Abstract
Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo1 gene. The variety of BK channels is correlated with the effects of BKα coexpression with auxiliary β (β1-β4) subunits, as well as newly defined γ subunits. Charybdotoxin (ChTX) blocks BK channel through physically occluding the K+-conduction pore. Human brain enriched β4 subunit (hβ4) alters the conductance-voltage curve, slows activation and deactivation time courses of BK channels. Its extracellular loop (hβ4-loop) specifically impedes ChTX to bind BK channel pore. However, the structure of β4 subunit’s extracellular loop and the molecular mechanism for gating kinetics, toxin sensitivity of BK channels regulated by β4 are still unclear. To address them, here, we first identified four disulfide bonds in hβ4-loop by mass spectroscopy and NMR techniques. Then we determined its three-dimensional solution structure, performed NMR titration and electrophysiological analysis, and found that residue Asn123 of β4 subunit regulated the gating and pharmacological characteristics of BK channel. Finally, by constructing structure models of BKα/β4 and thermodynamic double-mutant cycle analysis, we proposed that BKα subunit might interact with β4 subunit through the conserved residue Glu264(BKα) coupling with residue Asn123(β4).
Highlights
Large-conductance Ca2+- and voltage-dependent K+ (BK) channels display diverse biological functions while their pore-forming α subunit is coded by a single Slo[1] gene
Human β4 subunit is enriched in brain[1,2,7,8,9], which slows both activation and deactivation kinetics of BK channels[2,7], and causes negative shifts of the channels’ conductance-voltage (G-V) curves at high Ca2+ concentration (>10 μM), but it leads to positive shifts at a low Ca2+ concentration[10,11,12], suggesting that it plays a critical role in regulating neuronal excitability and neurotransmitter release[13]
Loop were known to form four disulfide bonds, which govern the topology of hβ4-loop, stabilize its conformation, and affect its biological functions[7]
Summary
Determination of disulfide bonds and solution structure of hβ4-loop. The eight cysteines of hβ4-. The chemical shifts of the Cβ atoms of residues Cys[54], Cys[68], Cys[72], Cys[76], Cys[84], Cys[113], Cys[119] and Cys[148] in hβ4-loop were assigned at 41.59 ppm, 42.32 ppm, 43.49 ppm, 39.69 ppm, 44.11 ppm, 44.40 ppm, 41.38 ppm and 44.92 ppm, respectively, indicating that all these cysteines form disulfide bonds. We further replaced Asn[123] with hydrophobic residues (Phe, Tyr, Leu, Val and Ile, group 2 mutations in Fig. 6c) and examined their effects with the application of 1 μM ChTX (Figs 6a and 7e–i) These mSlo1/β4 mutant channels display higher binding affinities to ChTX than mSlo1/β4 channel. Asn[123] in β4 subunit might interact with Glu[264] of BKα subunit through hydrogen-bond based on the properties of their side-chains in nature
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
