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.

Mechanical activation of angiotensin II type 1 receptors causes actin remodelling and myogenic responsiveness in skeletal muscle arterioles

“…As described previously (Nourian et al . ; Lee et al . ), total RNA was extracted from 1A arterioles using the Arcturus PicoPure RNA isolation kit (Applied Biosystems, Carlsbad, CA, USA) and on‐column DNase I (Qiagen, Valencia, CA, USA) to minimize contamination from genomic DNA.…”

Smooth Muscle Ion Channels and Regulation of Vascular Tone in Resistance Arteries and Arterioles

“…Put in more physiological terms, the high Ca 2+ setpoint in cremasteric arteriolar muscle cells means that the internal face of the membrane of these cells must be exposed to Ca 2+ concentrations on the order of 3 µM for any activity of the channels to be observed at negative membrane potentials (665, 672). A high Ca 2+ setpoint (~12 µmol/L) has also been measured in SMCs isolated from rat first-order cremaster arterioles (1593), and appears to arise from reduced expression of β1 subunits in these arteriolar SMCs relative to cerebral arteries (1589, 1593) and possible differences in expression of spliced variants (1098). It was also shown that siRNA knockdown of β1 subunit expression in SMCs isolated from cerebral arteries produced a phenotype (increased Ca 2+ setpoint) similar to what was observed in cremaster arteriolar SMCs (1593).…”

Caveolar disruption causes contraction of rat femoral arteries via reduced basal NO release and subsequent closure of BKCachannels

“…In contrast to cerebral resistance arteries, some other arteries do not display this tight spark-STOC coupling. In particular, arteries supplying skeletal muscle have BK Ca channels that have lower Ca 2+ sensitivity and that are not activated by sparks, and this is thought to be due to lower expression of the β 1 subunit relative to the α subunit as well as a lower overall level of α -subunit expression ( Yang et al, 2009 ; Yang et al, 2013 ; Nourian et al, 2014 ). Decreased activity results in a smaller contribution of BK Ca channels to the cell membrane potential and this may in turn permit higher levels of vascular resistance in the skeletal muscle circulation ( Jackson & Blair, 1998 ; Hill et al, 2010 ).…”