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 (Tb3+) and divalent metal cations (Ca2+, Sr2+, and Ba2+) on 86Rb+ influx into rabbit and human red blood cells. We found that Tb3+ at 15 and 25 μM was a non-competitive inhibitor of 86Rb+ influx suggesting that Tb3+ is not binding to the transport site. This result reduces the usefulness of Tb3+ as a potential probe for the Eout conformation (the conformation with the transport site facing extracellularly). Ba2+, Sr2+ and Ca2+, 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 Ein conformation. Chrysoidine and similar compounds may be useful as optical probes of the Ein conformation.