Uptake of Ca2+ Driven by the Membrane Potential in Energy-depleted Yeast Cells

Summary: The time-course of 45Ca2+ influx into yeast cells was measured under non-steady-state conditions obtained by preincubating the cells in a Ca2+-free medium containing glucose and buffer. Two components were distinguished: a saturable component which reached a steady-state after about 40 s of 45Ca2+ uptake and a linear increase in cellular 45Ca2+ starting after 60-90s. Using differential extraction methods it was determined that after 20s of uptake, 45Ca2+ was localized in the cytoplasmic pool and in bound form with no 45Ca2+ in the vacuole. After 3 min most of the cellular 45Ca2+ was concentrated in the vacuole and in bound form. The initial rate of 45Ca2+ uptake under non-steady-state conditions thus measured 45Ca2+ transport across the plasma membrane without interference by vacuolar uptake. The effect of membrane potential (δψ) on this transport was investigated in cells depleted of ATP. A high δψ was produced by preincubating the cells with trifluoperazine (TFP) and subsequently washing the cells free from TFP. Substantial 45Ca2+ influx was measured in the absence of metabolic energy in cells with a high δψ. Below a threshold value of −69·5 mV the logarithms of the initial rate of 45Ca2+ influx and of the steady-state level of the first component were linear with respect to δψ. It is suggested that 45Ca2+ influx across the plasma membrane is mediated by channels which open when δψ is below a threshold value. The results indicated that Ca2+ influx across the plasma membrane was driven electrophoretically by δψ.