Analysis of the structural determinants for voltage-dependent G protein modulation of synaptic Cav2 channels

[摘要] 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 by strong depolarizations. LCav2, an invertebrate homolog from the pulmonate snail Lymnaea stagnalis, serves a similar function as a mediator of transmitterrelease in the nervous system. To examine the G protein modulation capacity in invertebrates, LCav2 was cloned to a bicistronic expression vector pIRES2-EGFP and expressed in HEK293T cells. Although LCav2 was almost indistinguishable from mammalian Cav2.2 in biophysical characteristics observed in vitro, snail LCav2 channel lacked the property ofvoltage dependent G protein modulation. The structural elements essential for the voltagesensitivity to G protein modulation were explored by swapping the N-terminus and I-II linker regions of rCav2.2 channels into LCav2 calcium channels. Functional comparisons were also made using both mammalian and invertebrate homologs of G protein beta subunits, Gβ1. Neither the N-terminus or I-II linker region of Cav2.2 alone, nor the invertebrate G proteinbeta subunit was sufficient for voltage-dependent G protein modulation. Further analyses using chimeric channels and G protein subunits will be required to find the minimal structural determinants for voltage-dependent G protein modulation.

[发布机构] University of Waterloo

[关键词] Biology