Premium
Manipulation of smooth muscle BK Ca using subunit directed siRNA
Author(s) -
Yang Yan,
Ella Srikanth R,
Braun Andrew P,
Korthuis Ronald J,
Davis Michael J,
Hill Michael A
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.777.12
Subject(s) - protein subunit , transfection , gene knockdown , vascular smooth muscle , small interfering rna , chemistry , microbiology and biotechnology , biophysics , biology , endocrinology , biochemistry , smooth muscle , gene
Cremaster and cerebral vascular smooth muscle cells (VSMC) exhibit heterogeneity in large conductance Ca 2+ ‐activated K + channels (BK Ca ) partly due to differences in β1:α subunit ratio. To gain insight into BK Ca methods were developed for subunit‐specific knockdown of the channel. Using transient transfection approaches and small interfering RNAs (siRNA) either the α or β1 subunit was targeted in isolated arterioles. Control studies used fluorescently labeled siRNA or unrelated siRNA. After 2–3 days culture fluorescence images and whole cell K + currents were examined in dispersed VSMC. From functional data α‐subunit expression was reduced by ~60% in both vessels. Thus, at +70 mV, IBTX‐sensitive K + current density was significantly reduced after α‐subunit siRNA compared to control. Similarly, STOC frequency (at +20 mV) decreased following siRNA treatment while BK Ca opening by NS1619 or estrogen (E2) was decreased. Cells treated with β1‐subunit siRNA showed impaired responses to E2 with a greater effect in cerebral VSMCs compared to those of cremaster. Thus transient transfection and siRNA can be used to effectively decrease endogenous BK Ca activity in intact small arteries. Further, cerebral VSMCs treated with β1‐subunit siRNA exhibit a functional phenotype similar to untreated cremaster VSMCs, supporting the idea that differences in β1:α subunit ratio underlie observed heterogeneity in BK Ca activity.