Abstract
High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes. However, whether different partners have additive or cooperative functional effects, or whether some combinations of proteins may preclude assembly of others are largely unexplored topics. The large conductance Ca2+-and-voltage activated potassium channel (BK) is well-suited to discern nuanced differences in regulation arising from combinations of subunits. Here we examine whether assembly of two different classes of regulatory proteins, β and γ, in BK channels is exclusive or independent. Our results show that both γ1 and up to four β2-subunits can coexist in the same functional BK complex, with the gating shift caused by β2-subunits largely additive with that produced by the γ1-subunit(s). The multiplicity of β:γ combinations that can participate in a BK complex therefore allow a range of BK channels with distinct functional properties tuned by the specific stoichiometry of the contributing subunits.
Highlights
High resolution proteomics increasingly reveals that most native ion channels are assembled in macromolecular complexes
Using a b1 construct in which its N terminus was replaced by the b2-N terminus (b1/b2Nt) so that inactivation reports the presence of b1, we found that all BK channels resulting from coexpression of a þ b1/b2Nt þ g1 simultaneously contain both types of regulatory subunits (Supplementary Fig. 3)
The present work unambiguously shows that two different types of non-pore-forming regulatory subunits, b2 and g1, can coassemble in the same functional BK channel and independently regulate channel function
Summary
All errors are s.e.m. where the mean values of each component are defined by the slowest component, the tinact distribution is better fit by a fourcomponent than by a three-component Gaussian distribution (Fig. 3g,h, Supplementary Fig. 2), indicating that channels containing the g1-induced effect can contain one, two, three or four b2-subunits. The Vh arising from the simultaneous presence of g1 and b2DNt approximately reflects the sum of the independent effects of g1 and b2DNt alone These results indicate that, whatever the underlying molecular mechanism of the Vh shift produced by the g1-subunit, it is predominantly energetically independent of that produced by the b2-subunit. Using a b1 construct in which its N terminus was replaced by the b2-N terminus (b1/b2Nt) so that inactivation reports the presence of b1, we found that all BK channels resulting from coexpression of a þ b1/b2Nt þ g1 simultaneously contain both types of regulatory subunits (Supplementary Fig. 3)
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