Title:
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Two-pore domain potassium channels in arterial smooth muscle
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The membrane potential of arterial smooth cells, and consequently arterial tone, is. regulated by the activity of plasmalemmal K+ selective ion channels. K2P channels are responsible for background K+ (KB) conductances which contribute to generation of the resting membrane potential. They have been identified in arterial smooth muscle cells but their significance is poorly understood. A Ks conductance is expressed in rat femoral arterial smooth muscle cells (rFASMCs), and the aim of this project was to (i) determine the expression profile of K2P subunits in rat femoral artery and (ii) identify the molecular correlate of the KB conductance in rFASMCs. Using RT-PCR, we identified transcripts encoding TWIK-2, TASK-I, TASK-2, TREK-I, TREK-2 and THIK-I in total rat femoral artery RNA. TWIK-2, TASK-I, TASK-2 and TREK-I were further identified at the protein level in isolated FASMCs by antibody staining. Only TWIK-2 was detected at the cell surface, while TASK-I, TASK-2 and TREK-I had a predominantly intracellular localisation. Immunohistochemistry of femoral artery sections may also support the expression of TWIK-2, TASK-I and TASK-2 (but not TREK-I) in the endothelium. To allow comparison between cloned and native conductances, the open reading frames of rTWIK-2, rTASK-2 and rTREK-I were cloned by PCR amplification. The sequence encoding rTASK-2 was cloned de novo and submitted to the EBI gene data base (Accession no: AM229406). Functional expression ofrTWIK-2 and rTASK-2 was investigated in Xenopus 'laevis oocytes. rTWIK-2 did not exhibit strong expression in the Xenopus laevis expression system. Conversely, recombinant rTASK-2 channels formed K+ selective, extracellular pH sensitive ion channels. The basic pharmacological properties of cloned rTASK-2 channels were also investigated. Currents were insensitive to extracellular TEA, 4-AP and Cs+, but were sensitive to inhibition by Ba2+, Zn2+and quinidine. Inhibition by extracellular Ba2+was strongly time and voltage-dependent. Ba2+ inhibition of rTASK-2 was characterised by steady-state analysis and voltage pulses. Although we were unable to describe the functional correlate of the KB conductance in rFASMCs, this thesis has established the groundwork for further correlative studies. The information provided will assist in the identification of native TASK-2 conductances.
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