GTP-binding protein βγ subunits mediate presynaptic calcium current inhibition by GABA _B receptor

Abstract

A variety of GTP-binding protein (G protein)-coupled receptors are expressed at the nerve terminals of central synapses and play modulatory roles in transmitter release. At the calyx of Held, a rat auditory brainstem synapse, activation of presynaptic ␥-aminobutyric acid type B receptors (GABAB receptors) or metabotropic glutamate receptors inhibits presynaptic P͞Q-type Ca 2؉ channel currents via activation of G proteins, thereby attenuating transmitter release. To identify the heterotrimeric G protein subunits involved in this presynaptic inhibition, we loaded G protein ␤␥ subunits (G␤␥) directly into the calyceal nerve terminal through whole-cell patch pipettes. G␤␥ slowed the activation of presynaptic Ca 2؉ currents (IpCa) and attenuated its amplitude in a manner similar to the externally applied baclofen, a GABAB receptor agonist. The effects of both G␤␥ and baclofen were relieved after strong depolarization of the nerve terminal. In addition, G␤␥ partially occluded the inhibitory effect of baclofen on IpCa. In contrast, guanosine 5 -O-(3-thiotriphosphate)-bound Go␣ loaded into the calyx had no effect. Immunocytochemical examination revealed that the subtype of G proteins Go, but not the Gi, subtype, is expressed in the calyceal nerve terminal. These results suggest that presynaptic inhibition mediated by G protein-coupled receptors occurs primarily by means of the direct interaction of Go ␤␥ subunits with presynaptic Ca 2؉ channels.I n the central nervous system, synaptic transmission is regulated by presynaptic autoreceptors or heteroreceptors. These receptors are coupled by G proteins to various targets such as Ca 2ϩ channels, K ϩ channels, or the exocytotic machinery downstream of Ca 2ϩ influx (1). At a rat brainstem auditory synapse formed by a giant nerve terminal, the calyx of Held, metabotropic glutamate receptors (mGluRs), or ␥-aminobutyric acid type B receptors (GABA B receptors) primarily inhibit P͞Q-type Ca 2ϩ channels (2-4). The inhibitory effect of the GABA B receptor agonist baclofen on presynaptic Ca 2ϩ currents can be blocked by the GDP analog guanosine 5Ј-O-(2-thiodiphosphate) (GDP [␤S]) and occluded by the nonhydrolyzable GTP analog guanosine 5Ј-O-(3-thiotriphosphate) (GTP[␥S]), both loaded directly into the calyx, indicating that G proteins mediate the presynaptic inhibition (3). At the cell soma, heterotrimeric G proteins attenuate Ca 2ϩ currents acting either directly via a membrane-delimited pathway involving G protein ␤␥ subunits (G␤␥) or indirectly via second messengers (5, 6). At the nerve terminal, however, it is not known which mechanism underlies this presynaptic inhibition. At the calyx of Held, which can be visually identified in slice, it is possible to load various molecules into the nerve terminal through whole-cell recording pipette (3,7,8). Using this technique, we examined the effect of G␤␥ on I pCa . Also, because of its large structure, it was possible to use immunocytochemistry to determine the subtype of G proteins expressed in the calyceal nerve terminal. Our results...

Exogenous nerve growth factor attenuates opioid-induced inhibition of voltage-activated Ba2+ currents in rat sensory neurons

“…The βγ subunits of the G protein heterotrimer dissociate from the α subunit and mediate inhibition of VGCC by direct binding to the α subunit of the VGCC (Herlitze et al, 1996;Ikeda, 1996;Zamponi et al, 1997;Delmas et al, 1998;Zamponi and Snutch, 1998). Because the GABA B receptor has also been shown to inhibit VGCC by activating G o (Menon-Johansson et al, 1993;Kajikawa et al, 2001), the lack of effect of NGF on baclofen-mediated inhibition of I Ba in the present study indicates that NGF did not alter the function of G protein subunits or their coupling to VGCC. Further evidence for normal function downstream of the opioid receptor is the lack of effect of NGF on direct G protein activation with GTP-γ-S.…”

Heterogeneous Ca2+ influx along the adult calyx of held: A structural and computational study

“…This larger conductance set was used because it resulted in a narrower action potential with an appropriate undershoot, as compared to the weaker conductances. The Ca 2+ current, as measured by Borst and Sakmann (1998), was 2 nA at room temperature, and about 4.5 nA at 36°C (at −30 mV; also see Cuttle et al, 1998;Kajikawa et al, 2001;and Ishikawa et al, 2005). Depending on conditions (R a , g lk , and channel distribution), a Ca 2+ conductance of 0.003 S/cm 2 was sufficient to produce currents of 4.5 nA at −20 mV at 37°C.…”

“…The Na + channels and low-voltage activated K + channels (Kv1.1/Kv1.2/Kv1.6) are absent from the calyx itself (Dodson et al, 2003;Leao et al, 2005). However, the calyx was populated with high-voltage activated K + channels (Kv3.1; Dodson et al, 2003, Elezegari et al, 2003 and Ca 2+ channels (in adults, P-type; see Kajikawa et al, 2001 andIshikawa et al, 2005). The uniform model had an even density for each conductance in all calyx compartments.…”

Activation of metabotropic GABA receptors increases the energy barrier for vesicle fusion

“…The reduction of transmitter release is for the most part achieved by the closing of voltage-dependent Ca 2+ channels (VDCCs) (Dunlap and Fischbach, 1981). This conversion of VDCCs into the 'reluctantly opening state' occurs by G-protein bc-subunit-mediated signal transduction in a membrane-delimited pathway, as has been directly demonstrated for the calyx of Held (Kajikawa et al, 2001;Takahashi et al, 1998).…”