Large conductance calcium- and voltage-activated potassium (BK) channels are important to the normal function of vascular smooth muscle, where they are involved in a negative feedback mechanism to limit vasoconstriction and regulate blood pressure. BK channels in vascular smooth muscle are composed of four alpha pore forming subunits and four modulatory betal subunits. Betal knockout mice contain BK channels with reduced opening, increased vascular tone and hypertension. Previous studies suggest that betal alters channel gating mainly by two opposing effects. Betal reduces opening by increasing the energetic barrier for intrinsic gating, and promotes opening by stabilizing voltage sensor activation. The molecular basis underlying channel modulation by the betal subunit is unknown. Our preliminary experiments show that deletion of either of two small intracellular domains abolishes betal's ability to stabilize voltage sensor activation and increase channel opening. Our working hypothesis is that residues in these intracellular domains mediate interactions with BK channel alpha subunits that stabilize the voltage sensor activation and promote channel opening. The goal of this research is to identify critical residues within the betal intracellular domains that are required for stabilizing voltage sensor activation. The approach will involve generating deletions and point mutations within the betal intracellular domains. Mutant betal will be characterized by co-transfection with the alpha subunit in HEK293 cells and patch-clamp recording of currents through single BK channels, as well as multiple channels (macroscopic currents). Residues that are critical for channel modulation will be replaced with amino acids of different polarity, hydrophobicity and size to determine the nature of their interactions. The current dual-allosteric model for BK channel gating will serve as a framework for describing the effects of the mutant betal on the gating process. Our data will lead to a clearer picture of how the betal subunit modulates BK channel activity and may provide guidance in drug design directed toward enhancement of BK channel activities to treat hypertension. Relevance BK channels are important to the normal function of vascular smooth muscle, where they are involved in a negative feedback mechanism to limit vasoconstriction and regulate blood pressure. The goal of the proposed study is to understand how the betal subunit modulates BK channel activity. This information may provide guidance in drug design directed toward enhancement of BK channel activities to treat hypertension. ? ? ?
| Wang, Bin; Rothberg, Brad S; Brenner, Robert (2009) Mechanism of increased BK channel activation from a channel mutation that causes epilepsy. J Gen Physiol 133:283-94 |
| Seibold, Max A; Wang, Bin; Eng, Celeste et al. (2008) An african-specific functional polymorphism in KCNMB1 shows sex-specific association with asthma severity. Hum Mol Genet 17:2681-90 |
