Research ReportDifferential distribution of individual subunits of strongly inwardly rectifying potassium channels (Kir2 family) in rat brain
Introduction
Inwardly rectifying potassium (Kir) channels modulate important cellular functions in excitable and nonexcitable cells such as regulation of cellular excitability, setting of membrane potential, and secretion of neurotransmitters and hormones. The 15 mammalian gene products related to the Kir family have been classified into seven subfamilies (Kir1 to Kir7), all sharing a typical architecture with intracellular N- and C-terminal domains and two transmembrane regions flanking an extracellular pore region. This postulated membrane topology has recently been verified by the analysis of the crystal structure of a bacterial Kir channel homolog [14].
Kir channels with the most pronounced inward rectification belong to the Kir2 family (Kir2.1–Kir2.4). The first three members of the Kir2 family show a widespread expression in many different tissues and cell types, whereas Kir2.4 seems to be specific for neuronal cells. The ubiquitous expression of Kir2.1 is highlighted by the finding that deleterious mutations in Kir2.1 result in Andersen's syndrome, an autosomal dominant disease characterized by cardiac arrhythmias, periodic paralysis, and dysmorphic features [23]. Diversity among functional Kir2 channels is enhanced by heteromerization within the Kir2 family [24], [27]. Functional differences within the Kir2 family are mainly reflected by divergent basic biophysical properties such as single channel conductance, rectification, or barium block [3], [16], [34] and differential regulations by phosphorylation [2] or by other cellular signals such as arachidonic acid [17] or phosphatidyl inositol phosphates [7]. Therefore, the properties of homo- and heteromeric Kir2.x channels may strongly depend on the cellular coexpression of Kir2 subunits [3], [16]. In addition, the subcellular localization of Kir2 channels is differentially regulated by interacting proteins, which is nicely exemplified by the fact that PDZ binding motifs are present in Kir2.1–Kir2.3 but missing in Kir2.4 [8], [32], [33].
In general, all four Kir2 channels are widely distributed throughout the brain. In situ hybridization data [1], [4], [6], [13], [19], [32] show expression of Kir2.1–Kir2.3 in dentate gyrus, caudate putamen, piriform cortex, and in the olfactory bulb. Kir2.2 is strongly expressed also in thalamic nuclei, cerebellum, and brainstem, whereas Kir2.4 mRNA is predominantly found in motoneurons of cranial nerve nuclei and to a much lesser extent in other brain regions [32], [33]. Based on immunocytochemical data, we have recently hypothesized that several members of the Kir2 family may represent promising targets for new pharmacological strategies to treat basal ganglia disorders like Parkinson's disease [25]. Namely, the Kir2.3 and Kir2.4 subunits are strongly expressed in the key positions for basal ganglia control.
So far, only few studies investigated the distribution of Kir2 proteins in the mammalian brain by using specific antibodies [2], [22], [26], [30]. Therefore, the present report aims at a detailed examination of Kir2 protein localization in regions and cells related to important physiological and clinical functions of the central nervous system.
Section snippets
Antibody production, affinity purification, and characterization
Antibody production was described in a previous report [25]. Briefly, C-terminal fragments for all Kir2 proteins were expressed in E. coli, purified, and used for immunization of rabbits. The serum was cleaned of IgM and cross reactivity against other Kir channel subunits, affinity-purified, and finally characterized using Western blot, competitive ELISA, cell transfection experiments, and immunocytochemistry.
Immunocytochemistry
Adult Wistar rats (n = 5) were deeply anesthetized with ether and perfused
Specificity of anti-Kir2 antibodies
In this study, sequence-specific antibodies directed against unique sequences of all four members of the Kir2 family are used to display the regional, cellular, and subcellular distribution of Kir2.1–Kir2.4 channels in the rat brain. In a recent report, the antibodies were extensively characterized using Western blot with absorption controls, competitive ELISA, cell transfection experiments, and immunocytochemical methods verifying the antibody specificity [25]. Moreover, specific staining
Discussion
Monospecific antibodies allowed the detailed analysis of Kir2 protein localization in the rat central nervous system. The widespread presence of all four Kir2 channel subunits in the rat brain may indicate their important role in central signal processing and neural transmission. In general, our data agree with in situ hybridization experiments, and electrophysiological studies, confirming the basic distribution patterns of Kir2.1, Kir2.2, and Kir2.3 subunits, however, show some differences to
Acknowledgments
This work was supported by a grant from the Deutsche Forschungsgemeinschaft (Ve187/1-3). We are indebted to Dr. A. Brune, Dr. D. Eulitz, Dr. A. Thomzig, and Sema Ünsal for technical help and discussions on the manuscript. In addition, we would like to thank A. Kaphahn for editorial help.
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