(a) The surface-rendered XtOTOP3 structure shows a solvent accessible cavity (red) from the intracellular side at the interface between the two pores. The Gln-Asp/Asn-Tyr constriction triads are shown as yellow sticks. (b) Zoomed-in view of the inter-pore protein contacts on the extracellular half of the membrane, highlighting the conserved glutamate (Glu321, magenta sticks) buried within an interface formed mostly by hydrophobic residues (grey sticks). (c) Zoomed-in view of the surrounding residues of the intracellular cavity. The two hydrogen-bonded residues, Glu509 and His669, are shown as cyan sticks. (d) Sample traces of whole cell recordings (left) of mutations at Glu321 and His669, and their current densities (right). (e) The Asp509 neutralization mutants exhibit reduced desensitization rate. Top, Sample traces of patch clamp recordings in the outside-out configuration of the wild type XtOTOP3 and the Asp509 mutants. Bottom, Overlaid sample traces with mutant traces scaled to that of the wild-type. Data in d are shown as mean ± s.e.m. (n = 5 independent experiments). All recordings were measured at −100 mV with pHi = 7.4. The currents were elicited by changing pHo from 7.4 to 5.0.