GTP, dynamin and synaptic transmission

The goal of my professional work on the Ketogenic Diet is to identify the biophysical property altered by the diet that is responsible for its anti-epileptic properties.  Along with that, I would like to identify the link between cytoplasmic biochemistry and membrane electrophysiology.  My working hypothesis at the moment for both of these centers on the molecule GTP as described below.

GTP (guanosine-5'-triphosphate.) is an energy storage molecule very similar to ATP.  GTP is produced from GDP directly in the Krebs cycle by succinyl-CoA synthetase and it can also be produced when a high-energy phosphate is transferred from ATP to GTP via the enzyme  nucleoside diphosphokinase (NDPK).  GTP is converted back to GDP by many cellular processes, including PEPCK, which transfers a high-energy phosphate from GTP to oxaloacetate during the formation of PEP.  My model for the mechanism of action of the anti-epileptic effect of the Ketogenic Diet relies on this reaction.

Synaptic transmission, and the release of synaptic vesicles is a very complicated process that is not completely understood.  In general, synaptic transmission begins when an action potential invades a pre-synaptic terminal.  This leads to the opening of voltage-gated calcium channels and rise in pre-synaptic calcium concentrations.  Calcium causes synaptic vesicles to fuse with the extracellular terminal (exocytosis) and release neurotransmitter in to the synapse to bind at post-synaptic receptors.  In most synapses, there are a very limited number of vesicles that respond quickly to increases in calcium and undergo exocytosis.  If the frequency of action potentials is high, the synapse will quickly be depleted of fast-responding vesicles.  The number of fast-responding vesicles has been termed the readily releasable pool (RRP) of synaptic vesicles.  There are many ways to replenish the RRP, but the fastest way is to reclaim the vesicles that had recently fused.  This process is called endocytosis, which involves many proteins and enzymatic steps.  One critical step is the physical separation of the new vesicles membrane from the extracellular membrane. Dynamin is one enzyme involved in endocytosis and has been describes as a “pinchase” because it helps pinch the vesicle off from the plasma membrane.   The action of dynamin to pinch off a vesicle for endocytosis requires the conversion of GTP to GDP.  This has been demonstrated anatomically by electron microscopy, and physiologically using the patch clamp technique.

A series of paper on the effect of pre-synaptic GTP have recently been published.  They use similar methodology to directly change intracellular concentration of GTP in the synaptic terminal.  These experiments have been done at a synapse in the brain named the Calyx of Held.  This particular synapse has been the site of some exciting neurophysiological research because the pre-synaptic terminal is very large, and a path-clamp electrode can be attached to it.  A second patch clamp electrode can then be placed on the post-synaptic cell.  This gives researchers the ability to measure the electrical properties of both sides of the synapse as well as control the intracellular milieu of each one independently.  Using this method, a group from the University of Tokyo has shown that removing pre-synaptic GTP does not alter normal synaptic transmission.  However, during high frequency stimulation synaptic transmission falters faster without GTP than with it.  Further experiments investigated the time course of refilling of the readily releasable pool of synaptic vesicles after they were emptied.  Synapses without GTP refilled much more slowly that synapses with GTP present.    There appear to be multiple steps where GTP is active during the synaptic vesicle cycle.  But, buy directly measuring the fusion and budding of synaptic vesicles, the same group demonstrated that dynamin-mediated endocytosis to be the most important GTP utilizing step for re-filling of the RRP.  The dynamics of synaptic release is a very hot topic in the epilepsy research field, as well as in the neuroscience field as a whole.  Read how GTP and vesicle cycling fit in to my model of the anti-epileptic properties of the Ketogenic Diet here.

Vesicle endocytosis requires dynamin-dependent GTP hydrolysis at a fast CNS synapse. Yamashita T, Hige T, Takahashi T.  Science. 2005 Jan 7;307(5706):124-7.

Minimum essential factors required for vesicle mobilization at hippocampal synapses. Mozhayeva MG, Matos MF, Liu X, Kavalali ET. J Neurosci. 2004 Feb 18;24(7):1680-8. [PDF]

The role of GTP-binding protein activity in fast central synaptic transmission. Takahashi T, Hori T, Kajikawa Y, Tsujimoto T.  Science. 2000 Jul 21;289(5478):460-3.

Myristyl trimethyl ammonium bromide and octadecyl trimethyl ammonium bromide are surface-active small molecule dynamin inhibitors that block endocytosis mediated by dynamin I or dynamin II.     Quan A, McGeachie AB, Keating DJ, van Dam EM, Rusak J, Chau N, Malladi CS, Chen C, McCluskey A, Cousin MA, Robinson PJ.  Mol Pharmacol. 2007 Dec;72(6):1425-39.

Copyright 2011 Steve Kriegler