Kallure, G. S. | bioRxiv Science

bioRxiv2025

β2 and β3a regulatory subunits can coassemble in the same BK channels

Ca2+- and voltage-activated BK-type K+ channels are influenced profoundly by associated regulatory subunits, including {beta} subunits (Kcnmb1-4; {beta}1-{beta}4). Although overlap in expression of different BK {beta} subunits occurs in native tissues, whether they can coassemble in the same channel complex is not known. We coexpress {beta}2- and {beta}3a subunits together with BK and, through a combination of macroscopic and single channel recordings, along with quantitative pull-down of tagged subunits, test whether coassembly can occur. We evaluate two models: 1) random mixing in which {beta}2 and {beta}3a subunits co-assemble in the same channels, and 2) segregated in which {beta}2 and {beta}3a are found in separate complexes. Our results support the view that, for {beta}2 and {beta}3a, BK currents arise from the random, independent assembly of both subunits in the same channels. Single channel recordings directly confirm coassembly of {beta}2 and {beta}3a subunits in the same channels. Quantitative biochemical analysis of coexpression of tagged BK subunits also reveals that ternary complexes form.

biophysics↗

bioRxiv2023

High-resolution structures illuminate key principles underlying voltage and LRRC26 regulation of Slo1 channels

Multi-modal regulation of Slo1 channels by membrane voltage, intracellular calcium, and auxiliary subunits enables its pleiotropic physiological functions. Our understanding of how voltage impacts Slo1 conformational dynamics and the mechanisms by which auxiliary subunits, particularly of the LRRC (Leucine Rich Repeat containing) family of proteins, modulate its voltage gating remain unresolved. Here, we used single particle cryo-electron microscopy to determine structures of human Slo1 mutants which functionally stabilize the closed pore (F315A) or the activated voltage-sensor (R207A). Our structures, obtained under calcium-free conditions, reveal that a key step in voltage-sensing by Slo1 involves a rotameric flip of the voltage-sensing charges (R210 and R213) moving them by [~]6 [A] across a hydrophobic gasket. Next we obtained reconstructions of a complex of human Slo1 with the human LRRC26 ({gamma}1) subunit in absence of calcium. Together with extensive biochemical tests, we show that the extracellular domains of {gamma}1 form a ring of interlocked dominos that stabilizes the quaternary assembly of the complex and biases Slo1:{gamma}1 assembly towards high stoichiometric complexes. The transmembrane helix of {gamma}1 is kinked and tightly packed against the Slo1 voltage-sensor. We hypothesize that {gamma}1 subunits exert relatively small effects on early steps in voltage-gating but structurally stabilize non-S4 helices of Slo1 voltage-sensor which energetically facilitate conformational rearrangements that occur late in voltage stimulated transitions.

biophysics↗