Modulation of BK channels by the novel auxiliary subunit, LINGO2

BOON, PEI XIN (2024) Modulation of BK channels by the novel auxiliary subunit, LINGO2. Doctoral thesis, Dundalk Institute of Technology.

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Abstract

Large conductance Ca2+ activated K+ (BK) channels are ubiquitously expressed transmembrane proteins that govern smooth muscle and neuronal excitability (Latorre et al., 2010; Τao et al., 2017). They are activated by both Ca2+ and cellular depolarisation. The biophysical and pharmacological properties of the channels can be fine-tuned by their regulatory subunits, β1-4, γ1-4 and LINGO1 (Solaro and Lingle, 1992; Knaus et al., 1994; Xia et al., 1999; Gonzalez-Perez and Lingle, 2019; Dudem et al., 2020). The aims of this thesis were as follows: 1.TocharacterisethebiophysicalpropertiesofBK:LINGO2currents. 2. To investigate the effects of oxidation on BK:LINGO2 currents and determine the residues responsible for these effects. 3.ToidentifytheresiduesintheLINGO2tailresponsibleforinactivation. 4. To study the role of non-conserved and conserved charged residues in the cytosolic tail of LINGO2. 5. To examine the contribution of phenylalanine residues positioned in the transmembrane of LINGO2. HEK cells were transiently co-transfected with BK, LINGO2 and GFP cDNA (100 ng:500 ng:150 ng). Inside out patches were studied at 37oC, under voltage clamp, using the patch clamp technique with equimolar K+ pipette solutions. Site-directed mutagenesis were carried out to study the role of specific amino acids located in the transmembrane and cytosolic tail of LINGO2. A docking model of LINGO2 with BK was utilised to identify the potential binding partner for several LINGO2 residues in BK channels. The main findings of this thesis are: 1. LINGO2isanovelauxiliarysubunitofBKchannels. 2. BK:LINGO2 currents can be modulated by redox reactions and oxidation of M603 and M605 in the C-terminus of LINGO2 tail inhibits the inactivation. 3.TheconservednegativelychargedaminoacidsinLINGO2tailcontributetothe voltage-dependent activation in BK:LINGO2 channels. 4.Mutationoffourphenylalanineresidues(F550,F552,F558&F560)toalanine in the TM domain of LINGO2 reduced the negative shift in V1/2, suggesting that these residues are important in mediating the effects of LINGO2 on activation V1/2. 5.TheF552AmutantintheLINGO2TMregionabolishedinactivation,suggesting that this residue plays a critical role in positioning the LINGO2 tail to permit inactivation.

Item Type:	Thesis (Doctoral)
Subjects:	Science > Biology Science
Research Centres:	UNSPECIFIED
Depositing User:	Mark Hollywood
Date Deposited:	15 Jul 2024 12:01
Last Modified:	15 Jul 2024 12:01
License:	Creative Commons: Attribution-Noncommercial-Share Alike 4.0
URI:	https://eprints.dkit.ie/id/eprint/885

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