title: Anomalous levels of Cl- transporters cause a decrease of GABAergic inhibition in human peritumoral epileptic cortex.
abstract: OBJECTIVE
  Several factors contribute to epileptogenesis in patients with brain tumors, including reduced γ-aminobutyric acid (GABA)ergic inhibition. In particular, changes in Cl(-) homeostasis in peritumoral microenvironment, together with alterations of metabolism, are key processes leading to epileptogenesis in patients afflicted by glioma. It has been recently proposed that alterations of Cl(-) homeostasis could be involved in tumor cell migration and metastasis formation. In neurons, the regulation of intracellular Cl(-) concentration ([Cl(-) ](i) ) is mediated by NKCC1 and KCC2 transporters: NKCC1 increases while KCC2 decreases [Cl(-) ](i) . Experiments were thus designed to investigate whether, in human epileptic peritumoral cortex, alterations in the balance of NKCC1 and KCC2 activity may decrease the hyperpolarizing effects of GABA, thereby contributing to epileptogenesis in human brain tumors.
  
  
  METHODS
  Membranes from peritumoral cortical tissues of epileptic patients afflicted by gliomas (from II to IV WHO grade) and from cortical tissues of nonepileptic patients were injected into Xenopus oocytes leading to the incorporation of functional GABA(A) receptors. The GABA-evoked currents were recorded using standard two-microelectrode voltage-clamp technique. In addition, immunoblot analysis and immunohistochemical staining were carried out on membranes and tissues from the same patients.
  
  
  RESULTS
  We found that in oocytes injected with epileptic peritumoral cerebral cortex, the GABA-evoked currents had a more depolarized reversal potential (E(GABA) ) compared to those from nonepileptic healthy cortex. This difference of E(GABA) was abolished by the NKCC1 blocker bumetanide or unblocking of KCC2 with the Zn(2+) chelator TPEN. Moreover, Western blot analysis revealed an increased expression of NKCC1, and more modestly, of KCC2 transporters in epileptic peritumoral tissues compared to nonepileptic control tissues. In addition, NKCC1 immunoreactivity was strongly increased in peritumoral cortex with respect to nonepileptic cortex, with a prominent expression in neuronal cells.
  
  
  CONCLUSIONS
  We report that the positive shift of E(GABA) in epileptic peritumoral human cortex is due to an altered expression of NKCC1 and KCC2, perturbing Cl(-) homeostasis, which might lead to a consequent reduction in GABAergic inhibition. These findings point to a key role of Cl(-) transporters KCC2 and NKCC1 in tumor-related epilepsy, suggesting a more specific drug therapy and surgical approaches for the epileptic patients afflicted by brain tumors.
authors.affiliation: Pasteur Institute-Cenci Bolognetti Foundation, Department of Physiology and Pharmacology, University of Rome La Sapienza, Rome, Italy.
authors.forename: Luca
authors.initials: L
authors.lastname: Conti
authors.forename: Eleonora
authors.initials: E
authors.lastname: Palma
authors.forename: Cristina
authors.initials: C
authors.lastname: Roseti
authors.forename: Clotilde
authors.initials: C
authors.lastname: Lauro
authors.forename: Raffaela
authors.initials: R
authors.lastname: Cipriani
authors.forename: Marjolein
authors.initials: M
authors.lastname: de Groot
authors.forename: Eleonora
authors.initials: E
authors.lastname: Aronica
authors.forename: Cristina
authors.initials: C
authors.lastname: Limatola
mesh_terms: D000328:Adult; D000368:Aged; D000818:Animals; D001703:Biophysics; D001932:Brain Neoplasms / Q000150:complications; D002540:Cerebral Cortex / Q000378:metabolism*; D004558:Electric Stimulation; D004827:Epilepsy / Q000209:etiology / Q000473:pathology*; D005260:Female; D015972:Gene Expression Regulation, Neoplastic / Q000502:physiology; D005910:Glioma / Q000150:complications; D006801:Humans; D008297:Male; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D008845:Microinjections / Q000379:methods; D008875:Middle Aged; D009865:Oocytes; D018408:Patch-Clamp Techniques; D011963:Receptors, GABA-A / Q000378:metabolism; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters / Q000378:metabolism*; D014981:Xenopus; D055815:Young Adult; D005680:gamma-Aminobutyric Acid / Q000378:metabolism* / Q000494:pharmacology; D000096922:K Cl- Cotransporters
chemical_list: D011963:Receptors, GABA-A; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; D027981:Symporters; D005680:gamma-Aminobutyric Acid; D000096922:K Cl- Cotransporters; C578877:SLC12A2 protein, human