Amiloride-sensitive epithelial Na+ channel currents in surface cells of rat rectal colon

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Abstract
Surface cells of the mammalian distal colon are shown to molecularly express the amiloride-sensitive epithelial Na+ channel composed of three homologous subunits (α-, β-, and γ-ENaC). However, because basic electrophysiological properties of amiloride-sensitive Na+ channels expressed in these cells are largely unknown at the cellular level, functional evidence for the involvement of the subunits in the native channels is incomplete. Using electrophysiological techniques, we have now characterized functional properties of native ENaC in surface cells of rectal colon (RC) of rats fed a normal Na+ diet. Ussing chamber experiments showed that apical amiloride inhibited a basal short-circuit current in mucosal preparation of RC with an apparent half-inhibition constant ( Ki) value of 0.20 μM. RT-PCR analysis confirmed the presence of transcripts of α-, β-, and γ-rENaC in rectal mucosa. Whole cell patch-clamp experiments in surface cells of intact crypts acutely isolated from rectal mucosa identified an inward cationic current, which was inhibited by amiloride with a Ki value of 0.12 μM at a membrane potential of –64 mV, the inhibition being weakly voltage dependent. Conductance ratios of the currents were Li+ (1.8) > Na+ (1) >> K+ (≈0), respectively. Amiloride-sensitive current amplitude was almost the same at 15 or 150 mM extracellular Na+, suggesting a high Na+ affinity for current activation. These results are consistent with the hypothesis that a heterooligomer composed of α-, β-, and γ-ENaC may be the molecular basis of the native channels, which are responsible for amiloride-sensitive electrogenic Na+ absorption in rat rectal colon.