title: Large conductance Ca2+-activated K+ channel (BKCa) α-subunit splice variants in resistance arteries from rat cerebral and skeletal muscle vasculature.
abstract: 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 α-subunit. BKCa variants in the intracellular C terminus of the α-subunit, and their relative expression to total α-subunit, were examined by qPCR. Sequencing of RT-PCR products showed two α-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 α-BKCa) compared with cerebral vessels (16.5±0.9%). Further, a low level of BKCa SS4 α-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 β1-BKCa subunit with STREX or ZERO did not influence the dominant membrane expression of α-BKCa subunits, whereas in the absence of α-BKCa, a significant proportion of β1-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 α-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 α-BKCa expression, regardless of differences in splice variant expression.
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Zahra
authors.initials: Z
authors.lastname: Nourian
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
authors.forename: M Dennis
authors.initials: MD
authors.lastname: Leo
authors.affiliation: Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
authors.forename: Jonathan H
authors.initials: JH
authors.lastname: Jaggar
authors.affiliation: Department of Physiology and Pharmacology, University of Calgary, Calgary, Alberta, Canada.
authors.forename: Andrew P
authors.initials: AP
authors.lastname: Braun
authors.affiliation: Dalton Cardiovascular Research Center, University of Missouri, Columbia, Missouri, United States of America; Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Missouri, United States of America.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Hill
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D002462:Cell Membrane / Q000378:metabolism; D002536:Cerebral Arteries / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D003600:Cytosol / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D051036:Large-Conductance Calcium-Activated Potassium Channels / Q000235:genetics / Q000378:metabolism*; D008297:Male; D008969:Molecular Sequence Data; D018482:Muscle, Skeletal / Q000098:blood supply; D009928:Organ Specificity; D020033:Protein Isoforms / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport; D051381:Rats; D017207:Rats, Sprague-Dawley
chemical_list: D051036:Large-Conductance Calcium-Activated Potassium Channels; D020033:Protein Isoforms; D021122:Protein Subunits