A facile approach for the in vitro assembly of multimeric membrane transport proteins.

Membrane proteins such as ion channels and transporters are frequently homomeric. The homomeric nature raises important questions regarding coupling between subunits and complicates the application of techniques such as FRET or DEER spectroscopy. These challenges can be overcome if the subunits of a homomeric protein can be independently modified for functional or spectroscopic studies.

Combining in Vitro Folding with Cell Free Protein Synthesis for Membrane Protein Expression.

Cell free protein synthesis (CFPS) has emerged as a promising methodology for protein expression. While polypeptide production is very reliable and efficient using CFPS, the correct cotranslational folding of membrane proteins during CFPS is still a challenge. In this contribution, we describe a two-step protocol in which the integral membrane protein is initially expressed by CFPS as a precipitate followed by an in vitro folding procedure using lipid vesicles for converting the protein precipitate to the correctly folded protein.

Ion-binding properties of a K+ channel selectivity filter in different conformations.

K(+) channels are membrane proteins that selectively conduct K(+) ions across lipid bilayers. Many voltage-gated K(+) (KV) channels contain two gates, one at the bundle crossing on the intracellular side of the membrane and another in the selectivity filter. The gate at the bundle crossing is responsible for channel opening in response to a voltage stimulus, whereas the gate at the selectivity filter is responsible for C-type inactivation.

Engineering the glutamate transporter homologue GltPh using protein semisynthesis.

Glutamate transporters catalyze the concentrative uptake of glutamate from synapses and are essential for normal synaptic function. Despite extensive investigations of glutamate transporters, the mechanisms underlying substrate recognition, ion selectivity, and the coupling of substrate and ion transport are not well-understood. Deciphering these mechanisms requires the ability to precisely engineer the transporter.

Neurotransmitter transporters: structure meets function.

At synapses, sodium-coupled transporters remove released neurotransmitters, thereby recycling them and maintaining a low extracellular concentration of the neurotransmitter. The molecular mechanism underlying sodium-coupled neurotransmitter uptake is not completely understood. Several structures of homologs of human neurotransmitter transporters have been solved with X-ray crystallography.

Opposite movement of the external gate of a glutamate transporter homolog upon binding cotransported sodium compared with substrate.

Recently, a new model for glutamate uptake by glutamate transporters was proposed based on crystal structures of the bacterial glutamate transporter homolog Glt(Ph). It was proposed that hairpin two (HP2) functions as the extracellular gate and that Na(+) and glutamate binding closes HP2, thereby allowing for the translocation of the glutamate binding pocket across the membrane. However, the conformation of HP2 in the apo state and the Na(+) bound state is unknown.

Studies of ion channels using expressed protein ligation.

Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins.

GDNF and insulin cooperate to enhance the proliferation and differentiation of enteric crest-derived cells.

Previously we have shown that glial derived neurotrophic factor (GDNF) stimulates modest increases in the proliferation of avian enteric crest-derived cells and similar increases in the phosphorylation of the phosphoinositide 3-kinase (PI3K) downstream substrate Akt (Akt-P). In the present study we tested whether GDNF-independent increases in PI3K activation would be sufficient to support proliferation. We found that insulin induces a large increase in the phosphorylation of Akt and can initiate DNA synthesis in avian enteric crest-derived cells, but is unable to maintain proliferation over time in culture, measured by BrdU incorporation.

Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells.

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections.

Enteric neuroblasts require the phosphatidylinositol 3-kinase pathway for GDNF-stimulated proliferation.

The enteric nervous system (ENS) develops from neural crest cells that enter the gut, migrate, proliferate, and differentiate into neurons and glia. The growth factor glial-derived neurotrophic factor (GDNF) stimulates the proliferation and survival of enteric crest-derived cells. We investigated the intracellular signaling pathways activated by GDNF and their involvement in proliferation.