Cloning and Characterization of a Transmembrane-Type Serine Protease from Rat Kidney, a New Sodium Channel Activator

Abstract

We have cloned the gene of a new transmembrane-type serine protease from rat kidney, which activates sodium channels. The amino acid sequence deduced from a full-length cDNA revealed that transmembrane serine protease-1 (TMSP-1) is a member of the clan SA/family S1 of serine proteases, comprising a 30 amino acid prepropeptide, a mature form sequence of 274 amino acids starting with the Ile-Val-Gly-Gly-Gln motif, and a common catalytic triad of serine proteases. The hydrophobic amino acid sequence in the carboxy-terminus of this enzyme suggests that it is a glycosylphosphatidylinositol-anchored protein. As revealed by quantitative reverse transcription-polymerase chain reaction analysis, it is highly expressed in kidney, small intestine, and stomach, and moderately expressed in lung, thymus, spleen and skin. The recombinant protease had an optimal pH at 9.0, selectively cleaved synthetic peptide substrates of trypsin, and was inhibited by aprotinin, leupeptin and benzamidine. Immunohistochemical studies revealed that this protease is predominantly expressed in cells from collecting ducts of the renal medulla. We also demonstrate that a C-terminally truncated variant of TMSP-1 significantly activates the epithelial sodium channel, and that its mRNA levels are upregulated by aldosterone. These observations suggest that it is a new member of the trypsin-type transmembrane proteases, which regulate sodium balance by activating the epithelial sodium channel.

In vitro and in vivo regulation of transepithelial lung alveolar sodium transport by serine proteases

“…Mammalian homologs of Xenopus CAP1, such as mouse mCAP1 or human and rat prostasin, were also shown to activate ENaC in the Xenopus oocyte expression system (1,11,38,39). More recently, additional transmembrane serine proteases activating ENaC have been identified in mammals, including channelactivating protease 2 (CAP2) and channel-activating protease 3 (CAP3) cloned from the mpkCCD d4 mouse kidney cell line (37), TMPRSS3 from human inner ear (15), or TMSP-1 from rat kidney (27). The precise mechanism for protease-mediated activation of ENaC has not been elucidated.…”

Effect of neutrophil elastase and its inhibitor EPI-hNE4 on transepithelial sodium transport across normal and cystic fibrosis human nasal epithelial cells

“…It has been recently shown that ENaC channels expressed at the cell surface can be activated in vitro and in vivo by various trypsin-like serine proteases [ 10 - 14 , 17 , 27 ]. Membrane-bound Channel-Activating Proteases, which are co-expressed with ENaC in airway and alveolar epithelial cells [ 15 , 20 - 22 , 24 ] but also in other epithelial cells transporting Na + [ 12 , 14 ] have the ability to stimulate ENaC activity by increasing the channel opening probability, most likely through proteolytic cleavage of γ-ENaC subunit [ 9 , 16 ].…”

“…Mammalian homologs of Xenopus CAP1, such as mouse mCAP1 or human and rat prostasin, were also shown to activate ENaC in the Xenopus oocytes expression system [ 12 - 15 ]. More recently, additional transmembrane serine proteases activating ENaC have been identified in mammals, including channel-activating protease 2 (CAP2) and channel-activating protease 3 (CAP3) cloned from the mpkCCD d4 mouse kidney cell line [ 14 ], TMPRSS3 from human inner ear [ 16 ], or TMSP-1 from rat kidney [ 17 ]. The precise mechanism for protease-mediated activation of ENaC has not been fully elucidated, but it likely involves proteolytic cleavage of α- and γ-ENaC subunits [ 9 , 16 ].…”

“…The precise mechanism for protease-mediated activation of ENaC has not been fully elucidated, but it likely involves proteolytic cleavage of α- and γ-ENaC subunits [ 9 , 16 ]. Studies in Xenopus oocytes [ 13 , 14 , 17 ] or transfected mammalian cells [ 18 ] have demonstrated that trypsin-like serine proteases increase Na + transport by activating a population of near-silent channels rather than by promoting plasma membrane insertion of new channels. In mammals, the channel-activating proteases (CAP1,-2 and 3) are coexpressed with ENaC in epithelial tissues transporting Na + like renal collecting duct, lung, and colon [ 12 , 19 , 20 ].…”