Methods for investigating the activities of neuronal chloride transporters

Medina, Igor; Pisella, Lucie I.

doi:10.1016/b978-0-12-815318-5.00002-9

Cited by 6 publications

(6 citation statements)

References 85 publications

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“…To find out whether the variant affects the Cl − extrusion capability of the KCC2 protein, the functionality of KCC2 R231H was studied in in vitro heterologous expression models with two different approaches, gramicidin-perforated patch-clamp and

flux assay. The gramicidin-perforated patch-clamp recordings allow determining the E Gly , i.e., [Cl − ] in , as a function of KCC2 activity, while the

flux assay provides the estimation of the ion-transport efficacy of the transporter (Medina and Pisella, 2020 ). For both methods, heterologous expression in N2a cells utilizing the KCC2b splice variant instead of KCC2a was opted for, as KCC2b is the primary isoform in mature neurons, undergoing up to a 20-fold increase in expression in mice during cortical development as opposed to a two-fold increase for KCC2a (Uvarov et al, 2009 ; Markkanen et al, 2014 ).…”

Section: Resultsmentioning

confidence: 99%

“…These include the interference with cytosolic concentration of K + , effects of high input resistances on voltage-clamp quality, and the possibility for different holding potentials affecting the kinetics of Cl − currents, and, thus, also the [Cl − ] in . Secondly, in the

flux assay, the complexity of mechanisms affecting changes in the internal pH can cause significant variability in the observed fluorescence signals (Medina and Pisella, 2020 ). Despite their caveats, when combined, these methods nevertheless provide a strong means for assessing the functionality of KCC2, albeit, in possible future studies on the functionality of the variant, additional complementary methods should be considered.…”

Section: Discussionmentioning

confidence: 99%

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A novel pathogenic SLC12A5 missense variant in epilepsy of infancy with migrating focal seizures causes impaired KCC2 chloride extrusion

Järvelä,

Hamze,

Komulainen-Ebrahim

et al. 2024

Front. Mol. Neurosci.

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The potassium-chloride co-transporter 2, KCC2, is a neuron-specific ion transporter that plays a multifunctional role in neuronal development. In mature neurons, KCC2 maintains a low enough intracellular chloride concentration essential for inhibitory neurotransmission. During recent years, pathogenic variants in the KCC2 encoding gene SLC12A5 affecting the functionality or expression of the transporter protein have been described in several patients with epilepsy of infancy with migrating focal seizures (EIMFS), a devastating early-onset developmental and epileptic encephalopathy. In this study, we identified a novel recessively inherited SLC12A5 c.692G>A, p. (R231H) variant in a patient diagnosed with severe and drug-resistant EIMFS and profound intellectual disability. The functionality of the variant was assessed in vitro by means of gramicidin-perforated patch-clamp experiments and ammonium flux assay, both of which indicated a significant reduction in chloride extrusion. Based on surface immunolabeling, the variant showed a reduction in membrane expression. These findings implicate pathogenicity of the SLC12A5 variant that leads to impaired inhibitory neurotransmission, increasing probability for hyperexcitability and epileptogenesis.

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flux assay. The gramicidin-perforated patch-clamp recordings allow determining the E Gly , i.e., [Cl − ] in , as a function of KCC2 activity, while the

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A novel pathogenic SLC12A5 missense variant in epilepsy of infancy with migrating focal seizures causes impaired KCC2 chloride extrusion

Järvelä,

Hamze,

Komulainen-Ebrahim

et al. 2024

Front. Mol. Neurosci.

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“…To assess the developmental activity of KCC2 in cultured hippocampal neurons, we performed the NH4 + flux assay. This assay offers an estimation of the ion-transport efficacy of KCC2, providing valuable insights into its functionality across different developmental stages [26,27]. Changes in NH4 + -dependent intracellular pH (pH i ) were monitored using the ratiometric fluorescent probe [28] composed of pH-sensitive pHluorine [29] and pH-insensitive mCherry (Fig.…”

Section: Resultsmentioning

confidence: 99%

Regulation of Neuronal Chloride Homeostasis by Pro- and Mature Brain-Derived Neurotrophic Factor (BDNF) via KCC2 Cation-Chloride Cotransporters in Rat Cortical Neurons

Hamze,

Brier,

Buhler

et al. 2024

Preprint

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The strength of inhibitory neurotransmission depends on intracellular neuronal chloride concentration, primarily regulated by the activity of cation-chloride cotransporters NKCC1 and KCC2. Brain-derived neurotrophic factor (BDNF) influences the functioning of these co-transporters. BDNF is synthesized from precursor proteins (proBDNF), which undergo proteolytic cleavage to yield mature BDNF (mBDNF). While previous studies have indicated the involvement of BDNF signaling in the activity of KCC2, its specific mechanisms are unclear. We investigated the interplay between both forms of BDNF and chloride homeostasis in rat hippocampal neurons and in-utero electroporated cortices of rat pups, spanning the behavioral, cellular, and molecular levels. We found that both pro- and mBDNF play a comparable role in immature neurons by inhibiting the capacity of neurons to extrude chloride. Additionally, proBDNF increases the endocytosis of KCC2 while maintaining a depolarizing shift of EGABA in maturing neurons. Behaviorally, proBDNF-electroporated rat pups in the somatosensory cortex exhibit sensory deficits delayed huddling and cliff avoidance. These findings emphasize the role of BDNF signaling in regulating chloride transport through the modulation of KCC2. In summary, this study provides valuable insights into the intricate interplay between BDNF, chloride homeostasis, and inhibitory synaptic transmission, shedding light on the underlying cellular mechanisms involved.

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“…However, radionuclides are hazardous and becoming more difficult to source. So, alternative methods using fluorescent probes in mammalian cells, or whole-cell perforated patch-clamping, are often used (Medina and Pisella, 2020). These assays require that CCCs localise to the plasma membrane.…”

Section: Resultsmentioning

confidence: 99%

Protein modelling and thermodynamics reveal that plant cation-chloride cotransporters mediate potassium-chloride symport

Henderson,

Nourmohammadi,

Hrmova

2024

Preprint

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Plant cation-chloride-cotransporters (CCC) are proposed to be Na+–K+–2Cl–transporting proteins, although phylogenetically they are closer to K+-Cl–cotransporters (KCC). Conserved features of plant CCC and animal KCC include the presence of predicted K+ and Cl– binding sites, and the absence of a Na+ binding site. Here, we investigated grapevine (Vitis vinifera L.) VvCCC using protein structural modelling and heterologous expression. Our modelling data predicted that 3D folds of VvCCC were more similar to DrNKCC1, but sequences of ion binding sites resembled those of hKCC1. The measurements with VvCCC-injected Xenopus laevis oocytes with VvCCC, localising to plasma membranes, indicated that oocytes were depleted of intracellular Cl–, net 86Rb fluxes, which agreed with thermodynamic predictions for KCC co-transport. 86Rb uptake by VvCCC-injected oocytes was Cl–-dependent, did not require external Na+, and was partially inhibited by the non-specific CCC blocker bumetanide – these properties are typical of KCC transporters. A loop diuretic-insensitive Na+ conductance in VvCCC-injected oocytes may account for earlier observations of Na+ uptake by plant CCC proteins in oocytes. Our data suggest plant CCC proteins are likely to function as K+-Cl– cotransporters, which opens avenues to define their mechanistic and biophysical properties and roles in physiology.

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Methods for investigating the activities of neuronal chloride transporters

Cited by 6 publications

References 85 publications

A novel pathogenic SLC12A5 missense variant in epilepsy of infancy with migrating focal seizures causes impaired KCC2 chloride extrusion

A novel pathogenic SLC12A5 missense variant in epilepsy of infancy with migrating focal seizures causes impaired KCC2 chloride extrusion

Regulation of Neuronal Chloride Homeostasis by Pro- and Mature Brain-Derived Neurotrophic Factor (BDNF) via KCC2 Cation-Chloride Cotransporters in Rat Cortical Neurons

Protein modelling and thermodynamics reveal that plant cation-chloride cotransporters mediate potassium-chloride symport

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