Cell-specific expression of amiloride-sensitive, Na(+)-conducting ion channels in the kidney

Abstract

Amiloride-sensitive, electrogenic Na+ absorption across the distal nephron plays a vital role in regulating extracellular fluid volume and blood pressure. Recently, two amiloride-sensitive, Na(+)-conducting ion channel cDNAs were cloned. One, an epithelial Na(+)-selective channel (ENaC), is responsible for Na+ absorption throughout the distal nephron. The second, a guanosine 3',5'-cyclic monophosphate (cGMP)-inhibitable cation channel, is conductive to Na+ and Ca2+ and contributes to Na+ absorption across the inner medullary collecting duct (IMCD). As a first step toward understanding the segment-specific contributions(s) of cGMP-gated cation channels and ENaC to Na+ and Ca2+ uptake along the nephron, we used in situ reverse transcription-polymerase chain reaction (RT-PCR) hybridization, solution-phase RT-PCR, and Western blot analysis to examine the nephron and cell-specific expression of these channels in mouse kidney cell lines and/or dissected nephron segments. cGMP-gated cation channel mRNA was detected in proximal tubule, medullary thick ascending limb (mTAL), distal convoluted tubule (DCT), cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and IMCD. cGMP-gated cation channel protein was detected in DCT, CCD, and IMCD cell lines. These observations suggest that hormones that modulate intracellular cGMP levels may regulate Na+, and perhaps Ca2+, uptake throughout the nephron. mRNA for alpha-mENaC, a subunit of the mouse ENaC, was detected in mTAL, DCT, CCD, OMCD, and IMCD. Coexpression of alpha-mENaC and cGMP-gated cation channel mRNAs in mTAL, DCT, CCD, OMCD, and IMCD suggests that both channels may contribute to Na+ absorption in these nephron segments.

Characterization of an amiloride binding region in the α-subunit of ENaC

“…The initial characterization of wild-type ␣-ENaC expressed in CHO cells demonstrated that channels with properties similar to those seen in other expression systems and endogenous tissues could be expressed (6,14). Another interesting observation was that the P o of the channels fell into two distinct groups.…”

“…However, regardless of which channel type is expressed, one of the defining characteristics of all these channels is their block by the diuretic amiloride, a substituted pyrazinoylguanidine. The tertiary structure of all the subunits is similar: each subunit is predicted to span the membrane twice, to have a large extracellular loop, and to have short intracellular NH 2 and COOH termini (2,6). Because amiloride blocks the channel from the extracellular surface of the channel protein, it is possible that one or more of the extracellular loops could play a role in amiloride's interaction with and block of the channel by stabilizing amiloride in the channel pore.…”

Role of gammaENaC subunit in lung liquid clearance and electrolyte balance in newborn mice. Insights into perinatal adaptation and pseudohypoaldosteronism.

“…ENaC subunits are coexpressed in the distal renal tubule and collecting ducts (5,6). A role for ENaC in electrolyte and fluid balance is deduced from mutations of ENaC subunits that cause two diseases: pseudohypoaldosteroidism (PHA, Type 1) and Liddle's syndrome.…”

Distribution of transcellular calcium and sodium transport pathways along mouse distal nephron

“…The above-mentioned proteins have been localized to the distal nephron of rabbit (1,9,15,16,26,29,37) and rat (4, 5, 10, 22, 33-35, 39, 41) but only in part in mice [e.g., NKCC2 (32), NCC (23), ENaC (6,27), and CB (38)]. Previous studies revealed significant differences in the distribution patterns of salt and water transport proteins along the distal nephron between rabbits (1, 26) and rats (24,33,41).…”