Aus dem Institut für Anatomie
der Medizinischen Fakultät Charité
der Humboldt-Universität zu Berlin

DISSERTATION

Kir2 potassium channels in rat striatum are strategically localized
to control basal ganglia function

Zur Erlangung des akademischen Grades
Doctor medicinae (Dr. med.)

vorgelegt der Medizinischen Fakultät Charité
der Humboldt-Universität zu Berlin

von
Harald Prüß

aus Güstrow

Dekan: Prof. Dr. Joachim W. Dudenhausen

Gutachter:
1. Prof. Dr. A. Karschin
2. Prof. Dr. R. W. Veh
3. Prof. Dr. J. Roeper

eingereicht: 16.07.2003

Datum der Promotion: 15. 03 2004

Abstract

Parkinson’s disease is the most frequent movement disorder caused by loss of dopaminergic neurons in the midbrain. Intentions to avoid side effects of conventional therapy should aim to identify additional targets for potential pharmacological intervention. In principle, every step of a signal transduction cascade, such as presynaptic transmitter release, type and occupation of postsynaptic receptors, G protein-mediated effector mechanisms, and the alterations of pre- or postsynaptic potentials as determined by the local ion channel composition, have to be considered. Due to their diversity and their widespread but distinct localizations, potassium channels represent interesting candidates for new therapeutic strategies.

As a first step, the present report aimed to study the cellular and subcellular distribution of the individual members of the Kir2 family in the striatum, a group of proteins forming inwardly rectifying potassium channels. For this purpose polyclonal, monospecific, affinity purified antibodies against the less conserved carboxyterminal sequences from the Kir2.1, Kir2.2, Kir2.3, and Kir2.4 proteins were prepared. All subunits of the Kir2 family were detected on somata and dendrites of most striatal neurons. However, the distribution of two of them was not homogeneous. Striatal patch areas were largely devoid of the Kir2.3 protein, and the Kir2.4 subunit was most prominently expressed on the tonically active, giant cholinergic interneurons of the striatum. These two structures are among the key players in regulating dopaminergic and cholinergic neurotransmission within the striatum, and therefore are of major importance for the output of the basal ganglia. The heterogeneous localization of the Kir2.3 and the Kir2.4 subunits with respect to these strategic structures pinpoints these channel proteins as promising targets for future pharmacological efforts.

Keywords: Basal ganglia, inwardly rectifying potassium channels, Kir2, cholinergic interneuron, Parkinson’s disease

Zusammenfassung

Der Morbus Parkinson ist die häufigste Erkrankung der Basalganglien und wird durch einen Abbau der dopaminergen Neurone in der Substantia nigra des Mittelhirns verursacht. Um Wege zu finden, die Nebenwirkungen bisheriger Therapien dieser Erkrankung zu vermeiden, sollten neue Angriffspunkte für pharmakologische Interventionen gesucht werden. Prinzipiell ist dabei jeder Schritt einer Signaltransduktions-Kaskade zu prüfen. Dazu gehören präsynaptische Transmitterfreisetzung, G-Protein-gesteuerte Effektormechanismen oder Veränderungen prä- und postsynaptischer Potentiale, wie sie durch ein bestimmtes lokales Ionenkanalmuster festgelegt werden. Aufgrund ihrer enormen molekularen Vielfalt bei gleichzeitig weiter, aber spezifischer Verbreitung, stellen Kaliumkanäle interessante Angriffspunkte für neue therapeutische Strategien dar.

Die vorliegende Arbeit untersucht die zelluläre und subzelluläre Verteilung aller Mitglieder der Kir2-Familie, einer Gruppe von Proteinen, die einwärts-gleichrichtende Kaliumkanäle bildet. Zu diesem Zweck wurden polyklonale, monospezifische, affinitätsgereinigte Antikörper gegen den wenig konservierten carboxyterminalen Anteil der Kir2.1-, Kir2.2-, Kir2.3- und Kir2.4-Proteine hergestellt. Alle Untereinheiten der Kir2-Familie wurden an den Somata und Dendriten der meisten striatalen Neurone nachgewiesen. Zwei dieser Kanäle zeigten jedoch ein inhomogenes Verteilungsmuster: Das "patch"-Kompartiment des Striatums wurde von der Expression des Kir2.3-Kanals ausgespart, und das Kir2.4-Protein wurde am stärksten auf den tonisch aktiven, cholinergen striatalen Interneuronen exprimiert. Diese beiden Strukturen stellen die Schlüsselstellen für die Kontrolle und Regulation der dopaminergen und cholinergen Transmission im Striatum dar, weswegen ihnen eine zentrale Rolle für die efferenten Projektionen der Basalganglien zukommt. Die nachgewiesene heterogene Lokalisation der Kir2.3- und Kir2.4-Untereinheit an diesen strategisch relevanten Strukturen macht diese Kanäle zu viel versprechenden Angriffspunkten für zukünftige Pharmakotherapien.

Eigene Schlagworte: Basalganglien, einwärts gleichrichtende Kaliumkanäle, Kir2, cholinerges Interneuron, Morbus Parkinson

Table of contents

• 1.  Introduction
  • 1.1. Kir2 channels
    • 1.1.1. Potassium channels
    • 1.1.2. Kir – inward rectifier potassium channels
      • 1.1.2.1.  Kir subfamilies
    • 1.1.3. Kir2 subfamily
      • 1.1.3.1.  Electrophysiology
      • 1.1.3.2. Basic principles of inward rectification
      • 1.1.3.3.  Modulation
      • 1.1.3.4. Distribution of Kir2 channels in the brain
  • 1.2. Basal ganglia
    • 1.2.1. Principle neuron and interneurons
      • 1.2.1.1. Kir current in striatal spiny neurons
      • 1.2.1.2. Cholinergic interneurons
    • 1.2.2. Patch and matrix
    • 1.2.3. Understanding the basal ganglia circuitry
    • 1.2.4. Striatal regulation of movements
    • 1.2.5. The therapeutic dilemma
    • 1.2.6. Intention of the research
• 2. Materials and Methods
  • 2.1. Cloning
    • 2.1.1. Selection of sequences
    • 2.1.2. Polymerase chain reaction (PCR)
    • 2.1.3.  Restriction
    • 2.1.4. Agarose gel electrophoresis
    • 2.1.5. Determination of DNA concentration
    • 2.1.6.  Ligation
    • 2.1.7. Generation of competent cells
    • 2.1.8. Transformation
  • 2.2. Protein expression and purification
    • 2.2.1. Overexpression of fusion proteins
    • 2.2.2. Protein purification
    • 2.2.3. SDS-Polyacrylamide gel electrophoresis (SDS-PAGE)
    • 2.2.4. Preparative electrophoresis
    • 2.2.5. Determination of protein content
      • 2.2.5.1. BCA (Bicinchoninic acid) assay
      • 2.2.5.2. Bradford assay
  • 2.3. Raising of antibodies in rabbits
    • 2.3.1. Immunization and blood taking
    • 2.3.2. Determination of antibody titer, ELISA
    • 2.3.3. Competitive ELISA
  • 2.4. Purification of antibodies
    • 2.4.1. Removal of IgM
    • 2.4.2. Removal of cross reactivity
    • 2.4.3.  Affinity purification
    • 2.4.4. Chromatofocussing
    • 2.4.5. Concentration of antibodies
  • 2.5. Characterization of antibodies
    • 2.5.1. Analysis of specificity in Western Blots
      • 2.5.1.1. Preparation of brain homogenates
      • 2.5.1.2. Western Blotting
      • 2.5.1.3. Immune detection
      • 2.5.1.4. Visualization by use of alkaline phosphatase (aP)
    • 2.5.2.  Analysis of specificity by transfected cells
      • 2.5.2.1. Liposome-mediated transfection
      • 2.5.2.2. Detection of transfected cells by immunofluorescence
  • 2.6.  Immunocytochemistry
    • 2.6.1. Perfusion fixation of rat brains
    • 2.6.2. Rat brain slices for light microscopy
    • 2.6.3. Rat brain slices for fluorescence microscopy
    • 2.6.4. Coating of slides
    • 2.6.5. Cresyl violet staining
    • 2.6.6. Electron microscopy
      • 2.6.6.1. Immunoreaction
      • 2.6.6.2. Staining with the avidin-biotin method
      • 2.6.6.3. Gold-silver-enhancement
      • 2.6.6.4. Araldite embedding
      • 2.6.6.5. Contrasting the ultrathin sections
      • 2.6.6.6. Toluidin blue staining
    • 2.6.7. Histological analysis
• 3.  Results
  • 3.1. Preparation of monospecific and affinity-purified antibodies
    • 3.1.1. Comparison of the amino acid sequences
    • 3.1.2.  Recombinant fusion proteins
    • 3.1.3. Removal of IgM antibodies by gel-filtration
    • 3.1.4. Affinity purification
  • 3.2. Specificity of purified antibodies
    • 3.2.1. Cross reactivity
    • 3.2.2. Competitive ELISA
    • 3.2.3. Western blot of fusion proteins
    • 3.2.4. Western blot of rat brain homogenates
    • 3.2.5. Specificity of anti-Kir2.4 antibodies
    • 3.2.6. Antibody specificity in brain sections
  • 3.3. Distribution of Kir2 channels in the rat brain
    • 3.3.1. Olfactory system
    • 3.3.2.  Hippocampus
    • 3.3.3. Neocortex
    • 3.3.4. Basal ganglia and amygdala
    • 3.3.5. Thalamus
    • 3.3.6. Hypothalamus and habenula
    • 3.3.7.  Substantia nigra, ventral tegmental area (VTA), superior colliculus
    • 3.3.8. Cerebellum and spinal medulla
  • 3.4. Distribution of Kir2 channels in the striatum
    • 3.4.1. Kir2 subunits are differentially distributed in the rat striatum
    • 3.4.2. Anti-Kir2.2 antibodies stain striatal fiber bundles
    • 3.4.3.  Kir2.3 channels are predominantly localized in the matrix compartment
    • 3.4.4. Kir2.4 channel subunits are localized at cholinergic interneurons
• 4.  Discussion
  • 4.1. 
    • 4.1.1. Kir2 channel proteins are differentially expressed throughout the rat brain
    • 4.1.2. The Kir2.3 subunit is preferentially expressed in striatal matrix neurons
    • 4.1.3.  Kir2.4 immunoreactivity in the striatum is most prominently displayed by the giant cholinergic interneurons
    • 4.1.4. Can Kir channel subunits be targets for novel therapeutic strategies?
• Abbreviations
• Summary
• References
• Curriculum vitae
• Publications
• Danksagung
• Eidesstattliche Erklärung

Tables

• Table 1: Kir2 channel modulators.
• Table 2: vectors
• Table 3: Primer and restriction enzymes (restriction sites underlined)
• Table 4: ELISA
• Table 5: Distribution of individual Kir2 subunits in different brain regions
• Table 6: Striatal Kir2 channel expression, results from in situ hybridization experiments

Images

• Fig. 1: Alpha-subunits of distinct potassium channel classes. A) voltage-gated (Kv) K+ channels, B) inward rectifier (Kir) potassium channels (M1, M2: transmembrane helices, P: pore-forming region), C) ‘background’ or ‘leakage’ channels
• Fig. 2: Dendrogram of the Kir family. Subfamilies, subtypes and former names. (modified from Reimann and Ashcroft 1999)
• Fig. 3: Inwardly rectifying currents recorded from a Xenopus oocyte expressing a Kir channel. Experimental conditions with symmetrical K+ concentrations. A) Current traces elicited by voltage steps from 0 mV (holding potential) to +80, +40, 0, -40, -80, -120 and –160 mV. B) Current-voltage relationship of the peak currents recorded from the same oocyte.
• Fig. 4: Model of channel block by polyamines . A) Spermine and Mg2+ directly block the pore by binding at residue D172. B) E224 and E299 are discussed controversially: spermine binding at residue E224 leads to a high affinity blocking complex (Lee et al. 1999), or intermediate binding at E224+E299 level facilitates the entry of spermine but does not plug the pore (Kubo and Murata 2001).
• Fig. 5: Synopsis of Kir2 channel modulations. For detailed explanation of (a) to (r) see table 1.
• Fig. 6: Schematic representation of the neuronal circuitry underlying basal ganglia function. For details see text.
• Fig. 7: Amino acid sequences of Kir2 subfamily members. The primary structures of all rat Kir2 channel subunits demonstrate that sequence conservation is lowest in the carboxyterminal area. Sequences shared by all four subfamily members are labeled in red. The carboxy terminal sequences uses for antibody production are depicted in gray. M1, M2 transmembrane regions; P5 pore-forming region (adapted from Töpert 1998)
• Fig. 8: SDS-PAGE of purified fusion proteins Kir2.1-Kir2.4
• Fig. 9: Elution profile after passage through a superdex column. The green line represents the protein concentration and the red line the immunoreactivity to the corresponding Kir2 protein. Fractions selected on the basis of high antibody titer (24-36) are marked.
• Fig. 10: Affinity purification. A) Initial application of pQE40 protein only to absorb cross reactivity to the plasmid part of the expressed antigen. B) Varying conditions to find protein concentration with best antibody quantity.
• Fig. 11: No cross reactivity to other Kir subunits. Microtiter plates were coated with different proteins of Kir channels. Antibodies of the Kir2 subfamily exclusively detect their corresponding antigen and display no cross reactivity to other subunits. The horizontal line indicates extinction of BSA controls.
• Fig. 12: ELISA assays of all members of the Kir2 subfamily. Microtiter plates were coated with the respective recombinant protein. Preincubation of antibodies with the cognate antigen only decreases the immunoreactivity in a concentration-dependent manner. Preincubation with other antigens did not show any effect.
• Fig. 13: Specificity of anti-Kir2 antibodies. In Western blots Kir2 antibodies recognize only their corresponding fusion protein and none of the other Kir2 subfamily members.
• Fig. 14: Specificities of the anti-Kir2 antibodies as judged from Western blots. Homogenates from total rat brain membranes (10 µg/lane) were separated on 10% SDS-PAGE. All antibodies recognized single bands of the expected molecular weights (left lanes; with exception of Kir2.4, see next chapter) which were largely abolished subsequent to preincubation block with the cognate recombinant proteins (right lanes).
• Fig. 15A,B: Specificity of anti-Kir2.4 antibodies, transfection experiments. The specificity of the Kir2.4 antibodies was further verified by the immunofluorescence obtained with Kir2.4 transfected COS-7 cells (A). Non-transfected cells were negative (B).
• Fig. 15C,D: Specificity of anti-Kir2.4 antibodies. Western blots from transfected cells (C) displayed a double band (left lane), most probably due to the presence of phosphorylated species, within the expected range of molecular weights. Untransfected cells yielded blank blots (C, right lane). Western blots of heart homogenate (D) also show a protein band within the correct range.
• Fig. 16: Immunocytochemical labeling of the rat olfactory bulb confirms the specificity of the anti-Kir2 antibodies. For detailed analysis of the immunocytochemical specificities of the anti-Kir2 antibodies an area (boxed in A) containing the outer layers of the olfactory bulb was selected. Individual layers are evident at higher magnification in the section stained with cresyl violet (B). Immunoreactivities of individual members of the Kir2 subfamily (C-F) display specific and individual distribution patterns. Gl, glomerular layer; EPl, external plexiform layer; Mi, mitral cell layer; IPl, internal plexiform layer; Gr, granule cell layer.
• Fig. 17-I: Olfactory tubercle. For details see text. cc, corpus callosum; IG, induseum griseum; CPu, caudate putamen; AcbC, core part of the accumbens nucleus, AcbSh, shell part of the accumbens nucleus; Tu, olfactory tubercle; Pir, piriform cortex.
• Fig. 17-II: Hippocampus. Areas C-F are boxed ‘a’ in A. For details see text. CA1-CA3, cornu ammonis 1-3; DG, dentate gyrus; fi, fimbriae; MHb, medial habenula; LHb lateral habenula.
• Fig. 17-III: Cortex (A-E), habenula (F-J). Areas A-E are boxed ‘b’ and F-J corresponds to ‘c’ in Fig. 17-IIA. For details see text.
• Fig. 17-IV: Nucleus accumbens. For details see text. cc, corpus callosum; CPu, caudate putamen; AcbC, core part of the accumbens nucleus, AcbSh, shell part of the accumbens nucleus; Tu, olfactory tubercle; Pir, piriform cortex; aca, anterior commissure.
• Fig. 17-V: Thalamus. For details see text. Areas G and H are boxed in A. CPu, caudate putamen; GP, globus pallidus; ic, internal capsule; Rt, reticular nucleus; sm, stria medullaris; Pir, piriform cortex; LOT, lateral olfactory tract nucleus; SO, supraoptic nucleus; ox, optic chiasma.
• Fig. 17-VI: Substantia nigra. For details see text. MG, medial geniculate; CG, central gray; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; cp, cerebral peduncle; IP, interpeduncular nucleus.
• Fig. 17-VII: Mesencephalon. Adjacent sections of the oculomotor nucleus (G-J) are from the region boxed in A. For details see text. MG, medial geniculate; SuC, superior colliculus; CG, central gray; SNC, substantia nigra pars compacta; SNR, substantia nigra pars reticulata; VTA, ventral tegmental area; cp, cerebral peduncle; IP, interpeduncular nucleus; 3, oculomotor nucleus.
• Fig. 17-VIII: Cerebellum. Adjacent sections from the cerebellar cortex (C-F) and deep nuclei (G-J). For details see text. Med, medial cerebellar nucleus; Int, interposed cerebellar nucleus; Lat, lateral cerebellar nucleus.
• Fig. 17-IX: Medulla spinalis. For details see text. cu, cuneate fasciculus; lfu, lateral funiculus; vfu, ventral funiculus; 2-9, spinal cord layers.
• Fig. 18: Regional and cellular distribution of Kir2 subunits in the rat striatum. A series of six adjacent sections was used to analyze the immunocytochemical patterns of the individual members of the Kir2 family. Coronal sections were obtained shortly caudal to the optic chiasm and stained according to Klüver-Barrera for morphological examination (A,B). An area (boxed in A) containing parts of the caudate-putamen and the external pallidum was selected for further investigation. Distinct patterns of immunostaining were obtained for the individual Kir2 subunits (C-F). Note that Kir2.2-positive neurons are clustered in the pallidum (D, arrowheads) and that large neurons in the striatum display prominent staining (F, arrows) with the anti-Kir2.4 antibody. CPu, caudate-putamen; GP, globus pallidum externum; Pir, piriform cortex; sm, stria medullaris thalami.
• Fig. 19: Anti-Kir2.2 antibodies stain striatal fiber bundles. Using carefully selected conditions (for details see text) Kir2.2 immunoreactivity displayed a prominent labeling of fiber bundles within the striatum (A,B). At the electron microscopic level (C) the gold dots representing Kir2.2 channel distribution are found highly concentrated within the fiber bundles, suggesting the Kir2.2 subunit being sorted to the axonal membrane. Gold dots are sparse in the surrounding neuropil.
• Fig. 20: Kir2.3 channel subunit protein is inhomogeneously distributed throughout the striatum. Complete immunostained sections (A) of the rat forebrain, unlike sections with antigen competition (B), show Kir2.3 immunoreactivity in the striatum. Using selected conditions, an inhomogeneous distribution of the Kir2.3 subunit in the striatum becomes obvious (C). The pattern of reduced Kir2.3 staining seems similar to that of the striatal patch compartment as visualized via the immunocytochemical localization of µ-opiate receptors (D).
• Fig. 21: Striatal cell density. Adjacent sections stained according to Klüver-Barrera (A) and for the Kir2.3 subunit (B) demonstrate that the increased Kir2.3 immunoreactivity is not simply due to an increased density of nerve cells at this location (compare areas between the three arrowheads or between the four arrows). Note the identical localization of capillaries in (A) and (B).
• Fig. 22: Correspondence between the striatal patch compartments and areas with reduced Kir2.3 immunoreactivity. For direct correlation, areas of reduced Kir2.3 immunoreactivity (B,D) and the striatal patch compartment as visualized by µ-opiate receptor staining (A,C) are compared in adjacent sections. In (B,D) areas occupied by fiber bundles are hatched to avoid confusion. The patch compartment (as seen in A and C) is outlined in black to facilitate comparison.
• Fig. 23: Kir2.4 channel immunoreactivity is most prominent in large striatal interneurons. Immunoreactivity for the Kir2.4 subunit in the rat striatum is mostly somatodendritic and largely absent from the neuropil (A). Staining is weak, however, in the large population of small striatal neurons and prominent in only a few, very large cells (A, arrows; B), most likely representing the so-called ‘giant’ interneurons in the striatum. Immunoreactivity is abolished (C) subsequent to preincubation of anti-Kir2.4 antibodies with the cognate protein.
• Fig. 24: Immunofluorescence double labeling of large striatal interneurons for choline acetyltransferase (ChAT) and Kir2.4 immunoreactivity. Sections were treated simultaneously with monoclonal mouse anti-ChAT- and rabbit anti-Kir2.4-antibodies, followed by visualization with Oregon Green-labeled goat anti-mouse and Texas Red-labeled goat anti-rabbit secondary antibodies. The four ChAT-positive cholinergic neurons (A) express Kir2.4-immunoreactivity (B, arrowheads) as judged from their yellowish appearance (C) after double exposition for green and red fluorescence.
• Fig. 25: Double labeling of Kir2.4 and choline acetyltransferase at the electron microscopic level. Cholinergic interneurons are identified by their amorphous diaminobenzidine reaction product (A, arrows) in single- (A) or double-stained sections. Kir2 immunoreactivity is visualized by silver-intensified ultrasmall gold particles (for details see methods). Cholinergic interneurons display Kir2.2 (B) as well as Kir2.4 (C) immunoreactivity.
• Fig. 26: Ratios of patch neurons to matrix neurons across embryonic ages. Ratios above the solid horizontal line indicate a predominance of patch neuron birthdates and ratios below the line indicate a predominance of matrix neuron birthdates. Consequently, at the time of beginning Kir2.3 mRNA expression most of the neurons end up in the matrix compartment.