TRPC6 G757D Loss-of-Function Mutation Associates with FSGS

Abstract

FSGS is a CKD with heavy proteinuria that eventually progresses to ESRD. Hereditary forms of FSGS have been linked to mutations in the transient receptor potential cation channel, subfamily C, member 6 (TRPC6) gene encoding a nonselective cation channel. Most of these TRPC6 mutations cause a gain-of-function phenotype, leading to calcium-triggered podocyte cell death, but the underlying molecular mechanisms are unclear. We studied the molecular effect of disease-related mutations using tridimensional in silico modeling of tetrameric TRPC6. Our results indicated that G757 is localized in a domain forming a TRPC6-TRPC6 interface and predicted that the amino acid exchange G757D causes local steric hindrance and disruption of the channel complex. Notably, functional characterization of model interface domain mutants suggested a loss-of-function phenotype. We then characterized 19 human FSGS-related TRPC6 mutations, the majority of which caused gain-of-function mutations. However, five mutations (N125S, L395A, G757D, L780P, and R895L) caused a loss-of-function phenotype. Coexpression of wild-type TRPC6 and TRPC6 G757D, mimicking heterozygosity observed in patients, revealed a dominant negative effect of TRPC6 G757D. Our comprehensive analysis of human disease-causing TRPC6 mutations reveals loss of TRPC6 function as an additional concept of hereditary FSGS and provides molecular insights into the mechanism responsible for the loss-of-function phenotype of TRPC6 G757D in humans.

Changes in podocyte TRPC channels evoked by plasma and sera from patients with recurrent FSGS and by putative glomerular permeability factors

“…This markedly expands the significance of observations made more than a decade ago that well-characterized genetic forms of FSGS occur as a result of mutations that lead to a gain of TRPC6 function [29–33] or a loss of functional podocin [25,27,28]. Podocin and TRPC6 are functionally linked in foot processes where they are endogenously co-localized.…”

“…FSGS associated with TRPC6 mutations often presents with an adult onset, with an autosomal dominant mode of inheritance, and the mutant proteins frequently have a gain of function or increased surface expression when examined in heterologous expression systems [29–31]. It should be noted, however, that loss of activation by at least some gating modes has been seen with some TRPC6 mutations [32,33], including a dominant-negative mutation that resulted in complete loss of activation by G protein cascades and that occurred with an unusually early disease onset [33]. …”

Molecular basis for the sensitivity of TRP channels to polyunsaturated fatty acids

“…After seeding, cells were transfected with 2 μg plasmid DNA coding for wild type (WT) or mutant TRPC isoforms using calcium phosphate, as previously described (Riehle et al 2016). In brief, the transfection mixture contained 2 μg DNA; 112 μl H 2 O; 125 μl 2× BBS buffer containing 50 mM BES, 280 mM NaCl, and 1.5 mM Na 2 HPO 4 ; and 12.5 μl CaCl 2 (2.5 M).…”

“…Statistical significance was determined by unpaired t test using Origin software (OriginLab, Northampton, MA, USA) (Riehle et al 2016). …”

Hereditary Podocytopathies in Adults: The Next Generation

“…TRPC6 is expressed at the podocyte slit diaphragm, where it interacts with podocin and nephrin [12] . Several gain-of-function missense mutations cause an increase in intracellular calcium influx [5,12] , whereas others result in a loss-offunction phenotype [24] . This discovery unexpectedly suggested the implication of calcium signalling in the pathogenesis of FSGS.…”