CA2-HYDROXYTRYPTAMINE TYPE-3 RECEPTORS( PERMEABILITY OF CLONED AND NATIVE 5)

We have used single-cell imaging of fura-2-loaded cells to examine the Ca2+ signals evoked by activation of 5-hydroxytryptamine type 3 (5-HT 3) receptors in undifferentiated N1E-115 neuroblastoma cells and in hu man embryonic kidney (HEK) 293 cells transfected with either of the tw o cloned 5-HT3 receptor subunits. The selective 5-HT3 receptor agonist 1-(m-chlorophenyl)-biguanide (mCPBG) caused a concentration-dependent increase in the cytoplasmic Ca2+ concentration ([Ca2+](i)) in N1E-115 cells and in HEK 293 cells transfected with either the 5-HT3 A subuni t or the 5-HT3 As subunit. In each case, the [Ca2+](i) rise was steepl y dependent on the mCPBG concentration (n(H) = 2-4) and abolished by r emoval of extracellular Ca2+ or addition of ondansetron. Pretreatment of N1E-115 cells with thapsigargin, caffeine, and ryanodine to deplete intracellular Ca2+ stores had no effect on the mCPBG-evoked Ca2+ sign als, indicating that they result entirely from stimulated Ca2+ entry. The steep concentration-effect curves therefore are not a consequence of amplification of Ca2+ influx by Ca2+-induced Ca2+ release from intr acellular stores and probably reflect cooperative activation of 5-HT3 receptors by mCPBG. Depolarization of transfected HEK 293 cells with m edium containing increased K+ concentrations invariably failed to evok e an increase in [Ca2+](i), confirming the absence of voltage-gated Ca 2+ channels and indicating that the mCPBG-evoked rise in [Ca2+](i) res ults from Ca2+ permeation of 5-HT3 receptors. However, in N1E-115 cell s and transfected HEK 293 cells, both extracellular Na+ and K+ substan tially inhibited the Ca2+ influx evoked by activation of 5-HT3 recepto rs, possibly by inhibition of agonist binding or by competition with C a2+ for permeation of the channel. We conclude that 5-HT3 receptors ar e Ca2+ permeant, that the Ca2+ influx is sufficient to generate a sign ificant rise in [Ca2+](i), and that, because the A and As subunits beh ave similarly, conflicting electrophysiological analyses of Ca2+ curre nts cannot be explained by differences between these two subunits.