INHIBITION OF CA2+ UPTAKE IN NEUROSPORA-CRASSA BY LA3+ - A MECHANISTIC STUDY

Addition of 1 mM-LaCl3 to Neurospora crassa 30 s prior to the initiation of Ca-45(2+) uptake resulted in a dramatic inhibition of Ca2+ influx to 7% of the control value. The lanthanide Gd3+ and several other recognized Ca2+ channel blockers (ruthenium red, nifedipine, methoxyverapamil) failed to inhibit Ca2+ influx. Direct measurement of membrane potential (DELTApsi) with micro-electrodes revealed a La3+-induced depolarization of about 80 mV for 1 mM-La3+ in the presence of 1 mM-Ca2+. The depolarization is rapid and partially reversible on La3+ washout. The concentration-dependence of the depolarization can be described by a rectangular hyperbola with a K0.5 for La3+ = 0.11 mM. The La3+-induced depolarization is Ca2+-sensitive, decreasing as external Ca2+ increases. The inhibitory effect of Ca2+ also exhibits a hyperbolic concentration-dependence, with a K0.5 for Ca2+ = 2.5 mM for depolarization induced by 1 mM-La3+. While the flux data suggest a direct effect of La3+ on Ca2+ uptake, the electrophysiological data imply additional effects of La3+ on the membrane. Three hypotheses were considered: (1) La3 + interacts with K+ channels; (2) La3+ entry into cells carries a large depolarizing current; (3) La3+ inhibits the electrogenic H+-pump. Hypothesis (1) was eliminated by experiments showing that depolarization occurs regardless of whether the equilibrium potential for K+ is positive or negative of the resting value of DELTApsi. Hypotheses (2) and (3) remain possible, although La3+ influx of the magnitude required to generate the observed depolarization seems very unlikely. We conclude that La3+ should be deployed only with considerable caution as a blocker of plasma-membrane Ca2+ influx in fungi.