Neuronal K-Cl Cotransporter : Transcriptional Mechanisms of KCC2 Gene Regulation

K-Cl cotransporter 2 (KCC2) maintains a low intracellular Cl concentration required for fast hyperpolarizing responses of neurons to classical inhibitory neurotransmitters γ-aminobutyric acid (GABA) and glycine. Decreased Cl extrusion observed in genetically modified KCC2-deficient mice leads to depolarizing GABA responses, impaired brain inhibition, and as a consequence to epileptic seizures. Identification of mechanisms regulating activity of the SLC12A5 gene, which encodes the KCC2 cotransporter, in normal and pathological conditions is, thus, of extreme importance. Multiple reports have previously elucidated in details a spatio-temporal pattern of KCC2 expression. Among the characteristic features are an exclusive neuronal specificity, a dramatic upregulation during embryonic and early postnatal development, and a significant downregulation by neuronal trauma. Numerous studies confirmed these expressional features, however transcriptional mechanisms predetermining the SLC12A5 gene behaviour are still unknown. The aim of the presented thesis is to recognize such transcriptional mechanisms and, on their basis, to create a transcriptional model that would explain the established SLC12A5 gene behaviour. Up to recently, only one KCC2 transcript has been thought to exist. A particular novelty of the presented work is the identification of two SLC12A5 gene promoters (SLC12A5-1a and SLC12A5-1b) that produce at least two KCC2 isoforms (KCC2a and KCC2b) differing by their N-terminal parts. Even though a functional 86Rb+ assay reveals no significant difference between transport activities of the isoforms, consensus sites for several protein kinases, found in KCC2a but not in KCC2b, imply a distinct kinetic regulation. As a logical continuation, the current work presents a detailed analysis of the KCC2a and KCC2b expression patterns. This analysis shows an exclusively neuron-specific pattern and similar expression levels for both isoforms during embryonic and neonatal development in rodents. During subsequent postnatal development, the KCC2b expression dramatically increases, while KCC2a expression, depending on central nervous system (CNS) area, either remains at the same level or moderately decreases. In an attempt to explain both the neuronal specificity and the distinct expressional kinetics of the KCC2a and KCC2b isoforms during postnatal development, the corresponding SLC12A5-1a and SLC12A5-1b promoters have been subjected to a comprehensive bioinformatical analysis. Binding sites of several transcription factors (TFs), conserved in the mammalian SLC12A5 gene orthologs, have been identified that might shed light on the observed behaviour of the SLC12A5 gene. Possible roles of these TFs in the regulating of the SLC12A5 gene expression have been elucidated in subsequent experiments and are discussed in the current thesis.

Oikea ionikonsentraatio on välttämätön elimistön normaalissa toiminnassa. Monia ionien kuljettajia, kanavia ja pumppuja tarvitaan tähän. Häiriöt näiden proteiinien toiminnassa aiheuttavat erilaisia sairauksia. Tässä väitöskirjatyössä keskitytään KCC2 proteiiniin, joka on tarpeen kaliumin ja kloridin liikenteessä solukalvon läpi. KCC2 proteiinin väheneminen hiirillä johtaa hermosolujen hyperaktivaatioon, mikä puolestaan voi aiheuttaa epileptisiä kohtauksia. Hiiret, joilta KCC2 puuttuu, kuolevat pian syntymän jälkeen, koska ne eivät voi hengittää. Kyvyttömyys hengittää todennäköisimmin liittyy siihen, että neuronien hyperaktivaatio rikkoo hengitysteiden rytmiä ylläpitävän mekanismin. Toisin kuin monet muut elimistön proteiinit, KCC2 kuljetusproteiini ilmenee ainoastaan keskushermoston hermosoluissa. Tässä tutkimuksessa tutkittiin mekanismeja, jotka estävät KCC2 proteiinin tuotanto muissa soluissa kuin hermosoluissa. Kuten edellä mainittiin, tuotetun KCC2 proteiinin väheneminen aiheuttaa vakavia seurauksia. Tutkimuksessa myös pyrittiin vastaamaan kysymykseen, mitkä mekanismit ylläpitävät tämän kuljettajan tarvittavaa tasoa soluissa.