2nd State: Inward Open

What are the key structural elements involved in the inward open conformation?

Yes, it all comes back to the transmembrane helices again!

• TM1a – tilts by 45°, juts into where membrane is believed to be
• TM6b – tilts by 17°, away from binding pocket
• TMs 1b and 6a – tilt by 24° and 21° respectively towards scaffold domain leading to obstruction of molecules on the outside of the membrane
• Buttressing helices, TM2, TM7 and TM5

-   Bend due to presence of glycine or proline residues in the middle of the helices

-   TM7 in particular causes EL4 to move downwards, thus acting like a plug preventing further extracellular solvent entry

Figure 4: A video demonstrating the major structural features of the inward-open conformation

How does this conformation facilitate substrate binding?

The states of the 2 gates previously mentioned swap around in the inward open conformation:

• Thick extracellular gate = formed – through the interaction of TM1b/TM10, EL4/TM10, TM6a/TM11

The gate is so important that mutating just one residue (Arg30) causes severe loss of function of the NSS.

• Thick intracellular gate = disrupted – through disturbing TM1a/TM6b, TM1a/TM8

Deliberately effecting these interactions by mutation yields the inward-open state!

Figure 5.1: A view of the intracellular gate of the inward-open state; TM1a and TM6b move to facilitate the pertubation of the gate

This allows contact with the substrate binding site from the intracellular side.

We thought it would be useful to compare figure 5.1 with the schematic of the outward-open conformation from the same angle in figure 5.2. It clearly shows how the substrate binding site is a lot more inaccessible in this state due to the presence of the intracellular gate.

Figure 5.2: The outward-open state as seen from the intracellular side of the membrane

But your understanding of substrate binding site access would not be complete without discussing the role of TM helices once again:

• The intracellular cavity is lined with TM domains 1a, 5 and 8. 
• TM1b and TM6a, which are essential to the outward open configuration (look back at this section if you don’t believe us) become buried when sodium is no longer present.
• EL4 packs together with domains TM1b and TM7 on one side and TM3, TM8 and EL2 on the other via hydrogen bonds and hydrophobic interactions. This confirms our analogy of the plug. as it fully halts the passage of extracellular molecules.

Figure 6: A Pymol diagram highlighting the role of EL4 in blocking the extracellular pathway

We have encompassed changes in transmembrane helices AND gates in the description of the inward-open intermediate but there is one last thing to be assessed, what happens to the Na+ AND substrate sites?

TM1a and TM8 move away from each other when the inward-open state is being formed and, as these helices characterise the Na2 site, their separation is linked to sodium release from the Na2 site and subsequently the opening of the cytoplasmic gate (we said before that Na+ binding here stabilises the intracellular gate). The Na2 retains some sort of structural integrity which allows it to switch back to the outward-open state, otherwise the receptor would essentially inactivate itself!

Now obviously, whilst we need the second sodium site to be altered, the substrate binding site needs to remain undamaged. It is thought that the hinges for TM1 and TM6 are precisely positioned to ensure this.