(A) Key interaction sites for Na⁺ ions within the hTPC2 pore are the SF (blue shade, residues N653 and N654 from filter-II are highlighted), the central cavity (green shade, residue N687 from 6-TM-2 is highlighted; N305 is located in the same pore region on 6-TM-1), and the hydrophobic gate region (red shade; shown are the externally facing residues E695 from 6-TM-2 near the gate for clarity; R316 is located in the same region on 6-TM-1). (B) Close-up view of residues N305 and N687 from two subunits, a Na⁺ ion binds between the asparagine side chains from one subunit but can change to other hydrophilic side chains in the cavity hydrophilic cluster; additional ions can occupy these binding sites. (C) The cavity is frequently occupied by more than one Na⁺ ion at lower voltage regimes (purple bars show the mean occupancy from five repeat simulations ± SD at a voltage of −70 and −200 mV, respectively). The individual simulation results are shown in red. (D) Mutual information between the time when changes in SF and cavity binding site occupancy occur (the green bars show the average SSI from five repeat simulations ± SD at voltages of −350, −200, and −70 mV; individual simulation results in red). Substantial mutual information is seen in all voltage ranges for the hTPC2 wild type (the gray bars denote the maximum theoretical mutual information attainable ± SD, individual data points in blue). This finding signifies a considerable contribution of a distant knock-on mechanism to conduction efficiency. By contrast, in the SF mutant V652M/N653G, the knock-on mechanism is disrupted, likely by abolishing a distinct SF binding site, leading to a decreased Na⁺ conductance.