Long QT Syndrome-Associated Calmodulin Mutations and Their Interactions with the Kv7.1 Voltage-Gated Potassium Channel


McCormick, Liam ORCID: 0000-0001-9557-9403 (2022) Long QT Syndrome-Associated Calmodulin Mutations and Their Interactions with the Kv7.1 Voltage-Gated Potassium Channel. PhD thesis, University of Liverpool.

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Abstract

Calmodulin (CaM) is a highly conserved mediator of calcium (Ca2+) dependent signalling and modulates various cardiac ion channels. Genotyping has revealed several CaM mutations associated with long QT syndrome (LQTS). LQTS patients display prolonged ventricular recovery times (QT-interval), increasing their risk of incurring life-threatening cardiac events. Loss of function mutations to KV7.1 (producer of the slow delayed rectifier potassium current, IKs, a key ventricular repolarising current) are the largest contributor to congenital LQTS (>50 % of cases). CaM modulates KV7.1 to produce a Ca2+-sensitive IKs, KV7.1 mutations which reduce CaM binding contribute to LQTS pathology, but never have the consequences of LQTS-associated CaM mutations been determined on KV7.1 function. Here, novel data is presented characterising the biophysical and modulatory properties of four LQTS-associated C-lobe CaM mutants (D96V, N98I, D132H and D134H). In their Ca2+-free (apo) states, mutants display subtle structural differences compared to wild-type (WT) CaM, when Ca2+-bound, mutant conformations become more distinct (circular dichroism and HSQC NMR). CaM mutations reduce C-lobe Ca2+-binding affinity and protein stability (thermostability and proteolytic susceptibility). Mutant CaM proteins display reduced affinities for their isolated, C-terminal KV7.1-binding sites (helix A and B; HA & HB) and adopt distinct conformations to WT CaM when target-bound (isothermal titration calorimetry and HSQC NMR). Reductions in HB affinity are more substantial in Ca2+-saturating than apo conditions, reflecting the more distinct calcified structures of LQTS-CaM proteins. Using patch-clamp electrophysiology, modulatory effects of LQTS-CaM mutants on IKs were determined in transfected HEK293T cells expressing channel (KCNQ1 and KCNE1) and CaM proteins. WT-CaM modulated channels generate larger IKs in response to increased cytosolic Ca2+. All mutants reduce current density at systolic Ca2+-concentrations (1000 nM), within physiological time periods (0.35 s), indicating a direct QT-prolonging modulatory effect. The work presented in this thesis describe disruptions to the structure-function relationships of LQTS-associated CaM mutants. For the first time, structural perturbations to CaM have been shown to impede complex formation with KV7.1 and subsequently result in reduced IKs which is aberrantly regulated by changes in Ca2+. This provides insight into how CaM mutations may exacerbate the dominant cause of LQTS (reduced IKs), and promotes further study into the ever-growing library of CaM mutations identified in LQTS patients.

Item Type: Thesis (PhD)
Divisions: Faculty of Health and Life Sciences
Depositing User: Symplectic Admin
Date Deposited: 25 Aug 2023 12:16
Last Modified: 25 Aug 2023 12:16
DOI: 10.17638/03163426
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URI: https://livrepository.liverpool.ac.uk/id/eprint/3163426
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