Differential distribution of individual subunits of strongly inwardly rectifying potassium channels (Kir2 family) in rat brain

Abstract

Inwardly rectifying potassium (Kir) channels modulate cellular excitability, membrane potential, and secretion of neurotransmitters and hormones. Kir channels with the strongest inward rectification belong to the Kir2 family. In this report, polyclonal monospecific affinity-purified antibodies against the less conserved carboxy-terminal sequences of Kir2.1, Kir2.2, Kir2.3, and Kir2.4 were used to analyze the detailed distribution of all members of the Kir2 family in the rat central nervous system. Kir2 channel expression is detected in neurons but not in glial cells. Kir2 protein distribution confirms the basic mRNA localization pattern given by in situ hybridization. Kir2.1 is detected throughout the whole brain but in particular subsets of neurons with highest expression in olfactory bulb and superior colliculus. Kir2.2 immunoreactivity is primarily displayed in several forebrain nuclei, hypothalamus, cerebellum, and spinal cord. The Kir2.3 subunit is predominantly localized in olfactory bulb, basal ganglia, cortex, and cerebellar Purkinje cells. In contrast, Kir2.4-positive staining is detected at significantly lower levels in most neurons throughout the rat brain with highest expression in brainstem motoneurons. Thus, our data show a more widespread distribution of Kir2.4 than previously determined. In summary, the widespread presence of all four Kir2 channel subunits in the rat brain provides further evidence for their important role in central signal processing and neural transmission.

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.