(A) Cartoon model of the SWELL1 pore, with two subunits removed for clarity. A surface representation of the radial distance between the protein surface and the pore axis is shown in grey. Pore-facing residues R103, T48 and T44, and N-terminal coil (NTC) are labeled in pink. (B) Graph of van der Waals radii of the pore, plotted against distance along the pore axis. Locations of residues R103, T48, T44, and NTC are labeled along 2D plot. Grey box covers potential area the N-terminus might occupy. (C) Electrostatic surface potential of channel pore, viewed by vertical cross-section. Narrow constriction on the extracellular side of the channel is formed by a ring of R103 residues (yellow arrows). Calculated using APBS implemented by Pymol2.0 with potentials ranging from −10 kT (red) to +10 kT (blue). (D–E) Cells expressing heteromeric VRACs composed of mutant SWELL1-R103F + LRRC8C show reduced chloride selectivity and insensitivity to external ATP block. (D) For highly Cl- selective channels, the voltage at which there is no net current (Vrev) is close to the equilibrium potential for Cl- (in these experiments ECl = +9.75 mV; indicated by the dotted line). Vrev of currents mediated by SWELL1-R103F-containing channels (orange bar; +4.6 ± 1.0 mV (mean ± s.e.m., n = 6 cells from 3 separate transfections)) is significantly reduced compared to WT (blue bar; +8.8 ± 0.8 mV (n = 13 from 6 separate transfections); p = 0.003, Student’s t-test. (E) The percent block of whole cell leak subtracted hypotonic-induced currents by extracellular applied Na2ATP (2 mM) was determined at +100 mV. Outward WT SWELL1 + LRRC8C-mediated currents are blocked 72 ± 2% (mean ± s.e.m., n = 7 from 4 separate transfections; blue bar). Outward currents mediated by SWELL1-R103 + LRRC8C are not blocked by extracellular ATP (2 ± 3% (mean ± s.e.m., n = 5 from 3 separate transfections; orange bar); this difference is highly significant (p = 5.4e^-8, Student’s t-test). (F) Detailed view of coordination of NTC (purple). The NTC makes intrasubunit contacts with V157 on LH1 and a conserved Y382 at the kink between LH6 and LH7 of the TM4-LRR linker. Additionally, P22 of the NTC makes an intersubunit contact with a conserved P147 at the kink between TM2 and the TM2-TM3 linker of the neighboring subunit. (G–H) SWELL1-T5 is close to or part of the pore. (G) A cysteine mutation at SWELL1-T5 confers sensitivity to the polar MTS reagent MTSES applied extracellularly; maximum percent block of T5C-containing channels (red bars) by 3.33 mM MTSES was 74.2 ± 7.7% at −100 mV (left) and 50.4 ± 9.5% at +100 mV (right) (n = 5; mean ± s.e.m.; four separate transfections). The unmodifiable T5R-containing heteromeric channels (blue bars) are unaffected (n = 3 from 3 separate transfections; p=0.0009 at −100 mV and p=0.010 at +100 mV, Student’s t-test). (H) Relative permeability PI/PCl is enhanced by the T5R mutation. Reversal potentials in iodide (left) and chloride (right) 230 mOsm/kg solutions are shown for the number of cells from 3 to 7 separate transfections (WT-, T5C- and T5R-expressing cells were from 6, 4, and 3 transfections, respectively, in the Cl- condition, and 3, 3, and 3 transfections, respectively, in the I- condition). The Vrev of currents mediated by SWELL1-T5R + LRRC8C (blue) in I- solution was significantly more negative than either WT- (black) or T5C (red)-containing channels (p=0.0088 (**) and 0.0047 (***), respectively. Table: PI/PCl is shown as means with lower and upper 95% confidence intervals.