Distribution and expression of TREK-1, a two-pore-domain potassium channel, in the adult rat CNS
Section snippets
Animals
Adult male Wistar rats (200–250 g, Charles River, UK) were kept in a fixed 12-h/12-h light–dark cycle with food and water provided ad libitum. All procedures performed on animals during this study conformed to the UK Animals (Scientific Procedures) Act, 1986, and all efforts were made to minimise animal suffering and reduce the number of animals used in this study.
Chemicals
All chemicals were purchased from Merck (UK) unless otherwise stated. The bovine serum albumin (BSA) used in all experiments was of a
Western blot analysis
Under non-reducing conditions, affinity-purified TREK-1 antiserum detected a high-molecular-weight aggregate (100–500 kDa) in c-myc-tagged TREK-1-transfected cells (Fig. 1A, lanes b and c). The immunoreactivity was specific to transfected cells and not detected in untransfected wild-type cells (Fig. 1A, lanes a, d and f). The aggregate was partially resolved into a 56-kDa monomer and a 112-kDa dimer after treatment with either β-mercaptoethanol (Fig. 1A, lane g) or 8 M urea (Fig. 1A, lanes h and
Discussion
In these studies, we have used an antibody raised against a 17-amino-acid-containing peptide sequence that is present at the intracellular N-terminal of the human,29 rat (Chapman C. G., personal communication) and mouse TREK-1.8 Searching with this sequence in the GenEMBL database indicates that this 17-amino-acid sequence is unique to TREK-1. Western blots using cell lysates from human TREK-1-transfected cells were carried out in order to establish the specificity of the antibody. The blots
Conclusion
We have reported the distribution of TREK-1 potassium channel protein visualised with a polyclonal anti-peptide antibody. The distribution of TREK-1 potassium channels indicates that they are often expressed in GABAergic cells. The functional involvement of TREK-1 potassium channels in a number of neuronal systems, however, requires further investigations which will become more accessible once specific pharmacological modulators of these channels have been identified.
Acknowledgements
We would like to thank C. G. Chapman for information on the rat TREK-1 sequence.
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