Abstract
The properties and function of large-conductance calcium- and voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory β1-subunits. Although the short cytosolic N-terminal domain of the β1-subunit is important for controlling both BK channel trafficking and function, whether the same, or different, regions of the N terminus control these distinct processes remains unknown. Here we demonstrate that the first six N-terminal residues including Lys-3, Lys-4, and Leu-5 are critical for controlling functional regulation, but not trafficking, of BK channels. This membrane-distal region has features of an amphipathic helix that is predicted to control the orientation of the first transmembrane-spanning domain (TM1) of the β1-subunit. In contrast, a membrane-proximal leucine residue (Leu-17) controls trafficking without affecting functional coupling, an effect that is in part dependent on controlling efficient endoplasmic reticulum exit of the pore-forming α-subunit. Thus cell surface trafficking and functional coupling with BK channels are controlled by distinct domains of the β1-subunit N terminus.
Highlights
The properties and function of large-conductance calciumand voltage-activated potassium (BK) channels are modified by the tissue-specific expression of regulatory 1-subunits
To address the role of the intracellular N terminus of the 1-subunit in enhancing the cell surface expression of the poreforming ␣-subunit, we used a cell surface imaging assay to investigate the co-expression of 1-subunit deletion and sitedirected mutants with a C-terminal myc (-myc) tag (1-mycc) and the BK channel ␣-subunit ZERO variant with an extracellular N-terminal FLAG (FLAG-) tag
␣-subunit expression following membrane permeabilization) in anti-myc-positive HEK293 cells, we found that wild-type 1-subunits significantly enhanced the cell membrane surface expression of ZERO by more than 2-fold (232.3 Ϯ 22.7%, n ϭ 7; Fig. 1, A–C) in comparison with that of ZERO alone (100 Ϯ 7.8%, n ϭ 8)
Summary
Membrane-proximal domain of 1-subunit N terminus is required for enhanced trafficking of BK ␣-subunit ZERO variant. With the 1-L5A-mycc subunit, BK channel activation and deactivation were still significantly slowed to 1.8 Ϯ 0.1 and 5.9 Ϯ 0.9 ms, respectively (n ϭ 10) These values were similar to the effect of wild-type 1 As predicted by the structural modeling, mutation of Val-6 to alanine (1-V6A-mycc) resulted in a significant left shift in V0.5max (to Ϫ29.7 Ϯ 5.6 mV, n ϭ 10), comparable with that observed with wild-type 1-mycc Taken together, these data reveal that distinct residues on the 1-subunit N terminus control trafficking (membrane-proximal Leu-17) and functional coupling (membranedistal Lys-3, Lys-4, and Leu-5), respectively
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