At 0 mV, the SLC9C1 transporter (grey) is able to bind to Na + , as shown here by SSM-based electrophysiology and as evident from the open inward-facing cavity, but is inactive because the very long S4 in the VSD (cyan) is in the up-state, which pulls up the clamped ICH3–ICH4 (orange) and ICH5–ICH6 (purple) helices so that ICH7 physically restricts the transporter domains from moving (left). In particular, Lys939 (yellow) in ICH7 is making a number of polar interactions to the linker helix TM7, which needs to rotate during an elevator alternating-access mechanism 23 , 24 . In the inactive state, the CNBDs (brown) further contribute to homodimerization and inactivation by stabilization of the cytosolic regulatory network. Middle, the addition of cAMP shifts the voltage activation of sea urchin SLC9C1 by -20 mV 2 and primes SLC9C1 for activation by hyperpolarization. The structural basis for priming is that the CNBD domains become more flexible, disrupting interactions with the S4 helix and decreasing cytosolic domain–domain interactions between protomers, enabling the attached ICHs to move outwards, increasing the mobility of ICH7 and altering the positioning of the VSD. In particular, the S1–S3 helices start to rotate anti-clockwise around S4. Right, on the basis of the high structural similarity between the VSD in sea urchin SLC9C1 and VSD IV in human Na V 1.7, and the fact that the mutation of the gating-charge residue R803Q reduces the elemental gating charge from 3.1 to 2.0 e 0 (ref. 2 ), we propose that S4 will probably undergo a 10 A vertical displacement to the down-state in response to hyperpolarization. Such a large rearrangement would displace the ICH network attached to S4, therefore removing the constraints from ICH7 and enabling ion-exchange. The diagram was created using BioRender.com.
