期刊
PLOS ONE
卷 9, 期 6, 页码 -出版社
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0098863
关键词
-
资金
- National Institutes of Health [RO1 HL092241, P01 HL095486]
- Natural Sciences and Engineering Research Council of Canada [RGPIN/312240]
Previous studies report functional differences in large conductance Ca2+ activated-K+ channels (BKCa) of smooth muscle cells (VSMC) from rat cerebral and cremaster muscle resistance arteries. The present studies aimed to determine if this complexity in BKCa activity may, in part, be due to splice variants in the pore-forming alpha-subunit. BKCa variants in the intracellular C terminus of the alpha-subunit, and their relative expression to total alpha-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two alpha-subunit variants, ZERO and STREX, to be identical in cremaster and cerebral arteries. Levels of STREX mRNA expression were, however, significantly higher in cremaster VSMCs (28.9 +/- 4.2% of total alpha-BKCa) compared with cerebral vessels (16.5 +/- 0.9%). Further, a low level of BKCa SS4 alpha-subunit variant was seen in cerebral arteries, while undetectable in cremaster arteries. Protein biotinylation assays, in expression systems and arterial preparations, were used to determine whether differences in splice variant mRNA expression affect surface membrane/cytosolic location of the channel. In AD-293 and CHO-K1 cells, rat STREX was more likely to be located at the plasma membrane compared to ZERO, although the great majority of channel protein was in the membrane in both cases. Co-expression of beta 1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of alpha-BKCa subunits, whereas in the absence of alpha-BKCa, a significant proportion of beta 1-subunit remained cytosolic. Biotinylation assays of cremaster and cerebral arteries showed that differences in STREX/ZERO expression do not alter membrane/cytosolic distribution of the channel under basal conditions. These data, however, revealed that the amount of alpha-BKCa in cerebral arteries is approximately 20X higher than in cremaster vessels. Thus, the data support the major functional differences in BKCa activity in cremaster, as compared to cerebral VSMCs, being related to total alpha-BKCa expression, regardless of differences in splice variant expression.
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