| Abstract No.: | B-B2052 |
| Country: | Canada |
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| Title: | ANALYSES OF THE MODULATORY CAPACITY OF CAV2 CHANNELS BY G PROTEINS |
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| Authors/Affiliations: | 1 Xuan Huang*; 1 Patrick McCamphill; 1 Phuc Pham; 1 Danielle Nash; 1 Taylor Dawson; 1 David Spafford;
1 University of Waterloo, ON, Canada
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| Content: | Introduction: Specialized voltage-gated calcium channels in the Cav2 channel class (such as Cav2.2, N-type) mediate neurotransmitter release from presynaptic nerve terminals. Cav2.2 channels are exquisitely sensitive to inhibition by G protein-coupled receptors. The ubiquitous form of G protein modulation is a fast, membrane delimited, voltage-dependent form of regulation, which is relieved in proportion to the frequency of firing of action potentials. One can mimic this form of regulation using a strong depolarizing prepulse, which temporary relieves the direct inhibition of Cav2.2 channels by G protein βγ subunits. Mechanisms for a slower, voltage-independent form of G protein regulation have been identified in select cell types, such as mammalian sensory and sympathetic neurons.
LCav2, a molluscan (snail) homolog, serves a similar function as a mediator of transmitter release in the nervous system and is almost indistinguishable from mammalian Cav2.2 in biophysical characteristics observed in vitro. Interestingly, while lacking the more ubiquitous, voltage-dependent regulation, snail LCav2 channel predominantly bears likeness to the less common, voltage-independent form of G protein regulation observed in mammals. The dramatic difference between these homologs (Cav2.2 and LCav2), provides an opportunity to explore the structural features that govern their unique G protein regulation mechanisms.
Objectives: The objectives of our research includes: 1) evaluate the features of G protein regulation of LCav2 channels in vitro and in neurons. 2) employ LCav2 channel as a surrogate to evaluate the features responsible for voltage-dependent G protein regulation.
Materials and Methods: We have swapped major cytoplasmic regions of Cav2.2 into LCav2 to identify the critical binding sites for voltage-dependent, G protein regulation. Wild type and chimeric channels were evaluated by whole-cell patch clamp recording in HEK293T cell lines.
Results: Our experiments so far have revealed the following: a) mammalian Cav2.2 but not snail LCav2 channel is modulated by co-expression of a snail Gβ subunit. These data indicate that the difference in Gβ subunit is not the cause of the absence of voltage-dependent modulation in invertebrates; b) Swapping of the N-terminus, I-II linker, the II-III linker or C-terminus of Cav2.2 alone is enough to endow LCav2 channels with voltage-dependent modulation. The involvement of multiple cytoplasmic regions in voltage-dependent G protein regulation is consistent with recent findings (Agler et al. Neuron 2005 46(6):891-904). c) Voltage-independent modulation of LCav2 is irreversible, activated by GTP, is pertussis toxin sensitive and mimicked by cAMP activation in vitro and in neurons.
Conclusion: The modulation of synaptic calcium channels by G proteins appears as a universal phenomenon. Different species and neuronal types express unique forms of G protein regulation that provides specialized modulatory capacities.
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