On the voltage-dependent Ca2+ block of serotonin 5-HT3 receptors: A critical role of intracellular phosphates

Natively expressed serotonin 5-HT(3) receptors typically possess a negative-slope conductance region in their I-V curve, due to a voltage-dependent block by external Ca(2+) ions. However, in almost all studies performed with heterologously expressed 5-HT(3) receptors, this feature was not observed. Here we show that mere addition of ATP to the pipette solution is sufficient to reliably observe a voltage-dependent block in homomeric (h5-HT(3A)) and heteromeric (h5-HT(3AB)) receptors expressed in HEK293 cells. A similar block was observed with a plethora of molecules containing a phosphate moiety, thus excluding a role of phosphorylation. A substitution of three arginines in the intracellular vestibule of 5-HT(3A) with their counterpart residues from the 5-HT(3B) subunit (RRR-QDA) was previously shown to dramatically increase single channel conductance. We find this mutant to have a linear I-V curve that is unaffected by the presence of ATP, with a fractional Ca(2+) current (Pf%) that is reduced (1.8 +/- 0.2%) compared to that of the homomeric receptor (4.1 +/- 0.2%), and similar to that of the heteromeric form (2.0 +/- 0.3%). Moreover, whereas ATP decreased the Pf% of the homomeric receptor, this was not observed with the RRR-QDA mutant. Finally, ATP was found to be critical for voltage-dependent channel block also in hippocampal interneurons that natively express 5-HT(3) receptors. Taken together, our results indicate a novel mechanism by which ATP, and similar molecules, modulate 5-HT(3) receptors via interactions with the intracellular vestibule of the receptor.