LINGO proteins- novel inactivating regulatory subunits of BK channels

Kulkarni, Shruti G (0026) LINGO proteins- novel inactivating regulatory subunits of BK channels. Doctoral thesis, Dundalk Institute of Technology.

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Abstract

Large-conductance, voltage- and Ca2+-regulated potassium (BK) channels are widely expressed across several tissues including smooth muscle, brain, kidney and thymus (Butler et al. 1993; Garcia-Calvo et al. 1994; Pallanck and Ganetzky 1994). These channels consist of tetrameric subunits and associated auxiliary or subunits, which can modulate their biophysical and pharmacological properties (Orio et al. 2002; Gessner et al. 2005; Latorre et al. 2017). Since the four subunits are Leucine Rich Repeat (LRR) proteins, we examined if other LRR proteins such as LRR and Ig domain-containing, Nogo Receptor-interacting protein, LINGO1, and its subtypes modulated BK channels. The aim of this study was to investigate the interacting partners between BK and LINGO proteins. In order to achieve this, we 1) examined the effects of the LINGO tail peptides on the BK channels, 2) investigated the effect of LINGO1 tail residues on BK channel inactivation, 3) elucidated the role of net positive charge and positively charged residues in LINGO1 tail peptide- mediated inactivation of BK channels. Patch-clamp experiments in the inside-out configuration were performed with LINGO tail peptides to determine their effects on BK channels. Site-directed mutagenesis was used to examine if the BK pore mutants altered the interaction with LINGO1 tail peptide. Additionally, the transcriptional expression of the LINGO subtypes in murine bronchial tissue and brain was examined using RT-PCR. The key findings of my study are: 1. mRNA expression for all four LINGO subtypes was detected in murine airway and brain. 2. The synthetic tail peptides of LINGO1 and LINGO2 inactivated BK currents. However, LINGO4 tail peptide blocked but did not inactivate BK currents. 3. The net positive charge and the position of positive charges in the LINGO1 tail peptide affected its ability to inactivate BK channels. 8 4. LINGO1 tail peptide appears to insert itself into the pore of the BK channel and may compete with quaternary ammonium ions for a common binding site. 5. The positively charged residues of LINGO1 tail peptide may interact with negatively charged residues in the S6 helix of the BK channel. In conclusion, the results of this thesis indicated that the last 8-residues of LINGO1 protein mimic the inactivation of the full-length LINGO1 protein, co- expressed with BK . Furthermore, these data suggest that LINGO peptides interact with BK channels and inactivation may occur by a mechanism involving charged and uncharged residues of the BK channel pore.

Item Type:	Thesis (Doctoral)
Subjects:	Science > Biology Science
Research Centres:	UNSPECIFIED
Depositing User:	Mark Hollywood
Date Deposited:	01 Sep 2022 09:00
Last Modified:	01 Sep 2022 09:00
License:	Creative Commons: Attribution-Noncommercial-Share Alike 4.0
URI:	https://eprints.dkit.ie/id/eprint/791

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