Goal: To clarify how defined regions of the human P2X3 receptor govern channel opening and ion passage, using mainly electrophysiological measurements. Method: The cytoplasmic cap was probed by controlled heteromer formation: channels carrying only one C terminus or lacking the N-terminus were expressed. Two Electrode Voltage Clamp (TEVC) recorded current size and desensitisation. Ionic and hydrophobic contacts inside the cap were weakened or removed. Biochemical assays tracked the approach of the termini on activation. A cysteine-scanning series (I323–V334) in TM2 was made and tested with TEVC. Charges at three pore fenestration levels were changed, and a rat P2X5 TM2 domain was introduced into the hP2X3 channel. Results: A single C-terminus still allowed opening, but loss of the N-terminus terminated it, showing the cap’s asymmetrical control. Breaking key contacts in the cap accelerated desensitisation, confirming cap stabilisation. I323, T330, and V334 acted as gate points; V326 contributed to partial dehydration of ions, and G333 helped keep the pore wide. TM2 showed marked flexibility, matching molecular-dynamics data and its link to the cap. Positive clusters at outer fenestrations enhanced cation entry; Replacing the corresponding TM2 residues of P2X3 with those from P2X5 resulted in varied changes in ion permeation. Discussion: These observations give a biophysical picture of how the cap, TM2, and fenestrations together shape P2X3 activity, mapping structural features directly onto gating and permeation behaviour.
Linhan Cheng (Wed,) studied this question.