Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes

Abstract

BackgroundTRPC6 is a nonselective cation channel, and mutations of this gene are associated with FSGS. These mutations are associated with TRPC6 current amplitude amplification and/or delay of the channel inactivation (gain-of-function phenotype). However, the mechanism of the gain-of-function in TRPC6 activity has not yet been clearly solved.MethodsWe performed electrophysiologic, biochemical, and biophysical experiments to elucidate the molecular mechanism underlying calmodulin (CaM)-mediated Ca2+-dependent inactivation (CDI) of TRPC6. To address the pathophysiologic contribution of CDI, we assessed the actin filament organization in cultured mouse podocytes.ResultsBoth lobes of CaM helped induce CDI. Moreover, CaM binding to the TRPC6 CaM-binding domain (CBD) was Ca2+-dependent and exhibited a 1:2 (CaM/CBD) stoichiometry. The TRPC6 coiled-coil assembly, which brought two CBDs into adequate proximity, was essential for CDI. Deletion of the coiled-coil slowed CDI of TRPC6, indicating that the coiled-coil assembly configures both lobes of CaM binding on two CBDs to induce normal CDI. The FSGS-associated TRPC6 mutations within the coiled-coil severely delayed CDI and often increased TRPC6 current amplitudes. In cultured mouse podocytes, FSGS-associated channels and CaM mutations led to sustained Ca2+ elevations and a disorganized cytoskeleton.ConclusionsThe gain-of-function mechanism found in FSGS-causing mutations in TRPC6 can be explained by impairments of the CDI, caused by disruptions of TRPC’s coiled-coil assembly which is essential for CaM binding. The resulting excess Ca2+ may contribute to structural damage in the podocytes.

TRPC channels: Structure, function, regulation and recent advances in small molecular probes

“…Likewise, receptor agonist-evoked TRPC3 current was strongly inhibited by the rise in [Ca 2+ ] c (Thyagarajan et al, 2001). The mechanisms for the Ca 2+ -dependent inhibition may involve both CaM-dependent and CaMindependent processes (Polat et al, 2019;Shi et al, 2004) with the IC 50 of Ca 2+ inhibition of TRPC7 being ~10 time lower than that of TRPC6. Single channel studies revealed that millimolar extracellular Ca 2+ also suppressed the unitary conductance of TRPC6 and C7 at negative potentials, again with sensitivity higher for TRPC7 than C6 (Shi et al, 2004).…”

“…Single channel studies revealed that millimolar extracellular Ca 2+ also suppressed the unitary conductance of TRPC6 and C7 at negative potentials, again with sensitivity higher for TRPC7 than C6 (Shi et al, 2004). A recent study indicates that CaM mediates Ca 2+ -dependent inactivation of TRPC6 by altering the assembly of the C-terminal coiled-coil domain, where gain-of-function TRPC6 mutations have been found from patients with focal segmental glomerulosclerosis (FSGS) (Polat et al, 2019). For TRPC4 and C5, Ca 2+ was shown to either inhibit channel activation or accelerate current inactivation at concentrations higher than that needed to support the channel opening (Gross et al, 2009;Ordaz et al, 2005;Thakur et al, 2016).…”

Association of Genetic Variants at TRPC6 With Chemotherapy-Related Heart Failure

“…Given the indirect nature of GWAS, the known association of rare missense variants in TRPC6 with the genetic form of chronic kidney disease, focal segmental glomerular sclerosis (FSGS) ( 24 , 25 ), and the fact that FSGS can result from exposure to anthracyclines ( 26 ), we sequenced the coding regions of TRPC6 in 38 patients with CRHF. None of the confirmed causative FSGS mutations were identified in patients with CRHF.…”

Role of Transient Receptor Potential Canonical Channel 6 (TRPC6) in Diabetic Kidney Disease by Regulating Podocyte Actin Cytoskeleton Rearrangement

“…It is caused by the impairment of the Ca 2+ -dependent inactivation, resulting from the disruptions of TRPC6's coiled-coil assembly. The excessive Ca 2+ may contribute to structural damage in the podocytes [31]. Systematic analysis of TRPC6-related mutations showed that loss-of-function (LOF) of TRPC6 has the potential to induce disease [32], which may cause FSGS earlier than in GOF-type TRPC6 patients by interacting with actin cytoskeleton rearrangement during podocyte development [33].…”