Dataset Information

The glycosylation of the extracellular loop of ?2 subunits diversifies functional phenotypes of BK Channels.

ABSTRACT: Large-conductance Ca2+- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming ? subunit (Slo) and 4 types of auxiliary ? subunits or just a pore-forming ? subunit. Although multiple N-linked glycosylation sites in the extracellular loop of ? subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp recordings, we demonstrated that 3 sites in the extracellular loop of ?2 subunit are N-glycosylated (N-X-T/S at N88, N96 and N119). Glycosylation of these sites strongly and differentially regulate gating kinetics, outward rectification, toxin sensitivity and physical association between the ? and ?2 subunits. We constructed a model and used molecular dynamics (MD) to simulate how the glycosylation facilitates the association of ?/?2 subunits and modulates the dimension of the extracellular cavum above the pore of the channel, ultimately to modify biophysical and pharmacological properties of BK channels. Our results suggest that N-glycosylation of ?2 subunits plays crucial roles in imparting functional heterogeneity of BK channels, and is potentially involved in the pathological phenotypes of carbohydrate metabolic diseases.

SUBMITTER: Huang ZG

PROVIDER: S-EPMC5398656 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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The glycosylation of the extracellular loop of β2 subunits diversifies functional phenotypes of BK Channels.

Huang Zhi-Gang ZG Liu Hao-Wen HW Yan Zhen-Zhen ZZ Wang Sheng S Wang Lu-Yang LY Ding Jiu-Ping JP

Channels (Austin, Tex.) 20161003 2

Large-conductance Ca<sup>2+</sup>- and voltage-activated potassium (MaxiK or BK) channels are composed of a pore-forming α subunit (Slo) and 4 types of auxiliary β subunits or just a pore-forming α subunit. Although multiple N-linked glycosylation sites in the extracellular loop of β subunits have been identified, very little is known about how glycosylation influences the structure and function of BK channels. Using a combination of site-directed mutagenesis, western blot and patch-clamp record ...[more]

PMID: 27690717

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Project description:Voltage/Ca²?(i)-gated, large conductance K+ (BK) channels result from tetrameric association of ? (slo1) subunits. In most tissues, BK protein complexes include regulatory ? subunits that contain two transmembrane domains (TM1, TM2), an extracellular loop, and two short intracellular termini. Four BK ? types have been identified, each presenting a rather selective tissue-specific expression profile. Thus, BK ? modifies current phenotype to suit physiology in a tissue-specific manner. The smooth muscle-abundant BK ?1 drastically increases the channel's apparent Ca²?(i) sensitivity. The resulting phenotype is critical for BK channel activity to increase in response to Ca2+ levels reached near the channel during depolarization-induced Ca2+ influx and myocyte contraction. The eventual BK channel activation generates outward K+ currents that drive the membrane potential in the negative direction and eventually counteract depolarization-induced Ca2+ influx. The BK ?1 regions responsible for the characteristic phenotype of ?1-containing BK channels remain to be identified. We used patch-clamp electrophysiology on channels resulting from the combination of smooth muscle slo1 (cbv1) subunits with smooth muscle-abundant ?1, neuron-abundant ?4, or chimeras constructed by swapping ?1 and ?4 regions, and determined the contribution of specific ?1 regions to the BK phenotype. At Ca2+ levels found near the channel during myocyte contraction (10 µM), channel complexes that included chimeras having both TMs from ?1 and the remaining regions ("background") from ?4 showed a phenotype (V(half), ?(act), ?(deact)) identical to that of complexes containing wt ?1. This phenotype could not be evoked by complexes that included chimeras combining either ?1 TM1 or ?1 TM2 with a ?4 background. Likewise, ? "halves" (each including ?1 TM1 or ?1 TM2) resulting from interrupting the continuity of the EC loop failed to render the normal phenotype, indicating that physical connection between ?1 TMs via the EC loop is also necessary for proper channel function.

Dataset Information

The glycosylation of the extracellular loop of ?2 subunits diversifies functional phenotypes of BK Channels.

Publications

The glycosylation of the extracellular loop of β2 subunits diversifies functional phenotypes of BK Channels.

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