CAMP-activation of amiloride-sensitive Na+ channels from guinea-pig colon expressed in Xenopus oocytes.

Abstract

Guinea-pig distal colonic mRNA injection into Xenopus laevis oocytes resulted in expression of functional active epithelial Na+ channels in the oocyte plasma membrane. Poly(A)+ RNA was extracted from distal colonic mucosa of animals fed either a high-salt (HS) or a low-salt (LS) diet. The electrophysiological properties of the expressed amiloride-sensitive Na+ conductances were investigated by conventional two-electrode voltage-clamp and patch-clamp measurements. Injection of poly(A)+ RNA from HS-fed animals [from hereon referred to as HS-poly(A)+ RNA] into oocytes induced the expression of amiloride-sensitive Na+ conductances. On the other hand, oocytes injected with poly(A)+ RNA from LS-fed animals [LS-poly(A)+ RNA] expressed a markedly larger amount of amiloride-blockable Na+ conductances. LS-poly(A)+ RNA-induced conductances were completely inhibitable by amiloride with a Ki of 77 nM, and were also blocked by benzamil with a Ki of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 "mu"M), had no detectable effect on the Na+ conductances. Expressed amiloride-sensitive Na+ channels could be further activated by cAMP leading to nearly doubled clamp currents. When Na+ was replaced by K+, amiloride (1 "mu"M) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (Po) between 0.4 and 0.9, and slope conductances of 3. 8 pS for Na+ and 5.6 pS for Li+. The expressed channels showed to be highly selective for Na+ over K+ with a permeability ratio PNa/PK > 20. Amiloride (500 nM) reduced channel Po to values < 0.05. All these features make the guinea-pig distal colon of LS-fed animals an interesting mRNA source for the expression of highly amiloride-sensitive Na+ channels in Xenopus oocytes, which could provide new insights in the regulatory mechanism of these channels.