THIAMINE TRIPHOSPHATE ACTIVATES AN ANION CHANNEL OF LARGE UNIT CONDUCTANCE IN NEUROBLASTOMA-CELLS

In neuroblastoma cells, the intracellular thiamine triphosphate (TTP) concentration was found to be about 0.5 mu M, which is several times a bove the amount of cultured neurons or glial cells. In inside-out patc hes, addition of TTP (1 or 10 mu M) to the bath activated an anion cha nnel of large unit conductance (350-400 pS) in symmetrical 150 mM NaCl solution. The activation occurred after a delay of about 4 min and wa s not reversed when TTP was washed out. A possible explanation is that the channel has been irreversibly phosphorylated by TTP. The channel open probability (P-o) shows a bell-shaped behavior as a function of p ipette potential (V-p). P-o is maximal for -25 mV < V-p < 10 mV and st eeply decreases outside this potential range. From reversal potentials , permeability ratios of P-cl/P-Na = 20 and P-cl/P-gluconate = 3 were estimated. ATP (5 mM) at the cytoplasmic side of the channel decreased the mean single channel conductance by about 50%, but thiamine deriva tives did not affect unit conductance; 4,4'-diisothiocyanostilbene-2,2 '-disulfonic acid (0.1 mM) increased the flickering of the channel bet ween the open and closed state, finally leading to its closure. Additi on of oxythiamine (1 mM), a thiamine antimetabolite, to the pipette fi lling solution potentiates the time-dependent inactivation of the chan nel at V-p = -20 mV but had the opposite effect at +30 mV. This findin g corresponds to a shift of P-o towards more negative resting membrane potentials. These observations agree with our previous results showin g a modulation of chloride permeability by thiamine derivatives in mem brane vesicles from rat brain.