An Aldosterone Regulated Chicken Intestine Protein with High Affinity to Amiloride

Abstract The pattern of chicken intestine amiloride-binding proteins was determined using the photoreactive amiloride analogue 2′-methoxy-5′-nitrobenzamil (NMBA) and a polyclonal anti-amiloride antibody. At 10 −7 M, NMBA inhibits ∼62% of the Na + channel activity. At this concentration the amiloride analogue labels a number of membrane proteins, and in particular a 40–45 kDa polypeptide denoted ABP40. Incorporation of NMBA into ABP40 could be prevented by a 100-fold excess of benzamil, but not by a 1000-fold excess of 5-(N-ethyl-N-isopropyl)-amiloride. Labeling of ABP40 was intense in membranes derived from salt-deprived chickens and ∼5-fold weaker in membranes from salt-repleted animals. Because of its small size, ABP40 is not likely to be an avian Na + channel subunit, yet this amiloride-binding protein could be involved in the response to aldosterone.