1st State: Outward Open/Substrate Free

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

• TMs 1b, 2a and 6a – undergo hinge-like movements

These movements, particularly TM1b and 6a, in turn cause other movements:

• Extracellular loop 3 is moved by 2.8 Angstroms
• TM11 is moved by 2.2 Angstroms

These features, along with extracellular loop 4, can be seen on figure 1 below:

Figure 1: A Pymol representation of the outward-open conformation of LeuT highlighting the prominent features

• When we look at Figure 2 there are 2 very distinct regions, the core domain and the scaffold domain. 

Figure 2: A schematic representation of the core and scaffold domains (adapted from Krishnamurthy&Gouax 2012)

• In the outward open conformation the core and scaffold do NOT interact. This causes TM regions 1b and 6a to move outwards as they are not restrained.
• Thin extracellular gate - this feature is broken; the receptor is no longer blocked.
• 2 sodium sites: Na1 and Na2

 How is the thin extracellular gate disrupted?

• TMs 1b and 6a are linked to TMs 3 and 10 via an amino acid linking sequence BUT when this bridge is broken the conformation is "open".
• Like a dam breaking it causes an influx of surrounding solution, this:
• Disrupts salt bridges, between Arg30 and Arg404
• Rotates Phe 253 which contributes to exposure of the binding site

What is the importance of the 2 sodium sites present in this conformation?

The Na+ ions link the core and scaffold regions to maintain a thick intracellular gate, therefore:

• Thin extracellular gate = disrupted
• Thick intracellular gate = intact

 Where are the Na sites?

• Na1) Near the pivot points of TM1b and 6a – It is thought that Na+ binds before the substrate to stabilise these helices in outward open, allowing binding site access; contributes to rupturing of extracellular gate
• Na2) Near the intracellular part of TM1 – Na+ binding stabilises the intracellular closed conformation; keeps the intracellular gate closed

Figure 3: The location of Na+ binding sites