Feature: Mysteries of nerve function

In 2000 a group in the Netherlands discovered a key insight into nerve function, showing the protein Munc18 was essential to exocytosis. However, around the same time another group demonstrated that Munc18 could also impair exocytosis, leading to something of a dilemma as to the role the protein played.

Then, in 2007, prominent biochemist James Rothman in the United States reported a second, previously unrealised, binding site at the N-terminus of syntaxin for Munc18. Using a very nifty in vitro membrane fusion assay and a mutated form of syntaxin that could not bind Munc18 at the N-terminal site, his group showed blocked fusion at the plasma membrane.

This suggested that exocytosis could not occur and that binding to syntaxin through this region is critical for Munc18 function in neurotransmission. “This was a really exciting finding in the field of neural cell biology because it confirmed that Munc18 promotes SNARE complex formation and, more specifically, the actual fusion process at the neural cell surface,” Meunier said.

Around that time, Meunier was collaborating with protein structural biologist, Professor Jenny Martin, at UQ’s Institute for Molecular Bioscience (IMB), who was also working on Munc18. They decided to make a mutation in Munc18 that could block the interaction with syntaxin, and then see what happened in real cells by doing a series of rescue experiments.

This involved deleting the function of Munc18 by RNAi interference in a cultured neuroendocrine PC12 cell line and then adding back in either the normal Munc18 protein or the mutated version and seeing if the knocked-out function of exocytosis could be rescued.

What Meunier predicted was a lovely rescue with the wild type, or normal form, of the Munc18 protein and nothing whatsoever with the mutant.

“We were very excited about doing this, but what we actually saw was a bit of a blow. A fantastic rescue with both wild type and mutant!” In all experiments, the syntaxin protein made it happily to the plasma membrane and neuroexocytosis proceeded as normal.

Although Meunier and his long-term collaborators, Shuzo Sugita and Brett Collins, published their findings in two papers in 2009, the gloss had certainly gone off this theory of a dual role for Munc18 in neurotransmission.

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“So to try and find out just what was going on, we started searching through hundreds of mutations in Munc18, and we found that basically whenever you change something in Munc18 that affects the transporting of syntaxin protein to the cell surface, you also have an effect on exocytosis, and it is almost a straight correlation,” he says.

“So, we are now thinking that Munc 18 is there just to transport syntaxin to the cell surface to execute fusion and thus exocytosis. It may be also doing something else, but we now think that this is the main role of Munc18, as a chaperone for syntaxin.”

Work from several groups including Meunier’s now support this chaperoning function of Munc18. At the Hunter meeting, Meunier discussed his current focus in this work, which is elucidating the trafficking event that regulates Munc18 delivery of syntaxin to the plasma membrane.

“We have found some pivotal things about that mechanism,” Meunier said. Although he could not elaborate in detail as the work is yet to be published. However, he did say that they have identified some of the other intracellular regulators of this critical trafficking mechanism. “Basically, if you don’t have syntaxin on the surface you don’t get efficient SNARE formation and you can’t have neurotransmission.”

Meunier knows that to tease out a definitive answer to all these questions about Munc18’s role or roles in neural cells, he will have to find just the right mutant or mutants of Munc18.

Having done hundreds of mutations already, he is optimistic that some of the recent versions might be the ones to dissect out the two functions of Munc18 – delivery of syntaxin to the plasma membrane and the priming of exocytosis – and even better, determine whether they are independent or interrelated.

“It is certainly not an easy exercise,” adds Meunier. “It is so difficult, in fact, that some of my collaborators now doubt that Munc18 has any effect on exocytosis, although I still believe that Munc18 does something to the SNARE complex.

“However, I think that unless we can find a mutation that does not affect the trafficking, but affects neurotransmission, we cannot be 100 per cent sure, and that is what we are looking for at the moment.”