An eye-specific G beta subunit essential for termination of the phototransduction cascade.

Heterotrimeric G proteins couple various receptors to intracellular effector molecules. Although the role of the G alpha subunit in effector activation, guanine nucleotide exchange and GTP hydrolysis has been well studied, the cellular functions of the G beta subunits are less well understood. G beta gamma dimers bind G alpha subunits and anchor them to the membrane for presentation to the receptor. In specific systems, the G beta subunits have also been implicated in direct coupling to ion channels and to effector molecules. We have isolated Drosophila melanogaster mutants defective in an eye-specific G-protein beta-subunit (G beta e), and show here that the beta-subunit is essential for G-protein-receptor coupling in vivo. Remarkably, G beta mutants are also severely defective in the deactivation of the light response, demonstrating an essential role for the G beta subunit in terminating the active state of this signalling cascade.

Microinjection of the alpha-subunit of the G protein Go2, but not Go1, reduces a voltage-sensitive calcium current.

Calcium currents can be modulated by receptor activation of the GTP-binding protein G(o). We have determined whether the two forms of G(o), Go1 and Go2, differentially regulate calcium current magnitude. Using identified neurons of the pond snail Helisoma, we demonstrate that a high-voltage-activated (HVA) calcium current is reduced by addition of the neuropeptide Phe-Met-Arg-Phe-amide (FMRFamide) and that this inhibition is mediated by a pertussis toxin (PTX)-sensitive G protein pathway. Using this calcium current as an assay for G protein activity, we microinjected GTP gamma S-activated alpha-subunits of G proteins into neuronal somata. We demonstrate that the calcium current is differentially regulated by the two forms of alpha o. Microinjection of alpha o2*, but not alpha o1*, reduces calcium current magnitude.

Modulation of growth cone calcium current is mediated by a PTX-sensitive G protein.

Growth cones of isolated neurons B5 of Helisoma were voltage clamped in the whole-cell configuration. Depolarization of growth cones to -20 mV or greater activated a high-voltage-activated (HVA) calcium current. Addition of the neuropeptide FMRFamide (1 microM), which causes a presynaptic inhibition of synaptic transmission, reversibly reduced the calcium current magnitude. This inhibitory effect is mediated by a pertussis toxin (PTX)-sensitive G protein. Dialysis with the non-hydrolyzable GTP analogs GTP gamma S and Gpp(NH)p caused FMRFamide's effect to become irreversible. Dialysis with GDP beta S or preincubation with PTX prevented FMRFamide from reducing the calcium current. Thus, one role of growth cone G proteins is to modulate ion channels in growth cone membrane which in turn may control growth cone motility.

FMRFamide modulation of secretory machinery underlying presynaptic inhibition of synaptic transmission requires a pertussis toxin-sensitive G-protein.

The neuropeptide FMRFamide modulates synaptic transmission between identified neurons of the pond snail Helisoma trivolvis. FMRFamide causes a presynaptic inhibition of transmitter release by actions on ion channels and secretory machinery (Man-Son-Hing et al., 1989). The actions of FMRFamide on secretory machinery were studied using giant synapses that form between somata in culture. Using the calcium cage DM-nitrophen, synchronized, calcium-clamped release of neurotransmitter was promoted by UV photolysis. A series of UV flashes (15 msec duration) repeatedly promoted the transient synchronized release of neurotransmitter. Addition of FMRFamide reduced the magnitude of these flash-evoked inhibitory postsynaptic currents. Under conditions of synchronized transmitter release, FMRFamide modulates the secretory responsiveness to internal calcium. The release of neurotransmitter at somasoma synapses was determined to be quantal in nature. To test for the involvement of G-proteins in mediating the effects of FMRFamide on secretory machinery, the modulation of the frequency of miniature inhibitory postsynaptic currents (MIPSCs) was examined. Addition of FMRFamide reduced the frequency of MIPSCs without affecting intracellular free calcium measured with fura-2. Injection of a nonhydrolyzable analog of GTP, GTP gamma S, mimicked the effect of FMRFamide and reduced MIPSC frequency. Preinjection of the presynaptic soma with the A-protomer of pertussis toxin (PTX) prevented FMRFamide from reducing MIPSC frequency. Thus, a PTX-sensitive G-protein mediates the action of FMRFamide on secretory machinery. Similarly, preinjection of the presynaptic soma with PTX prevented FMRFamide from reducing the magnitude of action potential-evoked IPSC. Dose-response curves for the actions of FMRFamide on secretory machinery and calcium current were constructed and demonstrated that secretory machinery can be modulated at concentrations of FMRFamide (less than or equal to 10(-7) M) that do not affect calcium current magnitude. At a concentration of 10(-7) M FMRFamide, action potential-evoked synaptic transmission was reduced. Thus, synaptic transmission can be regulated by the modulation of secretory machinery, without a requirement for the modulation of ion channels.

A relation between synaptic specificity and the acquisition of presynaptic properties.

1. The specificity of synaptogenesis of identified adult neurons of Helisoma was determined in cell culture. Cholinergic neuron B5 indiscriminately forms the presynaptic element of chemical connections with novel cholinoceptive target neurons and muscle. By contrast, cholinergic neuron B19 is selective and discriminates between novel and appropriate target cells. Neuron B19 forms chemical connections with appropriate muscle targets only. 2. The acquisition of presynaptic properties independent of target contact was studied for both identified neurons. Functional connections form between neuron B5 and novel targets within seconds of contact, indicating that this cell has synthesized the presynaptic apparatus before target contact. In contrast, neuron B19 showed no evidence of possessing the ability to release neurotransmitter. 3. To further study the development of presynaptic properties, a model system of giant synaptic terminals was developed. The soma of neuron B5, acutely isolated from the nervous system is non-secretory. In conditions that prevent the extension of neurites, somata gain the ability to release neurotransmitter. This experimentally tractable system was used to study the calcium currents of presynaptic neuron B5. Acutely-isolated non-secretory somata contain two types of calcium currents: low-voltage-activated (LVA) and high-voltage-activated (HVA). The types of calcium currents in the soma change when B5 gains its secretory capacity. Secretory somata contain HVA calcium current only. 4. Neuron B5 was also plated in conditions which permit the extension of neurites. LVA and HVA calcium currents were maintained in its soma (non-secretory) but HVA calcium current only was maintained in its growth cones (secretory). Thus, B5 differentially regulates the presence of specific calcium currents in its membrane in relation to local secretory capacity without target-derived cues. 5. These data suggest that neuron B5 has an intrinsic program which generates presynaptic calcium channels and secretory apparatus prior to target contact. This autonomy of initial presynaptic development may underlie the lack of target cell discrimination exhibited by B5 in synaptogenesis.

A neuromodulator of synaptic transmission acts on the secretory apparatus as well as on ion channels.

The mechanisms that underlie synaptic plasticity have been largely inferred from electrophysiological studies performed at sites remote from synaptic terminals. Thus the mechanisms involved in plasticity at the secretory sites have remained ill-defined. We have now used somatic synapses of cultured Helisoma neurones to directly assess presynaptic ion conductances and study the secretory apparatus. At these synapses we determined the actions of a modulatory neuropeptide, Phe-Met-Arg-Phe-NH2 (FMRFa), on the release of the neurotransmitter acetylcholine (ACh). Using voltage- and calcium-clamp techniques, we have demonstrated that FMRFa causes a presynaptic inhibition of ACh release by (1) reducing the magnitude of the voltage-dependent calcium current, and (2) regulating the secretory apparatus. The photolabile calcium cage, nitr-5 (refs 3-8), was dialysed into the presynaptic cell. In response to ultraviolet light, calcium was released from nitr-5 and ACh secretion was stimulated. Under conditions of constant internal calcium, FMRFa reduced the rate of ACh release. Thus we conclude that FMRFa reduces the influx of calcium during the action potential and decreases the sensitivity of the secretory apparatus to elevated internal calcium, thereby contributing to a presynaptic inhibition of transmitter release.

Low- and high-voltage-activated calcium currents: their relationship to the site of neurotransmitter release in an identified neuron of Helisoma.

In this study we have characterized two types of Ca2+ currents in identified neuron B5 of Helisoma and have examined the relationship between these currents and neurotransmitter release. Neuron B5 contains low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca2+ currents. These currents have distinct electrophysiological and pharmacological properties. To gain access to the site of neurotransmitter release, we used a model system in which somata that do not extend neurites assume the role of neurotransmitter release. Before somata gain the ability to release neurotransmitter, they contain LVA and HVA Ca2+ currents. After 3 days of culture, when spherical somata have gained the secretory capacity, only the HVA Ca2+ current is present. Experiments were also performed when neurite extension was permitted. These data indicate that neurons with processes have a differential distribution of Ca2+ currents. The soma, which does not release neurotransmitter, contains both LVA and HVA Ca2+ currents, while distal secretory processes contain only HVA current.