An investigation into the molecular identity of the KATP channel of vascular smooth muscle
thesisposted on 15.12.2014, 10:30 by Helen. Kuhlman
The molecular identity of the KATP channel of vascular smooth muscle was investigated using a variety of methods. RT-PCR was used to investigate the expression of KATP subunit mRNA. Kir6.1, 6.2, and SUR 2B transcripts were detected in all vascular smooth muscle preparations, SUR 1 was detected in mesenteric artery. Expression at the protein level was determined, in femoral artery cells, using an immunohistochemical technique, visualizing stained cells with confocal microscopy. Antibodies specific for Kir 6.1, 6.2 and SUR 2B, 2A, subunits were used and all resulted in staining. Using a Xenopus expression system cloned KATP channel currents were characterized. Differences in channel activation, via metabolic poisoning, were found to be dependent on the SUR subunit. The compound PNU-37883A was found to be selective for the Kir subunit, with channels comprising Kir 6.1 being more sensitive to inhibition by this compound than Kir 6.2, Kir 6.x chimeras were constructed to identify a possible site of modulation/binding and a region of the C-terminus was identified which was important in the channels response to the compound. The differences in sensitivity of channels with Kir 6.1 and 6.2 are similar to those reported for vascular smooth muscle and skeletal or cardiac muscle, providing further evidence that the Kir 6.1 subunit is involved in forming the KATP channel of vascular smooth muscle. However, the other evidence provided here doesn't rule out the possibility of heteromultimeric KATP channel formation in vascular smooth muscle.