BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site

Chen, Guanxing; Li, Qin; Webb, Tim; Hollywood, Mark A.; Yan, Jiusheng

doi:10.1085/jgp.202213237

Cited by 2 publications

(2 citation statements)

References 38 publications

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“…For instance, the γ1 subunit is thought to profoundly affect voltage-gating with relatively modest effects on calcium regulation 14,33 while β1 exerts robust effects on calcium-regulation [34][35][36] but has relatively smaller effects on voltage-gating.…”

Section: Introductionmentioning

confidence: 99%

“…Deconvolving voltage and calcium dependent structural changes of Slo1 will be pivotal to not only obtain a more granular view of its allosteric gating, but they will also be crucial to understand how the auxiliary subunits affect gating transitions linked to one or both stimuli. For instance, the g1 subunit is thought to profoundly affect voltage-gating with relatively modest effects on calcium regulation 14,33 while b1 exerts robust effects on calcium-regulation 34–36 but has relatively smaller effects on voltage-gating.…”

Section: Introductionmentioning

confidence: 99%

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

Kallure,

Pal,

Zhou

et al. 2023

Preprint

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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 Å across a hydrophobic gasket. Next we obtained reconstructions of a complex of human Slo1 with the human LRRC26 (γ1) subunit in absence of calcium. Together with extensive biochemical tests, we show that the extracellular domains of γ1 form a ring of interlocked dominos that stabilizes the quaternary assembly of the complex and biases Slo1:γ1 assembly towards high stoichiometric complexes. The transmembrane helix of γ1 is kinked and tightly packed against the Slo1 voltage-sensor. We hypothesize that γ1 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.

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Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

Kallure,

Pal,

Zhou

et al. 2023

Preprint

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Concatenated tandem constructs of BK channels reveal three distinct types of subunit stoichiometry in channel modulation by the auxiliary γ subunit and mutations in the channel pore region

Chen

Yan

2023

Preprint

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The large-conductance, Ca2+-activated K+ (BK) channels consist of the Ca2+- and voltage-sensing and pore-forming α subunits and regulatory auxiliary β or γ subunits. Concatenated tandem constructs have been proved to be necessary and powerful in understanding the subunit stoichiometry of K+ channel gating and regulation by constraining the stoichiometry, organization, and mutation of individual subunits. However, because of its unique possession of an extra S0 transmembrane segment that puts the N- and C-termini on the opposite sides of the membrane, it is impossible to generate concatenated tandem subunit constructs of BK channels as those done with other K+ channels. In order to investigate the molecular mechanisms of the atypical all-or-none modulatory function of the γ subunits and the subunit stoichiometry of BK channel gating, we have taken a novel strategy and generated functional concatenated tandem constructs containing 2 or 4 copies of the BKα subunits by splicing BKα into 2 protein constructs that can be co-expressed to form functional channels. We observed that the BK channels formed by concatenated tandem constructs are similar to intact channels in voltage and Ca2+ gating properties. Interestingly, the BKγ1 subunit and mutations located at the S6 transmembrane segment and selectivity filter displayed 3 distinct types of subunit stoichiometry in BK channel modulation. By fusion of the BKγ1 subunit to the N-terminus of the concatenated tandem BKα constructs, we observed that a single molecule of BKγ1 subunit per tetrameric BKα channel is sufficient to fully modulate the channels. The Y294K, F303A, and L312A mutations at each BKα subunit has an incremental effect on BK channel gating. The V288A mutation at the selectivity filter produce an all-or-none effect but requires mutations at all 4 BKα subunits to have modulatory effect on channel gating, i.e., mutations at 1, 2, or 3 subunits have nearly no effect. Interestingly, the F307A mutation exhibited an atypical all-or-none modulatory function similar to that of the γ1 subunit. The diversity in subunit stoichiometry of BK channel modulation indicates distinct mechanisms in affecting BK channel gating. Overall, we have developed novel concatenated tandem constructs for BK channels and revealed three distinct types of subunit stoichiometry in BK channel modulation by the auxiliary γ subunit and mutations at the pore-lining S6 transmembrane segment and selectivity filter.

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BK channel modulation by positively charged peptides and auxiliary γ subunits mediated by the Ca2+-bowl site

Cited by 2 publications

References 38 publications

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

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

Concatenated tandem constructs of BK channels reveal three distinct types of subunit stoichiometry in channel modulation by the auxiliary γ subunit and mutations in the channel pore region

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