Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices

Khirug, Stanislav; Huttu, Kristiina; Ludwig, Anastasia; Smirnov, Sergei; Voipio, Juha; Rivera, Claudio; Kaila, Kai; Khiroug, Leonard

doi:10.1111/j.1460-9568.2005.03886.x

Cited by 147 publications

(205 citation statements)

References 38 publications

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“…5D). These data are in agreement with measurements of the ion transport efficacy of KCC2 in cortical neurons in vitro, which show strong up-regulation commencing at around P6, with near-maximum extrusion rates achieved at ∼P14-P16 (3,36). The high [Cl − ] i in immature neurons is generally thought to be maintained by Cl − uptake via NKCC1 (in the Introduction).…”

Section: Significancesupporting

confidence: 78%

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Sato

Artoni

Landi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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132

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Intracellular chloride ([Cl−] i ) and pH (pH i ) are fundamental regulators of neuronal excitability. They exert wide-ranging effects on synaptic signaling and plasticity and on development and disorders of the brain. The ideal technique to elucidate the underlying ionic mechanisms is quantitative and combined two-photon imaging of [Cl − ] i and pH i , but this has never been performed at the cellular level in vivo. Here, by using a genetically encoded fluorescent sensor that includes a spectroscopic reference (an element insensitive to Cl − and pH), we show that ratiometric imaging is strongly affected by the optical properties of the brain. We have designed a method that fully corrects for this source of error. Parallel measurements of [Cl − ] i and pH i at the single-cell level in the mouse cortex showed the in vivo presence of the widely discussed developmental fall in [Cl − ] i and the role of the K-Cl cotransporter KCC2 in this process. Then, we introduce a dynamic twophoton excitation protocol to simultaneously determine the changes of pH i and [Cl − ] i in response to hypercapnia and seizure activity.I ntracellular ion concentrations are controlled by plasmalemmal transporters and channels, which generate and dissipate ionic electrochemical gradients, respectively (1). In recent years, regulation of the intracellular Cl − concentration ([Cl − ] i ) in neurons has attracted lots of attention, because it is the main ion that carries current across GABA A (and also, glycine) receptors. Changes in [Cl − ] i exert an immediate effect on the reversal potential of GABAergic currents (E GABA ) and, thereby, on the properties of GABA A receptor-mediated transmission (2-4). The "ionic plasticity" of GABAergic signaling involves not only the passive flux of Cl − ions through membrane channels but also, a number of ion transporters that regulate [Cl − ] i . Furthermore, this mechanism is under the control of intracellular signaling cascades that regulate the expression patterns as well as functional properties of ion transporters and channels (5, 6). With regard to long-term ionic modulation of GABAergic transmission, a case in point is the decrease in [Cl − ] i that is generally thought to take place during maturation of most central neurons. According to this widely accepted scenario, the Na-K-2Cl cotransporter NKCC1 accumulates Cl − in immature neurons, thereby promoting depolarizing GABA responses (3, 7-9), which is followed by developmental upregulation of the neuron-specific K-Cl cotransporter KCC2 that is required for the generation of classical hyperpolarizing inhibitory postsynaptic potentials (IPSPs) (10).A wealth of electrophysiological evidence dating back to the work in vivo by Eccles and coworkers (11) has provided evidence for active regulation of [Cl − ] i in mammalian central neurons and its crucial effect on the driving force of Cl − in inhibitory synapses (1). However, thus far, there are no direct data on neuronal [Cl − ] i measured in vivo at the single-cell level in the living brain, and for in...

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Section: Significancesupporting

confidence: 78%

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Sato

Artoni

Landi

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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132

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“…5), so we next asked whether this residual protein was still capable of extruding Cl − . Functional changes in the Cl − extrusion capacity of KCC2 can be examined in the presence of an ionic load (8,26,27). We examined the function of KCC2 in Neto2-null mice by loading the neuronal soma with 50 mM Cl − (using a patch pipette).…”

Section: Resultsmentioning

confidence: 99%

Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons

Ivakine

Acton

Mahadevan

et al. 2013

Proc. Natl. Acad. Sci. U.S.A.

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KCC2 is a neuron-specific K + -Cl − cotransporter that is essential for Cl − homeostasis and fast inhibitory synaptic transmission in the mature CNS. Despite the critical role of KCC2 in neurons, the mechanisms regulating its function are not understood. Here, we show that KCC2 is critically regulated by the single-pass transmembrane protein neuropilin and tolloid like-2 (Neto2). Neto2 is required to maintain the normal abundance of KCC2 and specifically associates with the active oligomeric form of the transporter. Loss of the Neto2:KCC2 interaction reduced KCC2-mediated Cl − extrusion, resulting in decreased synaptic inhibition in hippocampal neurons. that makes fast GABA A Rmediated synaptic inhibition possible is maintained by the neuronspecific K + -Cl − cotransporter KCC2 (4), a member of the cationchloride cotransporter SLC12 gene family (5). KCC2 is essential for survival, as KCC2 knockout mice die immediately at birth due to respiratory failure (6). In the adult nervous system, decreased KCC2 expression correlates with neuropathic pain, spasticity following spinal cord injury, and epileptic seizures (5, 7-11).Based on the critical importance of KCC2 in the brain, an understanding of the mechanisms that promote and maintain KCC2 expression and efficacy is essential for designing therapeutic strategies to treat neurological disorders characterized by KCC2 dysfunction and the subsequent loss of inhibitory synaptic transmission. A major limitation in the development of these strategies is a lack of understanding regarding the cellular mechanisms regulating KCC2. In particular, KCC2 interacting proteins required for KCC2 transport and function in the mature CNS have not been identified, which represents a large gap in our fundamental knowledge regarding neuronal Cl − regulation and inhibitory synaptic transmission.Here, we identify neuropilin and tolloid like-2 (Neto2) as a KCC2 interacting protein in vivo and demonstrate that this interaction is required for normal neuronal Cl − homeostasis. Neto2 is a complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing transmembrane protein abundantly expressed in neurons (12), which is important for proper neurological function (13). CUB domains are evolutionarily conserved protein domains (14) that participate in protein-protein interactions (15, 16). In the present study, we performed an unbiased proteomic screen and discovered that Neto2 interacts with KCC2. We elucidated the role of this association and characterized its importance in the normal neurophysiological function of KCC2. Because Neto proteins were previously identified as auxiliary subunits of ionotropic glutamate receptors, including kainate and NMDA receptors (17-21), this study further extends the role of the Neto proteins to inhibitory synapses. ResultsNeto2 and KCC2 Interact in Vivo. Recent reports have demonstrated that Neto2 and its homologous protein Neto1 have important functions at excitatory synapses. Neto1 interacts with both the N-methyl D-aspartate receptor (NMDAR) and kainate receptor comp...

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“…Indeed, control measurements of somatic E GABA yielded a value of Ϫ40.7 Ϯ 0.41 mV (n ϭ 26), demonstrating that the somatic [Cl Ϫ ] i of CA1 pyramidal neurons was clamped at the [Cl Ϫ ] of the pipette. Thus, under these conditions, a negative deviation of the dendritic E GABA from the somatic value (i.e., ⌬E GABA ) is a quantitative measure of the efficacy of Cl Ϫ extrusion (Jarolimek et al, 1999;Khirug et al, 2005). Somatic and dendritic E GABA values were determined from the current-voltage rela-tions, obtained by sequentially clamping the membrane potential at different holding potentials with a 5 mV increment and a 10 s step interval.…”

Section: Methodsmentioning

confidence: 99%

“…Somatic and dendritic E GABA values were determined from the current-voltage rela-tions, obtained by sequentially clamping the membrane potential at different holding potentials with a 5 mV increment and a 10 s step interval. Caged GABA was photolyzed 50 ms after the start of each voltage step to evoke local GABA A receptor-mediated currents (Khirug et al, 2005). To this end, 1 mM DPNI-caged GABA dissolved in extracellular solution was delivered at a flow rate of 1-2 l/min via an UltraMicroPump II (WPI) equipped with a syringe and a 100 m inner tip diameter quartz needle (WPI) and locally photolyzed using the 375 nm output of a continuous emission diode laser (Excelsior 375; Spectra Physics).…”

Section: Methodsmentioning

confidence: 99%

“…To study the effect of blocking protein synthesis on the functional KCC2 pool, we used an electrophysiological assay to measure KCC2-mediated Cl Ϫ extrusion (Khirug et al, 2005;Li et al, 2007). Because of the similar results obtained with the two protein synthesis inhibitors in the biochemical assays (Fig.…”

Section: Total Block Of Protein Synthesis Does Not Affect Kcc2 Proteimentioning

confidence: 99%

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Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

et al. 2012

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The K-Cl cotransporter KCC2 plays a crucial role in neuronal chloride regulation. In mature central neurons, KCC2 is responsible for the low intracellular Cl Ϫ concentration ([Cl Ϫ ] i ) that forms the basis for hyperpolarizing GABA A receptor-mediated responses. Fast changes in KCC2 function and expression have been observed under various physiological and pathophysiological conditions. Here, we show that the application of protein synthesis inhibitors cycloheximide and emetine to acute rat hippocampal slices have no effect on total KCC2 protein level and K-Cl cotransporter function. Furthermore, blocking constitutive lysosomal degradation with leupeptin did not induce significant changes in KCC2 protein levels. These findings indicate a low basal turnover rate of the total KCC2 protein pool. In the presence of the glutamate receptor agonist NMDA, the total KCC2 protein level decreased to about 30% within 4 h, and this effect was blocked by calpeptin and MDL-28170, inhibitors of the calcium-activated protease calpain. Interictal-like activity induced by incubation of hippocampal slices in an Mg 2ϩ -free solution led to a fast reduction in KCC2-mediated Cl Ϫ transport efficacy in CA1 pyramidal neurons, which was paralleled by a decrease in both total and plasmalemmal KCC2 protein. These effects were blocked by the calpain inhibitor MDL-28170. Taken together, these findings show that calpain activation leads to cleavage of KCC2, thereby modulating GABAergic signaling.

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Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices

Cited by 147 publications

References 38 publications

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

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Distinct properties of functional KCC2 expression in immature mouse hippocampal neurons in culture and in acute slices

Cited by 147 publications

References 38 publications

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Simultaneous two-photon imaging of intracellular chloride concentration and pH in mouse pyramidal neurons in vivo

Neto2 is a KCC2 interacting protein required for neuronal Cl − regulation in hippocampal neurons

Activity-Dependent Cleavage of the K-Cl Cotransporter KCC2 Mediated by Calcium-Activated Protease Calpain

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Neto2 is a KCC2 interacting protein required for neuronal Cl ⁻ regulation in hippocampal neurons