Unravelling the molecular mechanism of inactivation of BK channels by the novel regulatory subunit - LINGO1

Al Kawadri, Yaly (2024) Unravelling the molecular mechanism of inactivation of BK channels by the novel regulatory subunit - LINGO1. Doctoral thesis, Dundalk Institute of Technology.

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Abstract

BK channels are present in excitable and non-excitable cells found throughout the body (Di Resta and Becchetti, 2010; Latorre et al., 2017; Tao et al., 2017) and are responsible for controlling diverse functions including action potential repolarisation, neurotransmission and airway hydration (Jin et al., 2000; Bengtson et al., 2021). Functional channels are formed from four identical α-subunits and each subunit is comprised of three functional domains called the voltage sensing, Ca2+ sensing and pore gate domains (Zhang et al., 2022). The biophysical and pharmacological properties of these channels are affected by splice variants and the presence of β,  and LINGO auxiliary subunits (Meera et al., 1996; Latorre et al., 2017; Dudem et al., 2020). These regulatory LINGO subunits are also comprised of three distinct domains which are the extracellular (ED), transmembrane (TMD) and tail domain (TD) (Dudem et al., 2020;2023). The co-expression of LINGO1 or LINGO2 proteins with BK channels resulted in inactivating currents and a negative shift in the half maximal voltage of activation (V1/2) in 100 nM Ca2+ by 50 mV and 30 mV, respectively (Dudem et al., 2020; 2023). Interestingly, LINGO1 also reduced the plasmalemmal expression of BK channels by more than 90% (Dudem et al., 2020) but LINGO2 did not (Dudem et al., 2023). The differences between LINGO1 and LINGO2 were exploited via using a set of six chimeras to identify the domains responsible for altering BK channels properties. Molecular docking models and site-directed mutagenesis were employed to identify the residues important for the inactivation and determine their contribution to the inactivation process. The inside-out configuration of the patch clamp technique was used on HEK cells co-expressing BK channels with either LINGO1, LINGO2 or their chimeras and GFP at 37°C to study the impact of the chimeras and mutations on BK channels behaviour. The aims of this study were to ascertain if the reduction in BK channel plasmalemmal expression and the negative shift in the activation V1/2 were linked with a specific domain of LINGO1. Investigate the impact of a putative electrostatic interaction between 329RKK331 in the S6/RCK1 linker in BK and E594 and E596 in the 9 juxta-membrane region of LINGO1 TD on the inactivation. Moreover, assess the contribution of the last eight residues in the TD to the inactivation process. The findings of the first chapter linked the ED of LINGO1 with the reduction in the plasmalemmal expression of BK channels while associated the TD of LINGO1-2 with setting the activation V1/2 of BK in 1 μM Ca2+. This chapter identified chimera 211 (211) as a useful tool to study the effects of LINGO1 on BK channels, showing similar biophysical properties to LINGO1 without reducing BK channel plasmalemmal expression. The subsequent chapter showed that removal of the positive charge of 329RKK331 residues in the S6/RCK1 linker resulted in a large negative shift in the activation V1/2 of BK channels in the absence and presence of wild type (WT) or mutated 211. In addition, this chapter linked E594 and E596 in the juxta-membrane region of 211 with the negative shift in the activation V1/2 induced by LINGO1 in 100 nM Ca2+. The results of this chapter also demonstrated that neutralising 329RKK331 residues in the BK S6/RCK1 linker and E594:E596, in the juxta-membrane region of LINGO failed to abolish the stability of inactivation. The final chapter indicated that the absence of all positively charged residues in the distal C-terminus of LINGO1 (R613:K614:K618) practically abolished inactivation. This chapter also attributed the reduction in the rate, stability and the steady-state voltage-dependency of the inactivation process to K618A mutant. The alanine scan targeting the hydrophobic residues in the distal eight residues of 211 TD revealed the modest contributions of hydrophobic interactions shown only at negative potentials in higher Ca2+ concentrations. These findings help in clarifying the mechanism by which LINGO1 activates BK channels at more negative potentials and inhibits K+ permeation. Therefore, these insights also can enhance our understanding of diseases linked to BK channel dysfunction and impairment in the regulation of BK channels.

Item Type:	Thesis (Doctoral)
Subjects:	Science > Biology Science
Research Centres:	UNSPECIFIED
Depositing User:	Mark Hollywood
Date Deposited:	17 Jan 2025 11:22
Last Modified:	17 Jan 2025 11:22
License:	Creative Commons: Attribution-Noncommercial-Share Alike 4.0
URI:	https://eprints.dkit.ie/id/eprint/899

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