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Download fileEnergetic and Structural Basis for Activation of the Epithelial Sodium Channel by Matriptase
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posted on 24.04.2012, 00:00 authored by Pradeep Kota, Agustin García-Caballero, Hong Dang, Martina Gentzsch, M. Jackson Stutts, Nikolay V. DokholyanLimited proteolysis, accomplished by endopeptidases,
is a ubiquitous
phenomenon underlying the regulation and activation of many enzymes,
receptors, and other proteins synthesized as inactive precursors.
Serine proteases make up one of the largest and most conserved families
of endopeptidases involved in diverse cellular activities, including
wound healing, blood coagulation, and immune responses. Heteromeric
α,β,γ-epithelial sodium channels (ENaC) associated
with diseases like cystic fibrosis and Liddle’s syndrome are
irreversibly stimulated by membrane-anchored proteases (MAPs) and
furin-like convertases. Matriptase/channel activating protease-3 (CAP3)
is one of the several MAPs that potently activate ENaC. Despite identification
of protease cleavage sites, the basis for the enhanced susceptibility
of α- and γ-ENaC to proteases remains elusive. Here, we
elucidate the energetic and structural bases for activation of ENaC
by CAP3. We find a region near the γ-ENaC furin site that has
previously not been identified as a critical cleavage site for CAP3-mediated
stimulation. We also report that CAP3 mediates cleavage of ENaC at
basic residues downstream of the furin site. Our results indicate
that surface proteases alone are sufficient to fully activate uncleaved
ENaC and explain how ENaC in epithelia expressing surface-active proteases
can appear refractory to soluble proteases. Our results support a
model in which proteases prime ENaC for activation by cleaving at
the furin site, and cleavage at downstream sites is accomplished by
membrane surface proteases or extracellular soluble proteases. On
the basis of our results, we propose a dynamics-driven “anglerfish”
mechanism that explains less stringent sequence requirements for substrate
recognition and cleavage by matriptase than by furin.