Biochemical and Biophysical Research Communications
Channel β2–4 subunits fail to substitute for β1 in sensitizing BK channels to lithocholate
Section snippets
Materials and methods
cRNA preparation and injection into Xenopus laevis oocytes. Full-length cDNA coding for cbv1 subunits (NCBI ID: AY330293) was cloned from freshly isolated rat cerebral artery myocytes as described [17], [18]. Cbv1 cDNA was cleaved from the cloning vector by BamHI (Invitrogen) and XhoI (Promega), and directly inserted into pOX for expression in Xenopus oocytes. pOX-cbv1 was linearized with NotI (Promega) and transcribed in vitro using T3 polymerase. BK β1 subunit cDNA inserted into the
Results and discussion
In order to study the effect of LC on recombinant BK channels of variant subunit composition, we first set to determine the phenotype of BK-mediated currents following injection of oocytes with BK channel-forming (cbv1) subunits with or without BK accessory (β1, β2, β3, or β4) proteins. Macroscopic currents were evoked from I/O patches by 200 ms-long, 10 mV depolarization steps from −150 to 150 mV (Vhold = 0 mV), with Ca2+free = 10 μM (Fig. 1A). As previously shown with other slo1 channels expressed in
Conclusion
Lithocholic acid-induced BK channels activation is conferred by the smooth muscle-abundant β1 subunit but not other β(2–4) subunits. Thus, LC and structural analogs represent a unique tool to probe the presence of functional β1-subunits and activate BK channel in tissues that highly express KCNMB1.
Acknowledgments
We deeply thank Maria Asuncion-Chin (UTHSC) and Aster Sigel (UCLA) for excellent technical assistance. This publication was made possible by NIH Grants R01 HL077424 (A.M.D.) and R01 HL054970 (L.T.). Its contents are solely the responsibility of the authors and do not necessary represent the official views of the NHLBI.
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