Potentiation of CNG channel gating by matrix metalloproteinases is governed by the turret region and intracellular disulfide bonds.

Abstract

Proteolytic modification of cyclic nucleotide gated (CNG) channel subunits by extracellular matrix metalloproteinases (MMP9 or MMP2) enhances the ligand sensitivity of native and recombinant channels. MMP modification of gating produced the sequential appearance of sub-populations having distinct ligand sensitivities, with a reduction in Hill slope consistent with emerging channel heterogeneity. Results demonstrated surprising state dependence: more rapid channel potentiation in sub-saturating ligand concentrations, compared to channels held open or held closed; and greater potentiation if channels were sequentially held closed then open, compared to open then closed. Channel potentiation generated by MMP9 activity was (1) reversed by intracellular DTT; (2) associated with high MW complexes on immunoblots, which were reduced by DTT; (3) absent for Cys-less CNGA1 (all cysteines mutated); (4) enhanced by a disease-associated mutation in CNGB3 (G558C); and (5) regulated by alternative splicing of CNGA3, which can toggle in/out a key cysteine within the N-terminal domain. In the absence of disulfide bonds, MMP9 activity instead reduced CNG channel ligand sensitivity. We have previously shown (Meighan et al., 2013) that CNGA subunit glycosylation can protect a sub-population of channels from MMP modification, but protection depends on the glycan site, which varies among paralogs and orthologs. We introduced an N339C mutation at the predicted cleavage site within the CNGA3 turret; MTSEA-biotin pretreatment of N339C attenuated MMP-dependent potentiation of gating. Furthermore, N339C channels exhibited increased susceptibility to modification by endogenous MMPs, producing subpopulations with different ligand sensitivities, which was prevented by MMP2/9 inhibitor. Together, these results suggest that CNG channel modification in the context of extracellular MMP activity is a multi-step process: sequential subunit proteolysis dependent on the extracellular turret (closed state) antecedent to potentiation resulting from cytoplasmic disulfide bond formation (open state).