Ion-regulatory proreins in neuronal development and communication

Brain function is critically dependent on the ionic homeostasis in both the extra- and intracellular compartment. The regulation of brain extracellular ionic composition mainly relies on active transport at blood brain and at blood cerebrospinal fluid interfaces whereas intracellular ion regulation is based on plasmalemmal transporters of neurons and glia. In addition, the latter mechanisms can generate physiologically as well as pathophysiologically significant extracellular ion transients. In this work I have studied molecular mechanisms and development of ion regulation and how these factors alter neuronal excitability and affect synaptic and non-synaptic transmission with a particular emphasis on intracellular pH and chloride (Cl-) regulation. Why is the regulation of acid-base equivalents (H+ and HCO3-) and Cl- of such interest and importance? First of all, GABAA-receptors are permeable to both HCO3- and Cl-. In the adult mammalian central nervous system (CNS) fast postsynaptic inhibition relies on GABAA-receptor mediated transmission. Today, excitatory effects of GABAA-receptors, both in mature neurons and during the early development, have been recognized and the significance of the dual actions of GABA on neuronal communication has become an interesting field of research. The transmembrane gradients of Cl- and HCO3- determine the reversal potential of GABAA-receptor mediated postsynaptic potentials and hence, the function of pH and Cl- regulatory proteins have profound consequences on GABAergic signaling and neuronal excitability. Secondly, perturbations in pH can cause a variety of changes in cellular function, many of them resulting from the interaction of protons with ionizable side chains of proteins. pH-mediated alterations of protein conformation in e.g. ion channels, transporters, and enzymes can powerfully modulate neurotransmission. In the context of pH homeostasis, the enzyme carbonic anhydrase (CA) needs to be taken into account in parallel with ion transporters: for CO2/HCO3- buffering to act in a fast manner, CO2 (de)hydration must be catalyzed by this enzyme. The acid-base equivalents that serve as substrates in the CO2 dehydration-hydration reaction are also engaged in many carrier and channel mediated ion movements. In such processes, CA activity is in key position to modulate transmembrane solute fluxes and their consequences. The bicarbonate transporters (BTs; SLC4) and the electroneutral cation-chloride cotransporters (CCCs; SLC12) belong the to large gene family of solute carriers (SLCs). In my work I have studied the physiological roles of the K+-Cl- cotransporter KCC2 (Slc12a5) and the Na+-driven Cl--HCO3- exchanger NCBE (Slc4a10) and the roles of these two ion transporters in the modualtion of neuronal communication and excitability in the rodent hippocampus. I have also examined the cellular localization and molecular basis of intracellular CA that has been shown to be essential for the generation of prolonged GABAergic excitation in the mature hippocampus. The results in my Thesis provide direct evidence for the view that the postnatal up-regulation of KCC2 accounts for the developmental shift from depolarizing to hyperpolarizing postsynaptic EGABA-A responses in rat hippocampal pyramidal neurons. The results also indicate that after KCC2 expression the developmental onset of excitatory GABAergic transmission upon intense GABAA-receptor stimulation depend on the expression of intrapyramidal CA, identified as the CA isoform VII. Studies on mice with targeted Slc4a10 gene disruption revealed an important role for NCBE in neuronal pH regulation and in pH-dependent modulation of neuronal excitability. Furthermore, this ion transporter is involved in the basolateral Na+ and HCO3- uptake in choroid plexus epithelial cells, and is thus likely to contribute to cerebrospinal fluid production.Väitöskirjassani tarkastelen hermosolujen ionisäätelyyn osallistuvien proteiinien toiminnan merkitystä hermosolujen välisessä kommunikaatiossa. Tutkimuksellinen painopiste kohdistuu kloridi-, protoni- ja bikarbonaatti-ioneihin sekä niiden säätelyyn osallistuvien solukalvon ionikuljettajiin ja hiilidioksidi-bikarbonaatti tasapainon säätelyyn osallistuvaan soluliman karboanhydraasi-entsyymiin. Tutkimuksessa on tarkasteltu näiden ionisäätelyproteiinien ilmentymistä yksilönkehityksen aikana, niiden vaikutusta hermosolujen ja hermosoluverkkojen synaptiseen ja ei-synaptiseen kommunikaatioon sekä kyseisten signalointimekanismien biofysikaalisia mekanismeja. Gamma-aminovoihappo (GABA) on täysikasvuisen nisäkkään aivojen pääasiallinen hermoimpulsseja ehkäisevä välittäjäaine. Siihen pohjautuva hermosolujen välinen nopea viestintä perustuu ns. A-tyypin GABA kanavien läpi kulkeviin kloridi- ja bikarbonaatti-ionien kantamiin sähkövirtoihin. Näiden ionien solunsisäisiä pitoisuuksia säätelevät proteiinit muovaavat GABA-välitteistä hermosoluviestintää. Väitöskirjassani esitetyt tutkimustulokset osoittavat, että tietyn kaliumvaraisen kloridikuljettajan (KCC2:n) ilmentyminen kehittyvissä aivoissa on edellytys hermoimpulsseja ehkäisevien, GABAA-kanavien kautta solusta ulos suuntautuvien virtojen synnylle. Myöhemmässä kehitysvaiheessa GABAA-kanavien voimakas aktivoituminen voi, esimerkiksi patofysiologisissa tilanteissa, johtaa solunsisäisen ja -ulkoisen ionitasapainon tilapäiseen häiriintymiseen, jolloin GABAA-kanavien tuottamat vasteet paradoksaalisesti synnyttävät hermoimpulsseja. Tällainen GABAA-välitteinen hermoärsytys edellyttää bikarbonaatti-ionien nopeaa tuottoa solunsisäisen karboanhydraasi-entsyymin avulla, mikä johtaa hermosoluja ärsyttävään hetkelliseen soluvälitilan kalium-pitoisuuden kasvuun. Tutkimukseni perusteella voidaan todeta, että karboanhydraasi-entsyymin ilmentyminen rotan hippokampus-aivoalueen pyramidihermosoluissa lisääntyy vasta syntymänjälkeisen kehityksen aikana ja on ratkaiseva tekijä tämän aivoalueen hermoverkon ärtyvyyden kasvussa. Lisäksi tutkin geenin Slc4a10 koodaamaa, natriumista riippuvaista bikarbonaatti-kuljettajaa (NCBE). Tulosten mukaan NCBE:lla on tärkeä osuus hippokampuksen pyramidisolujen pH-, ja sitä kautta myös ärtyvyyden, säätelyssä: poistogeenisten NCBE-hiirten hippokampuksen pyramidisolujen kyky poistaa protoni-ylijäämää oli selvästi heikentynyt ja vastaavasti kynnys epileptisen kohtauksen laukeamiselle oli huomattavasti kohonnut. Tutkimustulokset viittaavat pH-säätelymekanismien tärkeään rooliin hermosolujen ärtyvyyden kontrolloinnissa. On myös erittäin todennäköistä, että poistogeenisissä NCBE-hiirissä havaittu aivokammioiden tilavuuden radikaali pienentyminen on seurausta aivo-selkäydinnesteen tuotannon heikentymisestä. Havainnot tukevat ajatusta, että NCBE kuljettajaproteiinista riippuvaisella natriumin ja bikarbonaatin yhteiskuljetuksella aivojen suonipunosten epiteelisoluissa olisi merkittävä osuus aivo-selkäydinnesteen tuotannossa

GABA actions and ionic plasticity in epilepsy

Concepts of epilepsy, based on a simple change in neuronal excitation/inhibition balance, have subsided in face of recent insights into the large diversity and context-dependence of signaling mechanisms at the molecular, cellular and neuronal network level. GABAergic transmission exerts both seizure-suppressing and seizure-promoting actions. These two roles are prone to short-term and long-term alterations, evident both during epileptogenesis and during individual epileptiform events. The driving force of GABAergic currents is controlled by ion-regulatory molecules such as the neuronal K-Cl cotransporter KCC2 and cytosolic carbonic anhydrases. Accumulating evidence suggests that neuronal ion regulation is highly plastic, thereby contributing to the multiple roles ascribed to GABAergic signaling during epileptogenesis and epilepsy.Peer reviewe

Regulation of intracellular pH in salamander retinal rods

1. We measured intracellular pH (pHi) in rods isolated from the retina of the axolotl salamander, Ambystoma mexicanum, using the fluorescent indicator 2',7'-bis(carboxyethyl)- 5(and -6)-carboxyfluorescein (BCECF). 2. The light exposures associated with data acquisition had no marked effect on pH,. There was no sharp change between the value obtained from the first exposure of dark-adapted rods and subsequent readings. Increasing the acquisition frequency from 1 to 10 min-' either had no effect, or brought about a slow acidification, which was stopped or reversed when the low frequency was restored. 3. In nominally HCO3--free solution at pH 7 5, the rods had a steady-state pHi of 7'09 + 0-02 (n = 46) and a buffering power (,Bi) of 24 + 1 mm (pH unit)f' (n = 48). The buffering power was virtually constant in the pH range 6-6-8-0. In the same range, pHi depended linearly on perfusion pH (pHO) with regression coefficients of 0 4-0 5. 4. There were no significant differences between the inner and outer segment of intact rods as regards steady-state pH, or responses to experimental treatments. 5. Recovery from an intracellular acid load imposed by sodium propionate or an NH4Cl prepulse in nominally bicarbonate-free perfusate was completely blocked by decreasing the extracellular Nae concentration to 7 mm, and slowed by 86% by applying 1 mm amiloride. 6. Introduction of 2% C02-13 mm HC03- caused an alkalinization that was often preceded by a transient acidification. Steady-state pHi was on average 0.1 pH units higher than in nominally bicarbonate-free solution. The mean acid extrusion rate, calculated on the assumption that C02-HC03- behaves as an open system, was 19% higher (31 + 2 mm h-1) than in a solution buffered only by Hepes (26 + 2 mm h-). 7. In the presence of CO2-HC03-, 100 ,m 4,4'-diisothiocyanatostilbene-2,2'-disulphonic acid (DIDS) decreased the acid extrusion rate by 20% on average. Lowering the extracellular Clconcentration to 7 mm raised pH,, but did not significantly affect the acid extrusion rate. 8. We conclude that retinal rods regulate pHi by both Na+-H+ exchange and mechanism(s) involving HC03--Cl- exchange. In the present conditions, the Na+-H+ exchanger appears as the dominant mechanism for acid extrusion

Vasopressin excites interneurons to suppress hippocampal network activity across a broad span of brain maturity at birth

During birth in mammals, a pronounced surge of fetal peripheral stress hormones takes place to promote survival in the transition to the extrauterine environment. However, it is not known whether the hormonal signaling involves central pathways with direct protective effects on the perinatal brain. Here, we show that arginine vasopressin specifically activates interneurons to suppress spontaneous network events in the perinatal hippocampus. Experiments done on the altricial rat and precocial guinea pig neonate demonstrated that the effect of vasopressin is not dependent on the level of maturation (depolarizing vs. hyperpolarizing) of postsynaptic GABA(A) receptor actions. Thus, the fetal mammalian brain is equipped with an evolutionarily conserved mechanism well-suited to suppress energetically expensive correlated network events under conditions of reduced oxygen supply at birth.Peer reviewe

Neuronal carbonic anhydrase VII provides GABAergic excitatory drive to exacerbate febrile seizures

Peer reviewe

Carbonic anhydrase seven bundles filamentous actin and regulates dendritic spine morphology and density

Intracellular pH is a potent modulator of neuronal functions. By catalyzing (de)hydration of CO2, intracellular carbonic anhydrase (CA(i)) isoforms CA2 and CA7 contribute to neuronal pH buffering and dynamics. The presence of two highly active isoforms in neurons suggests that they may serve isozyme-specific functions unrelated to CO2-(de)hydration. Here, we show that CA7, unlike CA2, binds to filamentous actin, and its overexpression induces formation of thick actin bundles and membrane protrusions in fibroblasts. In CA7-overexpressing neurons, CA7 is enriched in dendritic spines, which leads to aberrant spine morphology. We identified amino acids unique to CA7 that are required for direct actin interactions, promoting actin filament bundling and spine targeting. Disruption of CA7 expression in neocortical neurons leads to higher spine density due to increased proportion of small spines. Thus, our work demonstrates highly distinct subcellular expression patterns of CA7 and CA2, and a novel, structural role of CA7.Peer reviewe