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Arrhythmia-associated calmodulin variants interact with KCNQ1 to confer aberrant membrane trafficking and function.

ABSTRACT: Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates ("calmodulinopathy"). As CaM regulates many key cardiac ion channels, an understanding of disease mechanism associated with CaM variant arrhythmias requires elucidating individual CaM variant effects on distinct channels. One key CaM regulatory target is the KCNQ1 (K_V7.1) voltage-gated potassium channel that carries the I_Ks current. Yet, relatively little is known as to how CaM variants interact with KCNQ1 or affect its function. Here, we take a multipronged approach employing a live-cell fluorescence resonance energy transfer binding assay, fluorescence trafficking assay, and functional electrophysiology to characterize >10 arrhythmia-associated CaM variants for effect on KCNQ1 CaM binding, membrane trafficking, and channel function. We identify one variant (G114W) that exhibits severely weakened binding to KCNQ1 but find that most other CaM variants interact with similar binding affinity to KCNQ1 when compared with CaM wild-type over physiological Ca²⁺ ranges. We further identify several CaM variants that affect KCNQ1 and I_Ks membrane trafficking and/or baseline current activation kinetics, thereby delineating KCNQ1 dysfunction in calmodulinopathy. Lastly, we identify CaM variants with no effect on KCNQ1 function. This study provides extensive functional data that reveal how CaM variants contribute to creating a proarrhythmic substrate by causing abnormal KCNQ1 membrane trafficking and current conduction. We find that CaM variant regulation of KCNQ1 is not uniform with effects varying from benign to significant loss of function, suggesting how CaM variants predispose patients to arrhythmia via the dysregulation of multiple cardiac ion channels. Classification: Biological, Health, and Medical Sciences, Physiology.

SUBMITTER: Kang PW

PROVIDER: S-EPMC10642763 | biostudies-literature | 2023 Nov

REPOSITORIES: biostudies-literature

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Arrhythmia-associated calmodulin variants interact with KCNQ1 to confer aberrant membrane trafficking and function.

Kang Po Wei PW Woodbury Lucy L Angsutararux Paweorn P Sambare Namit N Shi Jingyi J Marras Martina M Abella Carlota C Bedi Anish A Zinn DeShawn D Cui Jianmin J Silva Jonathan R JR

PNAS nexus 20231014 11

Missense variants in calmodulin (CaM) predispose patients to arrhythmias associated with high mortality rates ("calmodulinopathy"). As CaM regulates many key cardiac ion channels, an understanding of disease mechanism associated with CaM variant arrhythmias requires elucidating individual CaM variant effects on distinct channels. One key CaM regulatory target is the KCNQ1 (K<sub>V</sub>7.1) voltage-gated potassium channel that carries the I<sub>Ks</sub> current. Yet, relatively little is known a ...[more]

PMID: 37965565

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Project description:As a member of the transient receptor potential (TRP) superfamily of ion channels, TRPV5 is a unique Ca2+-selective channel important for active reabsorption of Ca2+ in the kidney. TRPV5-mediated Ca2+ entry into the cell is controlled by a negative feedback mechanism, in which calmodulin (CaM) blocks the TRPV5 pore upon Ca2+ binding. Combining microscopy techniques and biochemical assays, the present study uncovered an auxiliary role for CaM in the regulation of human (h)TRPV5 intracellular trafficking. Overexpressed hTRPV5 was mainly localised to the endoplasmic reticulum (ER) and associated with peripheral ER tubules. Limiting expression using the HEK293 TET-off system revealed that hTRPV5 trafficked through the endocytic recycling pathway. CaM co-localised with hTRPV5 at intracellular sites and overexpression of CaM slowed hTRPV5 exit from the ER. In accordance, CaM binding-disrupting truncations of the TRPV5 C-terminus (698X) or knockdown of endogenous CaM by small interfering RNA resulted in an increased fraction of TRPV5 that localised to the plasma membrane. hTRPV5 expressing cells had an increased intracellular Ca2+ concentration upon knockdown of CaM. The protein abundance of the Ca2+ impermeable hTRPV5-D542 mutant is also regulated by CaM, which suggests that the mode of action is independent of disrupted intracellular calcium concentrations. In conclusion, our study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, modulating TRPV5 intracellular trafficking. KEY POINTS: The renal Ca2+ channel TRPV5 is a crucial player in maintenance of the body's Ca2+ homeostasis. Ca2+ transport through TRPV5 is controlled by single channel activity, as well as TRPV5 plasma membrane abundance. Calmodulin (CaM) co-localised with TRPV5 at intracellular sites and retained TRPV5 in the endoplasmic reticulum. Disrupted CaM-TRPV5 binding or knockdown of endogenous CaM by small interfering RNA (siRNA) resulted in an increased TRPV5 plasma membrane abundance. Knockdown of endogenous CaM by siRNA resulted in increased intracellular Ca2+ concentrations. The regulation of TRPV5 trafficking by CaM is independent of the effect of CaM on intracellular Ca2+ concentrations. This study reveals a novel role for CaM in Ca2+-dependent TRPV5 regulation, next to its ability to directly block the TRPV5 channel pore, by modulating TRPV5 trafficking in the secretory pathway.

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Arrhythmia-associated calmodulin variants interact with KCNQ1 to confer aberrant membrane trafficking and function.

Publications

Arrhythmia-associated calmodulin variants interact with KCNQ1 to confer aberrant membrane trafficking and function.

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