Meyer B. Jackson

cGMP and S-nitrosylation: two routes for modulation of neuronal excitability by NO

The nitric oxide (NO)-cGMP signaling cascade has been implicated in synaptic plasticity and, more broadly, in the control of many forms of electrical activity. This raises the issue of how these second messengers regulate ion channels. The field of ion-channel modulation is dominated by G proteins; NO and cGMP are often treated as poor cousins. However, recent advances surveyed here could change this perception. A surprising new dimension to NO signaling is the direct cGMP-independent action of NO on channel proteins through S-nitrosylation. The existence of two effector pathways has important functional implications, expanding and enriching the possibilities for modulating neuronal excitability.

The Sigma Receptor as a Ligand-Regulated Auxiliary Potassium Channel Subunit

The sigma receptor is a novel protein that mediates the modulation of ion channels by psychotropic drugs through a unique transduction mechanism depending neither on G proteins nor protein phosphorylation. The present study investigated sigma receptor signal transduction by reconstituting responses in Xenopus oocytes. Sigma receptors modulated voltage-gated K+ channels (Kv1.4 or Kv1.5) in different ways in the presence and absence of ligands. Association between Kv1.4 channels and sigma receptors was demonstrated by coimmunoprecipitation. These results indicate a novel mechanism of signal transduction dependent on protein-protein interactions. Domain accessibility experiments suggested a structure for the sigma receptor with two cytoplasmic termini and two membrane-spanning segments. The ligand-independent effects on channels suggest that sigma receptors serve as auxiliary subunits to voltage-gated K+ channels with distinct functional interactions, depending on the presence or absence of ligand.

The hallucinogen N,N-dimethyltryptamine (DMT) is an endogenous sigma-1 receptor regulator.

The sigma-1 receptor is widely distributed in the central nervous system and periphery. Originally mischaracterized as an opioid receptor, the sigma-1 receptor binds a vast number of synthetic compounds but does not bind opioid peptides; it is currently considered an orphan receptor. The sigma-1 receptor pharmacophore includes an alkylamine core, also found in the endogenous compound N,N-dimethyltryptamine (DMT). DMT acts as a hallucinogen, but its receptor target has been unclear. DMT bound to sigma-1 receptors and inhibited voltage-gated sodium ion (Na+) channels in both native cardiac myocytes and heterologous cells that express sigma-1 receptors. DMT induced hypermobility in wild-type mice but not in sigma-1 receptor knockout mice. These biochemical, physiological, and behavioral experiments indicate that DMT is an endogenous agonist for the sigma-1 receptor.

Capacitance steps and fusion pores of small and large-dense-core vesicles in nerve terminals

The vesicles that package neurotransmitters fall into two distinct classes, large dense-core vesicles (LDCVs) and small synaptic vesicles, the coexistence of which is widespread in nerve terminals. High resolution capacitance recording reveals unitary steps proportional to vesicle size. Measurements of capacitance steps during LDCV and secretory granule fusion in endocrine and immune cells have provided important insights into exocytosis; however, extending these measurements to small synaptic vesicles has proven difficult. Here we report single vesicle capacitance steps in posterior pituitary nerve terminals. These nerve terminals contain neuropeptide-laden LDCVs, as well as microvesicles. Microvesicles are similar to synaptic vesicles in size, morphology and molecular composition, but their contents are unknown. Capacitance steps of two characteristic sizes, corresponding with microvesicles and LDCVs, were detected in patches of nerve terminal membrane. Both types of vesicles fuse in response to depolarization-induced Ca2+ entry. Both undergo a reversible fusion process commonly referred to as ‘kiss-and-run’, but only rarely. Fusion pores seen during microvesicle kiss-and-run have a conductance of 19 pS, 11 times smaller than LDCV fusion pores. Thus, LDCVs and microvesicles use structurally different intermediates during exocytosis.

Different domains of synaptotagmin control the choice between kiss-and-run and full fusion

Exocytosis—the release of the contents of a vesicle—proceeds by two mechanisms. Full fusion occurs when the vesicle and plasma membranes merge. Alternatively, in what is termed kiss-and-run, vesicles can release transmitter during transient contacts with the plasma membrane. Little is known at the molecular level about how the choice between these two pathways is regulated. Here we report amperometric recordings of catecholamine efflux through individual fusion pores. Transfection with synaptotagmin (Syt) IV increased the frequency and duration of kiss-and-run events, but left their amplitude unchanged. Endogenous Syt IV, induced by forskolin treatment, had a similar effect. Full fusion was inhibited by mutation of a Ca2+ ligand in the C2A domain of Syt I; kiss-and-run was inhibited by mutation of a homologous Ca2+ ligand in the C2B domain of Syt IV. The Ca2+ sensitivity for full fusion was 5-fold higher with Syt I than Syt IV, but for kiss-and-run the Ca2+ sensitivities differed by a factor of only two. Syt thus regulates the choice between full fusion and kiss-and-run, with Ca2+ binding to the C2A and C2B domains playing an important role in this choice.

Fusion pore dynamics are regulated by synaptotagmin*t-SNARE interactions

Exocytosis involves the formation of a fusion pore that connects the lumen of secretory vesicles with the extracellular space. Exocytosis from neurons and neuroendocrine cells is tightly regulated by intracellular [Ca2+] and occurs rapidly, but the molecular events that mediate the opening and subsequent dilation of fusion pores remain to be determined. A putative Ca2+ sensor for release, synaptotagmin I (syt), binds directly to syntaxin and SNAP-25, which are components of a conserved membrane fusion complex. Here, we show that Ca2+-triggered syt*SNAP-25 interactions occur rapidly. The tandem C2 domains of syt cooperate to mediate binding to syntaxin/SNAP-25; lengthening the linker that connects C2A and C2B selectively disrupts this interaction. Expression of the linker mutants in PC12 cells results in graded reductions in the stability of fusion pores. Thus, the final step of Ca2+-triggered exocytosis is regulated, at least in part, by direct contacts between syt and SNAP-25/syntaxin.

Adaptation of Ca2+-Triggered Exocytosis in Presynaptic Terminals

Rapid increases in Ca2+ concentration, produced by photolysis of caged Ca2+, triggered exocytosis in squid nerve terminals. This exocytosis was transient in nature, decaying with a time constant of approximately 30 ms. The decay could not be explained by a decline in presynaptic Ca2+ concentration, depletion of synaptic vesicles, or desensitization of postsynaptic receptors. Experiments in which Ca2+ was increased either in a series of steps or continuously at different rates suggested that the decay is caused by adaptation of the exocytotic Ca2+ receptor to higher levels of Ca2+. This adjustable sensitivity to Ca2+ represents a novel property of the triggering mechanism that can be used to evaluate molecular models of exocytosis. Adaptation can limit the amount of transmitter released by a nerve terminal and permit the speed of a presynaptic Ca2+ rise to serve as a critical determinant of synaptic efficacy.

A unique amino acid of the Drosophila GABA receptor with influence on drug sensitivity by two mechanisms.

1. The Drosophila gene Rdl (resistance to dieldrin) encodes a GABA receptor. An alanine‐to‐serine mutation in this gene at residue 302 confers resistance to cyclodiene insecticides and picrotoxin. Patch clamp analysis of GABA receptors in cultured neurons from wild type and mutant Drosophila was undertaken to investigate the biophysical basis of resistance. 2. In cultured neurons from both wild type and mutant strains, GABA activated a channel that reversed near 0 mV in symmetrical chloride. GABA dose‐response characteristics of wild type and mutant receptors were very similar. 3. GABA responses in neurons from the mutant strains showed reduced sensitivity to the GABA antagonists picrotoxin, lindane and t‐butyl‐bicyclophosphorothionate. Resistance ratios were 116, 970 and 9 for the three blockers, respectively. Inhibition increased with blocker concentration in a manner consistent with saturation of a single binding site. 4. The mutation reduced the single channel conductance by 5% for inward current and 17% for outward current. The single channel current was approximately 60% lower for outward current than for inward current in both wild type and mutant. 5. Open and closed times were both well fitted by the sum of two exponentials. Resistance was associated with longer open times and shorter closed times, reflecting a net stabilization of the channel open state by a factor of approximately five. 6. The mutation was associated with a marked reduction in the rate of GABA‐induced desensitization, and a net destabilization of the desensitized conformation by a factor of 29. 7. The Rdl mutation manifests resistance through two different mechanisms. (a) The mutation weakens drug binding to the antagonist‐favoured (desensitized) conformation by a structural change at the drug binding site. (b) The mutation destabilizes the antagonist‐favoured conformation in an allosteric sense. The global association of a single amino acid replacement with cyclodiene resistance suggests that the resistance phenotype depends on changes in both of these properties, and that insecticides have selected residue 302 of Rdl for replacement because of its unique ability to influence both of these functions. 8. The location of alanine 302 in the sequence of the Rdl gene product supports a mechanism of action in which convulsants such as picrotoxin bind within the channel lumen, where they induce a rapid conformational change to the desensitized state.

Successive openings of the same acetylcholine receptor channel are correlated in open time.

Previous analysis of single-channel current records has shown that both the opening and closing transitions of chemically activated ion channels are operated by fast and slow kinetic processes. The fast component in the kinetics of channel opening has been interpreted as the reopening of a channel that has just closed. The fast component in the kinetics of channel closure has many possible explanations and is therefore more difficult to interpret. We can gain insight into the closing process by asking whether the lifetimes of successive openings of an acetylcholine receptor channel are correlated in open-state lifetime. Five kinetic models of channel closure are considered. Two of these models predict uncorrelated open-state lifetimes, one predicts correlated open-state lifetimes, and for two others a range of behavior is possible. Acetylcholine receptor channel data from cultured rat muscle are analyzed to show that open-state lifetimes are correlated, eliminating two models of channel gating.

Synaptotagmin-IV modulates synaptic function and long-term potentiation by regulating BDNF release

Synaptotagmin-IV (syt-IV) is a membrane trafficking protein that influences learning and memory, but its localization and role in synaptic function remain unclear. We found that syt-IV localized to brain-derived neurotrophic factor (BDNF)-containing vesicles in hippocampal neurons. Syt-IV/BDNF–harboring vesicles underwent exocytosis in both axons and dendrites, and syt-IV inhibited BDNF release at both sites. Knockout of syt-IV increased, and overexpression decreased, the rate of synaptic vesicle exocytosis from presynaptic terminals indirectly via changes in postsynaptic release of BDNF. Thus, postsynaptic syt-IV regulates the trans-synaptic action of BDNF to control presynaptic vesicle dynamics. Furthermore, selective loss of presynaptic syt-IV increased spontaneous quantal release, whereas a loss of postsynaptic syt-IV increased quantal amplitude. Finally, syt-IV knockout mice showed enhanced long-term potentiation (LTP), which depended entirely on disinhibition of BDNF release. Thus, regulation of BDNF secretion by syt-IV emerges as a mechanism for maintaining synaptic strength in a useful range during LTP.