P. T. Choy

Solving the α-Conotoxin Folding Problem: Efficient Selenium-Directed On-Resin Generation of More Potent and Stable Nicotinic Acetylcholine Receptor Antagonists

Alpha-conotoxins are tightly folded miniproteins that antagonize nicotinic acetylcholine receptors (nAChR) with high specificity for diverse subtypes. Here we report the use of selenocysteine in a supported phase method to direct native folding and produce alpha-conotoxins efficiently with improved biophysical properties. By replacing complementary cysteine pairs with selenocysteine pairs on an amphiphilic resin, we were able to chemically direct all five structural subclasses of alpha-conotoxins exclusively into their native folds. X-ray analysis at 1.4 A resolution of alpha-selenoconotoxin PnIA confirmed the isosteric character of the diselenide bond and the integrity of the alpha-conotoxin fold. The alpha-selenoconotoxins exhibited similar or improved potency at rat diaphragm muscle and alpha3beta4, alpha7, and alpha1beta1 deltagamma nAChRs expressed in Xenopus oocytes plus improved disulfide bond scrambling stability in plasma. Together, these results underpin the development of more stable and potent nicotinic antagonists suitable for new drug therapies, and highlight the application of selenocysteine technology more broadly to disulfide-bonded peptides and proteins.

Neuromuscular synapses mediate motor axon branching and motoneuron survival during the embryonic period of programmed cell death

The embryonic period of motoneuron programmed cell death (PCD) is marked by transient motor axon branching, but the role of neuromuscular synapses in regulating motoneuron number and axonal branching is not known. Here, we test whether neuromuscular synapses are required for the quantitative association between reduced skeletal muscle contraction, increased motor neurite branching, and increased motoneuron survival. We achieved this by comparing agrin and rapsyn mutant mice that lack acetylcholine receptor (AChR) clusters. There were significant reductions in nerve-evoked skeletal muscle contraction, increases in intramuscular axonal branching, and increases in spinal motoneuron survival in agrin and rapsyn mutant mice compared with their wild-type littermates at embryonic day 18.5 (E18.5). The maximum nerve-evoked skeletal muscle contraction was reduced a further 17% in agrin mutants than in rapsyn mutants. This correlated to an increase in motor axon branch extension and number that was 38% more in agrin mutants than in rapsyn mutants. This suggests that specializations of the neuromuscular synapse that ensure efficient synaptic transmission and muscle contraction are also vital mediators of motor axon branching. However, these increases in motor axon branching did not correlate with increases in motoneuron survival when comparing agrin and rapsyn mutants. Thus, agrin-induced synaptic specializations are required for skeletal muscle to effectively control motoneuron numbers during embryonic development.

Role of calcium and vesicle-docking proteins in remobilising dormant neuromuscular junctions in desert frogs

Despite prolonged immobility the desert frog, Cyclorana alboguttata, suffers little impairment in muscle function. To determine compensatory mechanisms at neuromuscular junctions, transmitter release was examined along primary terminals in C. alboguttata iliofibularis muscle. Using extracellular recording we found the amplitudes of evoked endplate currents were significantly smaller in dormant frogs. In active frogs we identified two negatively sloping proximal–distal gradients of transmitter frequency and quantal content; a shallow proximal–distal gradient with low probability of transmitter release (<0.2) and a second much steeper proximal–distal gradient for quantal content with high probability release sites (>0.6). During aestivation, only a shallow gradient was identified. The high probability release sites in control frogs were inhibited during aestivation by a mechanism that could be reversed by (1) increasing the extracellular calcium concentration, and (2) increasing the frequency of stimulation. This suggests that transmitter vesicles are available during aestivation but not released. We quantified expression of messenger RNA transcripts coding for the transmitter vesicle-docking proteins synaptotagmin 1, syntaxin 1B and UNC-13. All three were rare transcripts maintained at control values during aestivation. Neuromuscular remobilisation after dormancy in C. alboguttata is more likely a product of rapidly reversible physiologic mechanisms than reorganisations of the neuromuscular transcriptome.

A low-cost CMOS neurological sensor array

Current methods used to study neural communication have not been able to achieve both good spatial and temporal resolution of recordings. There are two ways to record synaptic potentials from nerve endings: recordings using single or dual intracellular or extra cellular metal electrodes give good temporal resolution but poor spatial resolution, and recording activity with fluorescent dyes gives good spatial resolution but poor temporal resolution. Such medical research activity in the area of neurological signal detection has thus identified a requirement for the design of a CMOS circuit that contains an array of independent sensors. As both spatial and temporal distribution of acquired data is required in this application, the circuit must be capable of continuous measurement of synaptic potentials from an array of points on a tissue sample, with a 10 μm separation between sensor points. The major requirement for the circuit is that it is capable of sensing synaptic potentials of the order of several mV, with a resolution of 0.05 mV. For data recording purposes, the circuit must amplify these synaptic potentials and digitise them together with their locations in the sensor array. Finally, the circuit must be biologically inert, to avoid specimen deterioration. This paper presents the design of a prototype single-chip circuit, which provides a 6 x 3 array of independent synaptic potential sensors. The signal from each of the sensors is amplified and time-multiplexed into an on-chip A/D converter. The circuit provides an 8-bit synaptic potential value, together with an 8-bit field containing array location and trigger signals suitable for external data acquisition instrumentation. Our test circuit is implemented in a low-cost 0.5 um, 5 V CMOS process. The fabricated die is mounted in a standard 40 pin DIP ceramic package, with no lid to allow direct contact of the die surface with the tissue sample. The only post-processing step required for these packages is to encapsulate the exposed bond wires to ensure that the device is biologically inert. No further processing of the silicon die is required. Both the circuit design and the chip performance will be presented in the seminar.