Matrix Metalloproteinase-9 Increases the Ligand Sensitivity of CNG Channels: Evidence for Sequential Subunit Modification

Abstract

Matrix metalloproteinases (MMPs) are a family of over 20 cell-surface and secreted, Zn2+- and Ca2+-dependent endopeptidases. Two closely related gelatinase MMPs, −9 and −2, have been implicated in both normal retinal processes and in several retinal diseases. MMPs are classically associated with tissue remodeling, but have been shown to influence ion channel function—either indirectly (e.g., through modification of the pericellular environment) or directly (e.g., by proteolysis of channel subunits). Because MMPs have been localized within the interphotoreceptor matrix, we hypothesized that MMP catalytic activity can influence the function of photoreceptor cyclic nucleotide-gated (CNG) channels. Cone and rod CNG channels were individually expressed in Xenopus oocytes and examined via patch-clamp recording in the inside-out configuration. Extracellular exposure to MMP-9 or −2 produced a profound increase in the efficacy of cAMP and the apparent cGMP affinity for both homomeric (CNGA3, K1/2,GMP, mean ± S.E.M: MMP960min = 0.62 μM ± 0.21; control60min = 12.6 μM ± 1.7) and heteromeric (CNGA3+CNGB3, K1/2,GMP: MMP980min = 2.2 μM ± 0.6; control80min = 16.4 μM ± 1.2) channel compositions. The gating changes required MMP-catalytic activity, exhibited complex state-dependence and were attenuated by N-glycosylation in the pore turret of CNGA subunits. Furthermore, MMP exposure resulted in decreased abundance of the full length CNGA3 subunit on immunoblots, and a concomitant increase in putative degradation products, consistent with proteolysis of core-channel subunits. Analyses of cGMP dose-response curves and current fluctuations suggest that MMPs generate channel subpopulations by sequentially modifying individual CNG channel subunits, where the apparent affinity for cGMP is governed by the total number of modified subunits (from 0 to 4). Together, these results provide evidence that MMPs can incrementally regulate the ligand sensitivity of CNG channels by proteolytic modification of CNGA subunits.