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 K_i of 77 nM, and were also blocked by benzamil with a K _i of 1.8 nM. 5-(N-Ethyl-N-isopropyl)-amiloride (EIPA), even in high doses (25 µM), 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 µM) showed no effect on the clamp current. Single-channel analysis revealed slow gating behaviour, open probabilities (P_o) 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 P_naP_K> 20. Amiloride (500 nM) reduced channel P_o to values < 0.05. All these features make the guineapig 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.