Mechanism of α-conotoxin Vc1.1 inhibition of Cav2.2 channel

Huynh, Thuan G. (2013). Mechanism of α-conotoxin Vc1.1 inhibition of Cav2.2 channel PhD Thesis, Queensland Brain Institute, The University of Queensland.

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Author Huynh, Thuan G.
Thesis Title Mechanism of α-conotoxin Vc1.1 inhibition of Cav2.2 channel
Formatted title
Mechanism of α-conotoxin Vc1.1 inhibition of Cav2.2 channel
School, Centre or Institute Queensland Brain Institute
Institution The University of Queensland
Publication date 2013
Thesis type PhD Thesis
Supervisor David Adams
Joe Lynch
Total pages 107
Total colour pages 23
Total black and white pages 84
Language eng
Subjects 110902 Cellular Nervous System
111501 Basic Pharmacology
1115 Pharmacology and Pharmaceutical Sciences
1116 Medical Physiology
111601 Cell Physiology
1109 Neurosciences
Formatted abstract
Pain sensation can be divided into acute and chronic pain. Acute pain results from soft tissue injury or inflammation, and its function is to protect the body from further injury. Chronic pain is a sustained painful sensation that occurs for longer periods of time, independently of its cause.

Currently, opioid drugs like morphine are the analgesic drugs most commonly used to alleviate pain. Opioids and compounds, such as the GABAB receptor (GABABR) agonist baclofen, work through similar pathways by binding to specific G protein-coupled receptors (GPCR) to activate Gβγ subunits. These subsequently bind to Cav2.2 channels to inhibit nociception. However, opioids and baclofen have unwanted side effects and their prolonged use leads to the development of tolerance, making them less effective. Therefore, new types of analgesic drugs are urgently needed. One promising candidate for such a drug is α-conotoxin Vc1.1, which originates from the venom of a predatory marine cone snail. Vc1.1 has not only been shown to potently relieve mechanical allodynia in animal pain models, but studies have also revealed it has more beneficial properties than conventional drugs. For example, it prolonged anti-nociception for several weeks and even accelerated functional recovery of injured neurons after injury.

In my laboratory, we have shown that analgesic α-conotoxin Vc1.1 indirectly inhibits Cav2.2 channels via GABABR activation in rat dorsal root ganglion (DRG) neurons. A subsequent study, in which siRNA was used to knock down GABABR in rat DRG neurons, supported GABABR’s essential role in mediating Vc1.1 inhibition of Cav2.2 channels.
In the first part of my research, I explored the Vc1.1 inhibitory pathway. GABABR-mediated inhibition of Cav2.2 channels (α1B, α2δ1 and β3) was successfully reconstituted in human embryonic kidney 293 (HEK293) cells. In addition, by transfecting different GABABR subunit cDNA concentrations relative to the constant Cav2.2 channel expression, I showed that the onset of Vc1.1 time course inhibition of Cav2.2 currents was significantly influenced by GABABR expression levels. In contrast, baclofen produced similar Cav2.2 current inhibition in low and high GABABRs expressing cells. This finding suggests that the GABABR and Cav2.2 channel need to be in close proximity with each other in the Vc1.1 inhibitory pathway.

Using the whole-cell patch-clamp technique, I characterised the Cav2.2 channel kinetics in the presence of GABA, baclofen and α-conotoxin Vc1.1. Vc1.1 shifted the voltage-dependence of Cav2.2 current activation to a more negative potential and increased the time course of Cav2.2 current activation in the cells transfected with GABAB subunits. After pre-treating GABABR transfected HEK293 cells with pertussis toxin (PTX) to antagonise G protein coupling to the GABABR, I showed that Vc1.1 inhibits Cav2.2 channels coupled to GABABRs and that this is dependent on PTX-sensitive Gi/o proteins. I also showed that Vc1.1 inhibition of Cav2.2 channels via GABABR is voltage-independent (VI). However, the splice variant Cav2.2-e371a, which has been shown to mediate VI inhibition of Cav2.2 currents, did not appear to be involved.

In the second part of my research project, various GABABR mutants were constructed and used to elucidate Vc1.1 inhibition of Cav2.2 channels. By introducing a point mutation from an arginine to aspartic acid at position 576 in the GABAB2 subunit, which has been shown to abolish baclofen’s activation of G proteins but not couple to the GABABR, I demonstrated that Vc1.1 signalling does not work through the conventional baclofen-mediated Cav2.2 inhibition pathway.

By using various GABABR mutants with or without their PCT domain, I showed that Vc1.1 inhibition of Cav2.2 channels via the GABABR is dependent on the PCT domain of the GABAB1a subunit. In addition, I discovered that by adding Ca2+ to the intracellular recording solution, Vc1.1 inhibition of Cav2.2 currents can be increased.

In conclusion, these findings support the idea that Vc1.1 inhibits Cav2.2 channels via a novel mechanism that is different from that of GABA- and baclofen-mediated modulation of Cav2.2 channels. This novel mechanism requires PTX–sensitive Gi/o proteins and is dependent on the GABAB1a subunit’s PCT domain, which may be in close proximity to the Cav2.2 channel. Overall, these findings provide important insights into how the GABABR contributes to Cav2.2 channel inhibition, and show a new mechanism of α-conotoxin Vc1.1 inhibition of nociceptive transmission.
Keyword Analgesic
GABAB receptor
G proteins
HEK293 cells

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Created: Thu, 17 Oct 2013, 12:51:28 EST by Thuan Huynh on behalf of Scholarly Communication and Digitisation Service