Research reportHeterogeneous expression of voltage-gated potassium channels of the shaker family (Kv1) in oligodendrocyte progenitors
Introduction
In excitable cells, such as neurones and muscle cells, voltage-gated potassium channels are essential elements for the generation of action potentials [3]. Moreover, these channels play an important role during development. Overexpression of voltage-gated K+ channels is shown to alter neuronal differentiation [22]. In glial cells, inhibition of delayed rectifying K+ currents due to the activation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA)/kainate receptors prevents lineage progression of oligodendrocyte progenitor cells 14, 31.
At least 11 main gene families of K+ channels have been molecularly characterized (Kv1 to Kv9, KvLQT and EAG) 9, 19, 45. Each family consists of several distinct members. The largest group is formed by the Kv1 channels originally cloned from the fruitfly Drosophila melanogaster[37]. The sequences of seven genes of this particular family have been published so far 9, 24. Localization at the protein and at the mRNA level has been determined by either immunohistochemistry or in situ hybridization. The members of this family are found in all regions of the brain at different levels of expression [55].
K+ channels are formed by tetrameric protein complexes delineating a central K+ ion selective pore. The existence of homomeric as well as heteromeric ion channels has been described 9, 44, 49. In addition, K+ channel complexes can associate with intracellularly localized proteins, β subunits, which can modulate the biophysical properties of the ion pore [39]. Electrophysiological and pharmacological properties of Kv1 channels have been investigated in several heterologous expression systems (for reviews, see Ref. [20]).
The oligodendrocyte lineage is characterized by several subsequent differentiation stages 12, 53. The most immature form are bipotential progenitor cells which can either develop into astrocytes or oligodendrocytes depending on growth factor conditions. After commitment to the oligodendrocyte lineage, immature oligodendrocytes are characterized by the expression of defined glycolipids. Mature oligodendrocytes express myelin-specific proteins like the myelin basic protein, the myelin-associated glycoprotein or the proteolipid protein. During lineage progression, cell morphology changes from a simple bi- or tripolar shape to a highly complex cell with several highly branched processes.
Progenitor cells of the oligodendrocyte lineage express outwardly rectifying K+ currents which have been characterized using biophysical and pharmacological tools both in vitro [52]as well as in situ [5]. In both preparations, cells displayed the same developmentally regulated expression of K+ currents during differentiation suggesting a dominating role of the lineage program over culture conditions. It has been shown that inhibition of these currents significantly reduces the proliferation rate 14, 31. Whereas progenitors express mainly delayed rectifying and A-type K+ currents, mature oligodendrocytes exhibit a high potassium conductance without strong voltage dependence 27, 51. Delayed rectifying K+ currents have also been described for the myelinating cells of the peripheral nervous system (PNS), the Schwann cells 57, 58.
The aim of this study was to (1) identify the expression pattern of shaker-type Kv1 genes in glial progenitors obtained from embryonic mouse brain and (2) to investigate how Kv1 channels contribute to the whole-cell conductance of progenitors. Expression of Kv1.1 to Kv1.6 genes was investigated at the mRNA level by use of single-cell reverse transcription–mediated polymerase chain reaction (RT-PCR) and at the protein level by use of immunocytochemistry with Kv1-subtype specific antibodies. Progenitors were identified by their expression of voltage-gated K+ currents as determined by patch-clamp recording in the whole-cell mode, by antigen expression and by their characteristic morphological features. A combination of voltage-step protocols during patch-clamp recording and application of selective K+ channel blockers was used to determine the contribution of individual Kv1 channels to the whole-cell conductance.
Section snippets
Cell culture
Primary glial cell cultures were obtained from 14 to 15 days old embryos of NMRI mice as described previously [54]. After 5 days in culture neurones were eliminated by complement-dependent immunocytolysis using the monoclonal antibody M5 directed against a neuronal surface antigen. After 4–8 days, loosely attached microglial cells were removed and, by vigorously shaking, glial progenitor cells were harvested. Cells were plated on a layer of astrocytes at coverslips coated with poly-l-lysine.
Results
The molecular identity of voltage-gated K+ channels of the Kv1-subfamily has been determined in cultured glial progenitor cells derived from embryonic mouse brain. Single-cell RT-PCR was applied to analyze the simultaneous activity of Kv1-channel genes. Kv1 channel proteins were visualized by immunocytochemistry using subtype-specific polyclonal antibodies. Whole-cell patch-clamp recordings and pharmacological tools were applied to investigate the contribution of K+ channel proteins to the
Discussion
Delayed rectifying K+ currents of glial progenitors in the CNS 5, 52and of Schwann cells in the PNS 57, 58have been examined in detail previously. The molecular identity, however, of K+ channel proteins which contribute to the whole cell conductance of CNS progenitor cells is poorly understood. So far, the Kv1 (shaker-type) family is the largest among all delayed rectifier K+ channel groups. This study describes the expression of the Kv1.1 to Kv1.6 channel family members in glial progenitors
Acknowledgements
The authors wish to thank Prof. Olaf Pongs, Zentrum für Molekulare Neurobiologie Hamburg, Germany, for stimulating discussions and providing Kv1 antibodies. The expert technical assistance of Sibylle Just and Brigitte Hunger and the contribution of Tobias Schmidt-Gönner in part of the patch-clamp analysis is gratefully acknowledged. We also thank Andrea Töppel and Christian Witt (Genetical Cardiology, MDC) for introducing the capillary electrophoresis technique. K.S. was supported by a
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