2 homomers. Co-expression of Neto1 with GluK2/GluK5 receptors further enhanced these effects, essentially eliminating desensitization at m glutamate concentrations without altering the EC50 for activation by glutamate. In addition, a prominent rebound current was observed upon removal of the agonist. The rate of recovery from desensitization was increased to the same degree by Neto1 for both homomeric GluK2 and heteromeric GluK2/GluK5 receptors. Expression of Neto1 with GluK1/GluK5, GluK3/GluK5 or GluK2/GluK4 receptors produced qualitatively similar effects on whole-cell currents, suggesting that the impact of Neto1 on the desensitization properties of heteromeric receptors was not subunit dependent. These results provide greater insight into the functional effects of the auxiliary subunit Neto1 on both homomeric and heteromeric KARs. Alteration of the characteristics of desensitization at both sub-maximal and saturating glutamate concentrations could influence the responsiveness of these receptors to repeated stimuli. As a result, assembly of KARs with the Neto auxiliary subunits could change the kinetic properties of the neuronal response to glutamatergic input.
Cholinergic modulation of neuronal excitability and recurrent excitation-inhibition in prefrontal cortex circuits: implications for gamma oscillations
Cholinergic neuromodulation in neocortical networks is required for gamma oscillatory activity associated with working memory and other cognitive processes. Importantly, the cholinergic agonist carbachol (CCh) induces gamma oscillations in vitro, via mechanisms that may be shared with in vivo gamma oscillations and that are consistent with the pyramidal interneuron network gamma (PING) model. In PING oscillations, pyramidal cells (PCs), driven by asynchronous excitatory input, recruit parvalbumin-positive fast-spiking interneurons (FSNs), which then synchronize the PCs via feedback inhibition. Whereas the PING model is favoured by current data, how cholinergic neuromodulation contributes to gamma oscillation production is poorly understood. We thus studied the effects of cholinergic modulation on circuit components of the PING model in mouse medial prefrontal cortex (mPFC) brain slices. CCh depolarized and evoked action potential firing in a fraction of PCs and increased excitatory synaptic input onto FSNs. In synaptically connected pairs, CCh reduced the short-term depression at FSN–PC and PC–FSN synapses, equalizing synaptic strength during repetitive presynaptic firing while simultaneously increasing the failure probability. Interestingly, when PCs or FSNs fired in response to gamma frequency oscillatory inputs, CCh increased the firing probability per cycle. Combined with the equalization of synaptic strength, an increase by CCh in the fraction of neurons recruited per oscillation cycle may support oscillatory synchrony of similar strength during relatively long oscillation episodes such as those observed during working memory tasks, suggesting a significant functional impact of cholinergic modulation of mPFC circuit components crucial for the PING model.
Cyclooxygenase-2, prostaglandin E2 glycerol ester and nitric oxide are involved in muscarine-induced presynaptic enhancement at the vertebrate neuromuscular junction
Previous work has demonstrated that activation of muscarinic acetylcholine receptors at the lizard neuromuscular junction (NMJ) induces a biphasic modulation of evoked neurotransmitter release: an initial depression followed by a delayed enhancement. The depression is mediated by the release of the endocannabinoid 2-arachidonylglycerol (2-AG) from the muscle and its binding to cannabinoid type 1 receptors on the motor nerve terminal. The work presented here suggests that the delayed enhancement of neurotransmitter release is mediated by cyclooxygenase-2 (COX-2) as it converts 2-AG to the glycerol ester of prostaglandin E2 (PGE2-G). Using immunofluorescence, COX-2 was detected in the perisynaptic Schwann cells (PSCs) surrounding the NMJ. Pretreatment with either of the selective COX-2 inhibitors, nimesulide or DuP 697, prevents the delayed increase in endplate potential (EPP) amplitude normally produced by muscarine. In keeping with its putative role as a mediator of the delayed muscarinic effect, PGE2-G enhances evoked neurotransmitter release. Specifically, PGE2-G increases the amplitude of EPPs without altering that of spontaneous miniature EPPs. As shown previously for the muscarinic effect, the enhancement of evoked neurotransmitter release by PGE2-G depends on nitric oxide (NO) as the response is abolished by application of either NG-nitro-l-arginine methyl ester (l-NAME), an inhibitor of NO synthesis, or carboxy-PTIO, a chelator of NO. Intriguingly, the enhancement is not prevented by AH6809, a prostaglandin receptor antagonist, but is blocked by capsazepine, a TRPV1 and TRPM8 receptor antagonist. Taken together, these results suggest that the conversion of 2-AG to PGE2-G by COX-2 underlies the muscarine-induced enhancement of neurotransmitter release at the vertebrate NMJ.
Acute inhibition of diacylglycerol lipase blocks endocannabinoid-mediated retrograde signalling: evidence for on-demand biosynthesis of 2-arachidonoylglycerol
The endocannabinoid (eCB) 2-arachidonoylglycerol (2-AG) produced by diacylglycerol lipase (DGL) is one of the best-characterized retrograde messengers at central synapses. It has been thought that 2-AG is produced ‘on demand' upon activation of postsynaptic neurons. However, recent studies propose that 2-AG is pre-synthesized by DGL and stored in neurons, and that 2-AG is released from such ‘pre-formed pools' without the participation of DGL. To address whether the 2-AG source for retrograde signalling is the on-demand biosynthesis by DGL or the mobilization from pre-formed pools, we examined the effects of acute pharmacological inhibition of DGL by a novel potent DGL inhibitor, OMDM-188, on retrograde eCB signalling triggered by Ca2+ elevation, Gq/11 protein-coupled receptor activation or synergy of these two stimuli in postsynaptic neurons. We found that pretreatment for 1 h with OMDM-188 effectively blocked depolarization-induced suppression of inhibition (DSI), a purely Ca2+-dependent form of eCB signalling, in slices from the hippocampus, striatum and cerebellum. We also found that at parallel fibre–Purkinje cell synapses in the cerebellum OMDM-188 abolished synaptically induced retrograde eCB signalling, which is known to be caused by the synergy of postsynaptic Ca2+ elevation and group I metabotropic glutamate receptor (I-mGluR) activation. Moreover, brief OMDM-188 treatments for several minutes were sufficient to suppress both DSI and the I-mGluR-induced retrograde eCB signalling in cultured hippocampal neurons. These results are consistent with the hypothesis that 2-AG for synaptic retrograde signalling is supplied as a result of on-demand biosynthesis by DGL rather than mobilization from presumptive pre-formed pools.
Neuronal major histocompatibility complex class I molecules are implicated in the generation of asymmetries in hippocampal circuitry
Left–right asymmetry is a fundamental feature of higher-order brain function; however, the molecular basis of brain asymmetry has remained unclear. We have recently demonstrated asymmetries in hippocampal circuitry resulting from the asymmetrical allocation of NMDA receptor (NMDAR) subunit GluR2 (NR2B) in pyramidal cell synapses. This asymmetrical allocation of 2 subunits affects the properties of NMDARs and generates two populations of synapses, ‘2-dominant' and ‘2-non-dominant' synapses, according to the hemispheric origin of presynaptic inputs and cell polarity of the postsynaptic neurone. To identify key regulators for generating asymmetries, we analysed the hippocampus of 2-microglobulin (2m)-deficient mice lacking cell surface expression of major histocompatibility complex class I (MHCI). Although MHCI proteins are well known in the immune system, accumulating evidence indicates that MHCI proteins are expressed in the brain and are required for activity-dependent refinement of neuronal connections and normal synaptic plasticity. We found that 2m proteins were localised in hippocampal synapses in wild-type mice. NMDA EPSCs in 2m-deficient hippocampal synapses receiving inputs from both hemispheres showed similar sensitivity to Ro 25-6981, an 2 subunit-selective antagonist, with those in ‘2-dominant' synapses for both the apical and basal synapses of pyramidal neurones. The structural features of the 2m-deficient synapse in addition to the relationship between the stimulation frequency and synaptic plasticity were also comparable to those of ‘2-dominant' synapses. These observations indicate that the 2m-deficient hippocampus lacks ‘2-non-dominant' synapses and circuit asymmetries. Our findings provide evidence supporting a critical role of MHCI molecules for generating asymmetries in hippocampal circuitry.
We recently described a new form of neural integration and firing in a subset of interneurons, in which evoking hundreds of action potentials over tens of seconds to minutes produces a sudden barrage of action potentials lasting about a minute beyond the inciting stimulation. During this persistent firing, action potentials are generated in the distal axon and propagate retrogradely to the soma. To distinguish this from other forms of persistent firing, we refer to it here as ‘retroaxonal barrage firing', or ‘barrage firing' for short. Its induction is blocked by chemical inhibitors of gap junctions and curiously, stimulation of one interneuron in some cases triggers barrage firing in a nearby, unstimulated interneuron. Beyond these clues, the mechanisms of barrage firing are unknown. Here we report new results related to these mechanisms. Induction of barrage firing was blocked by lowering extracellular calcium, as long as normal action potential threshold was maintained, and it was inhibited by blocking L-type voltage-gated calcium channels. Despite its calcium dependence, barrage firing was not prevented by inhibiting chemical synaptic transmission. Furthermore, loading the stimulated/recorded interneuron with BAPTA did not block barrage firing, suggesting that the required calcium entry occurs in other cells. Finally, barrage firing was normal in mice with deletion of the primary gene for neuronal gap junctions (connexin36), suggesting that non-neuronal gap junctions may be involved. Together, these findings suggest that barrage firing is probably triggered by a multicellular mechanism involving calcium signalling and gap junctions, but operating independently of chemical synaptic transmission.
The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold ‘bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3–7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (~45% decrease in the total current density measured 200 ms after onset of a voltage step from –78 to –2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na+ channels from inactivation. Depolarization and the number of inactivated Na+ channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.