Introduction

LeuT is presently the best model for the study of neurotransmitter sodium symporters (NSS). The study of different conformations of the NSS is a great help in understanding how they form complexes with various drugs, and therefore help with therapeutic research. Knowledge about the outward-open and inward-open conformations of LeuT and their mechanisms is developed recently. 

This blog will take you through some details about the outward-open and inward-open conformations of LeuT. We hope this helps you understand!

The following are some background information to help you go through the blog.

What is NSS?

An NSS is a membrane-integral transporter involved in the process of synaptic transmission, and it transports a neurotransmitter across the membrane, where sodium ions are co-transported to facilitate the transport of the neurotransmitter against the concentration gradient.In humans, they have important roles in arresting neurotransmissions of:

• Serotonin
• Dopamine
• Noradrenaline
• Glycine
• GABA (c-aminobutyric acid)

Figure A- Mechanism of NSS. Conformations focussed in this blog are coloured. (Adapted from Krishnamurthy et al, 2012)

The NSSs are targets of therapeutic drugs which include tricyclic antidepressants (TCAs) and

selective serotonin re-uptake inhibitors (SSRIs) such as Prozac, as well as addictive substances such as cocaine and amphetamines. [3]

What is LeuT?

LeuT, a member of the NSS family of transport proteins, is found in animals and prokaryotes. In this research, LeuT^k is used instead of LeuT. LeuT^k, obtained from Aquifex aeolicus (K288A) is a variant of LeuT which is wild-type-like. LeuT^k structure mediates the gating of leucine rather although being highly similar to Tryptophan-bound LeuT structure. [1]

Why is LeuT used?

LeuT is the only member of the family for which we have high-resolution structural information. In recent years, the characteristic protein fold of LeuT, including an inverted structural repeat, has been found in several other transporter families – suggesting a common ancestor. [2] 

Aim of research:

To provide insight into the transport mechanism of neurotransmitter transporters in which two substrate-free X-ray crystal structures of LeuT^k, a bacterial homologue are provided. A detailed study the outward-open and inward-open states of LeuT^k is carried out. One structure shows the amino acid transporter LeuT with its empty substrate site exposed to the extracellular medium and the other with the site exposed to the cytoplasm. 

Relevance of this research:

Crystal structures of the bacterial homologue, LeuT, in substrate-bound outward-occluded

and competitive inhibitor-bound outward-facing states have advanced our mechanistic understanding of neurotransmitter sodium symporters but have left fundamental questions unanswered. These conformational changes in LeuT are not restricted to the NSS family, rather they are also found in other families with similar LeuT protein fold. vSGLT and Mhp1 are two proteins representing these other families. In these structures, the bundle conformation is similar to models proposed for LeuT and the serotonin transporter [4, 5] but different from the new inward-open structure. Mhp1 was crystallized in both inward-open and outward-open conformations [6, 7] and the bundle conformation was identical in both. Thus, here suggests the conformational mechanism of transport can be different between families with the same fold.