Involvement of gamma‐adducin in the regulation of cytoskeleton and membrane trafficking of the BK channel in rat cerebrovascular smooth muscle cells
Authors: Wang Shaoxun , Fan Fan , Pabbidi Mallikarjuna R. , Li Longyang , Mims Paige N. , Zhang Chao , Roman Richard J. 
Published: November 2023

We recently identified a region on chromosome 1 containing 15 genes, including gamma‐adducin (Add3), that causes the impaired myogenic response of the middle cerebral artery (MCA) and autoregulation of cerebral blood flow (CBF) in Fawn Hood Hypertensive (FHH) rats. We also found an inactivating K572Q mutation in FHH rats in the Add3 gene in this region. In addition, we have reported that the activity of the large‐conductance calcium‐activated potassium channel (BK) is elevated in this strain. In the present studies, we examined whether Add3 plays a role in the regulation of cerebral vascular function and explored the mechanisms involved. The expression of Add3 is markedly reduced in cerebral vessels of FHH vs. FHH.Add3 transgenic rats and FHH.1BN congenic rats in which the small region of the 15 genes contained chromosome 1 from BN rats was transferred into the FHH background. A newly designed 27‐mer Add3 DsiRNA blocked the expression of the Add3 protein in a HEK293 cell line that was stably transfected with BKα. The potassium currents were 3‐fold elevated in the primary smooth muscle cells (SMC) isolated from the cerebral vessels of FHH rats in comparison to those from FHH.Add3 and FHH.1BN rats, and in Add3 DsiRNA treated HEK293 BKα cells, as well as in a rat aorta smooth muscle cell (RASM) line that was transfected with Add3 DsiRNA. The high potassium currents were blocked by IBTX, a BK channel inhibitor. The expression of the BK α‐subunit was enhanced in the membrane of primary cerebral vascular SMCs isolated from FHH rats in comparison to from FHH.Add3 and FHH.1BN rats by western blot. A stronger fluorescence at the membrane in these cells was also detected by immunocytochemistry. The expression of p‐PKC, a potential transducer of the signaling cascade leading to myosin light chain (MLC) phosphorylation, was significantly reduced in cerebral vessels of FHH vs. FHH.1BN rats. The structure of F‐actin was disrupted in RASM cells treated with Add3 DsiRNA and in the primary cerebrovascular SMCs isolated from FHH rats in comparison to untreated RASM cells and SMCs of FHH.Add3 and FHH.1BN rats. Irregular, punctate and polygonal F‐actin networks were detected in the primary cerebrovascular SMCs of FHH rats, which has been referred to as cross‐linked actin networks (CLANs) that may affect vascular elastic behavior by modulation of the phosphorylation of MLC. In summary, these results suggest that Add3 is involved in membrane trafficking of the BK channel and in regulation of cytoskeletal activity by modulation of F‐actin structure and phosphorylation of MLC via PKC signaling pathway in rat cerebrovascular smooth muscle cells. It supports our hypothesis that Add3 is a viable candidate gene that may play a causal role in the impaired myogenic response of MCA and autoregulation of CBF in FHH rats and contributes to the development of cerebral vascular injury.

Support or Funding Information

This study was supported by grants HL36279 (RJR) and DK104184 (RJR), AG050049 (FF), P20GM104357 (RJR and FF) from the National Institutes of Health; 16GRNT31200036 (FF) from the American Heart Association. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.