Dataset Information

Glycosylation of β1 subunit plays a pivotal role in the toxin sensitivity and activation of BK channels.

ABSTRACT:

Background

The accessory β1 subunits, regulating the pharmacological and biophysical properties of BK channels, always undergo post-translational modifications, especially glycosylation. To date, it remains elusive whether the glycosylation contributes to the regulation of BK channels by β1 subunits.

Methods

Herein, we combined the electrophysiological approach with molecular mutations and biochemical manipulation to investigate the function roles of N-glycosylation in β1 subunits.

Results

The results show that deglycosylation of β1 subunits through double-site mutations (β1 N80A/N142A or β1 N80Q/N142Q) could significantly increase the inhibitory potency of iberiotoxin, a specific BK channel blocker. The deglycosylated channels also have a different sensitivity to martentoxin, another BK channel modulator with some remarkable effects as reported before. On the contrary to enhancing effects of martentoxin on glycosylated BK channels under the presence of cytoplasmic Ca²⁺, deglycosylated channels were not affected by the toxin. However, the deglycosylated channels were surprisingly inhibited by martentoxin under the absence of cytoplasmic Ca²⁺, while the glycosylated channels were not inhibited under this same condition. In addition, wild type BK (α+β1) channels treated with PNGase F also showed the same trend of pharmacological results to the mutants. Similar to this modulation of glycosylation on BK channel pharmacology, the deglycosylated forms of the channels were activated at a faster speed than the glycosylated ones. However, the V_1/2 and slope were not changed by the glycosylation.

Conclusion

The present study reveals that glycosylation is an indispensable determinant of the modulation of β1-subunit on BK channel pharmacology and its activation. The loss of glycosylation of β1 subunits could lead to the dysfunction of BK channel, resulting in a pathological state.

SUBMITTER: Wang X

PROVIDER: S-EPMC8183112 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Large-conductance (BK-type) Ca(2+)-activated potassium channels are activated by membrane depolarization and cytoplasmic Ca(2+). BK channels are expressed in a broad variety of cells and have a corresponding diversity in properties. Underlying much of the functional diversity is a family of four tissue-specific accessory subunits (beta1-beta4). Biophysical characterization has shown that the beta4 subunit confers properties of the so-called "type II" BK channel isotypes seen in brain. These properties include slow gating kinetics and resistance to iberiotoxin and charybdotoxin blockade. In addition, the beta4 subunit reduces the apparent voltage sensitivity of channel activation and has complex effects on apparent Ca(2+) sensitivity. Specifically, channel activity at low Ca(2+) is inhibited, while at high Ca(2+), activity is enhanced. The goal of this study is to understand the mechanism underlying beta4 subunit action in the context of a dual allosteric model for BK channel gating. We observed that beta4's most profound effect is a decrease in P(o) (at least 11-fold) in the absence of calcium binding and voltage sensor activation. However, beta4 promotes channel opening by increasing voltage dependence of P(o)-V relations at negative membrane potentials. In the context of the dual allosteric model for BK channels, we find these properties are explained by distinct and opposing actions of beta4 on BK channels. beta4 reduces channel opening by decreasing the intrinsic gating equilibrium (L(0)), and decreasing the allosteric coupling between calcium binding and voltage sensor activation (E). However, beta4 has a compensatory effect on channel opening following depolarization by shifting open channel voltage sensor activation (Vh(o)) to more negative membrane potentials. The consequence is that beta4 causes a net positive shift of the G-V relationship (relative to alpha subunit alone) at low calcium. At higher calcium, the contribution by Vh(o) and an increase in allosteric coupling to Ca(2+) binding (C) promotes a negative G-V shift of alpha+beta4 channels as compared to alpha subunits alone. This manner of modulation predicts that type II BK channels are downregulated by beta4 at resting voltages through effects on L(0). However, beta4 confers a compensatory effect on voltage sensor activation that increases channel opening during depolarization.