Extracellular terbium and divalent cation effects on the red blood cell Na pump and chrysoidine effects on the renal Na pump

We examined the effect of extracellular terbium (Tb 3+) and divalent metal cations (Ca 2+, Sr 2+, and Ba 2+) on 86Rb + influx into rabbit and human red blood cells. We found that Tb 3+ at 15 and 25 μM was a non-competitive inhibitor of 86Rb + influx suggesting that Tb 3+ is not binding to the transport site. This result reduces the usefulness of Tb 3+ as a potential probe for the E out conformation (the conformation with the transport site facing extracellularly). Ba 2+, Sr 2+ and Ca 2+, at concentrations > 50 mM, had minimal effects on Rb + influx into red blood cells (1 mM Rb-out). This suggests that the outside transport site is very specific for monovalent cations over divalent cations, in contrast to the inside transport site. We also found that chrysoidine (4-phenylazo- m-phenylenediamine) competes with Na + for ATPase activity and K + for pNPPase activity suggesting it is binding to the E in conformation. Chrysoidine and similar compounds may be useful as optical probes of the E in conformation.