A pore-blocking hydrophobic motif at the cytoplasmic aperture of the closed-state Nav1.7 channel is disrupted by the erythromelalgia-associated F1449V mutation.
Sodium channel Na(v)1.7 has recently elicited considerable interest as a key contributor to human pain. Gain-of-function mutations of Na(v)1.7 produce painful disorders, whereas loss-of-function Na(v)1.7 mutations produce insensitivity to pain. The inherited erythromelalgia Na(v)1.7/F1449V mutation, within the C terminus of domain III/transmembrane helix S6, shifts channel activation by -7.2 mV and accelerates time to peak, leading to nociceptor hyperexcitability. We constructed a homology model of Na(v)1.7, based on the KcsA potassium channel crystal structure, which identifies four phylogenetically conserved aromatic residues that correspond to DIII/F1449 at the C-terminal end of each of the four S6 helices. The model predicted that changes in side-chain size of residue 1449 alter the pore's cytoplasmic aperture diameter and reshape inter-domain contact surfaces that contribute to closed state stabilization. To test this hypothesis, we compared activation of wild-type and mutant Na(v)1.7 channels F1449V/L/Y/W by whole cell patch clamp analysis. All but the F1449V mutation conserve the voltage dependence of activation. Compared with wild type, time to peak was shorter in F1449V, similar in F1449L, but longer for F1449Y and F1449W, suggesting that a bulky, hydrophobic residue is necessary for normal activation. We also substituted the corresponding aromatic residue of S6 in each domain individually with valine, to mimic the naturally occurring Na(v)1.7 mutation. We show that DII/F960V and DIII/F1449V, but not DI/Y405V or DIV/F1752V, regulate Na(v)1.7 activation, consistent with well established conformational changes in DII and DIII. We propose that the four aromatic residues contribute to the gate at the cytoplasmic pore aperture, and that their ring side chains form a hydrophobic plug which stabilizes the closed state of Na(v)1.7.
Lampert
Angelika
A
Department of Neurology and Center for Neuroscience & Regeneration Research, Yale University School of Medicine, New Haven, CT 06510, USA.
O'Reilly
Andrias O
AO
Dib-Hajj
Sulayman D
SD
Tyrrell
Lynda
L
Wallace
B A
BA
Waxman
Stephen G
SG
D020816:Amino Acid Motifs; D019943:Amino Acid Substitution; D002460:Cell Line; D004916:Erythromelalgia / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D008954:Models, Biological; D020125:Mutation, Missense; D062556:NAV1.7 Voltage-Gated Sodium Channel; D009619:Nociceptors / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary / Q000235:genetics; D017434:Protein Structure, Tertiary / Q000235:genetics; D015222:Sodium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D062556:NAV1.7 Voltage-Gated Sodium Channel; C486866:SCN9A protein, human; D015222:Sodium Channels