The leucine-rich repeat domains of BK channel auxiliary γ subunits regulate their expression, trafficking, and channel-modulation functions

Guanxing Chen,Qin Li,Jiusheng Yan

doi:10.1016/j.jbc.2022.101664

Guanxing Chen, Qin Li + Show 1 more

Open Access

https://doi.org/10.1016/j.jbc.2022.101664

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Abstract

As high-conductance calcium- and voltage-dependent potassium channels, BK channels consist of pore-forming, voltage-, and Ca2+-sensing α and auxiliary subunits. The leucine-rich repeat (LRR) domain–containing auxiliary γ subunits potently modulate the voltage dependence of BK channel activation. Despite their dominant size in whole protein masses, the function of the LRR domain in BK channel γ subunits is unknown. We here investigated the function of these LRR domains in BK channel modulation by the auxiliary γ1–3 (LRRC26, LRRC52, and LRRC55) subunits. Using cell surface protein immunoprecipitation, we validated the predicted extracellular localization of the LRR domains. We then refined the structural models of mature proteins on the membrane via molecular dynamic simulations. By replacement of the LRR domain with extracellular regions or domains of non-LRR proteins, we found that the LRR domain is nonessential for the maximal channel-gating modulatory effect but is necessary for the all-or-none phenomenon of BK channel modulation by the γ1 subunit. Mutational and enzymatic blockade of N-glycosylation in the γ1–3 subunits resulted in a reduction or loss of BK channel modulation by γ subunits. Finally, by analyzing their expression in whole cells and on the plasma membrane, we found that blockade of N-glycosylation drastically reduced total expression of the γ2 subunit and the cell surface expression of the γ1 and γ3 subunits. We conclude that the LRR domains play key roles in the regulation of the expression, cell surface trafficking, and channel-modulation functions of the BK channel γ subunits.

Highlights

The large-conductance, voltage- and calcium-activated potassium (BK, or KCa1.1) channels are ubiquitously expressed and critically involved in various cellular and physiological processes, such as regulation of neuron firing and transmission [1, 2], motor coordination [3], learning and memory [4, 5], circadian rhythmicity [6, 7], the contractile tone of almost all types of smooth muscle cells [8, 9], and resting K+ efflux in
The four BK channel γ subunits are a group of leucine-rich repeat (LRR)–containing (LRRC) membrane proteins that consist of the γ1 (LRRC26), γ2 (LRRC52), γ3 (LRRC55), and γ4 (LRRC38) subunits [16, 17]
Structural models of BK channel γ subunits refined by molecular dynamic simulation The BK channel γ subunits each contain an N-terminal short hydrophobic segment, an LRR domain, a single TM segment, and a short C-terminal tail (Fig. S1)

Summary

Introduction

The large-conductance, voltage- and calcium-activated potassium (BK, or KCa1.1) channels are ubiquitously expressed and critically involved in various cellular and physiological processes, such as regulation of neuron firing and transmission [1, 2], motor coordination [3], learning and memory [4, 5], circadian rhythmicity [6, 7], the contractile tone of almost all types of smooth muscle cells [8, 9], and resting K+ efflux in. To determine the effect of N-glycosylation on the γ2 subunit’s function in BK channel regulation, we measured the voltage dependence of BK channel activation by patch-clamp recording of the BK channels on inside-out membrane patches of HEK-293 cells coexpressing BKα and the γ2 WT and glycosylation-null (N→Q) single-, double-, or triple-site mutants.

Results

Conclusion