Neurotransmitter release is initiated by influx of Ca2+ through voltage-gated Ca2+ channels, within 200 microseconds of the action potential arriving at the synaptic terminal, as the Ca2+ concentration increases from 100 nM to > 200 microM. Exocytosis requires high Ca2+ concentration, with a threshold of 20-50 microM and half-maximal activation at 190 microM. The synaptic membrane proteins syntaxin, 25K synaptosome-associated protein (SNAP25), and vesicle-associated membrane protein (VAMP)/synaptobrevin, are thought to form a synaptic core complex which mediates vesicle docking and membrane fusion. Synaptotagmin may be the low-affinity Ca(2+)-sensor, but other Ca(2+)-sensors are involved as residual neurotransmission persists in synaptotagmin-null mutants. Syntaxin binds to N-type Ca2+ channels at a site in the intracellular loop connecting domains II and III. Here we describe Ca(2+)-dependent interaction of this site with syntaxin and SNAP25 which has a biphasic dependence on Ca2+, with maximal binding at 20 microM free Ca2+, near the threshold for transmitter release. Ca(2+)-dependent interaction of Ca2+ channels with the synaptic core complex may be important for Ca(2+)-dependent docking and fusion of synaptic vesicles.

The binding of cGMP to the noncatalytic sites on two isoforms of the phosphodiesterase (PDE) from mammalian rod outer segments has been characterized to evaluate their role in regulating PDE during phototransduction. Nonactivated, membrane-associated PDE (PDE-M, ␣␤␥ 2 ) has one exchangeable site for cGMP binding; endogenous cGMP remains nonexchangeable at the second site. Non-activated, soluble PDE (PDE-S, ␣␤␥ 2 ␦) can release and bind cGMP at both noncatalytic sites; the ␦ subunit is likely responsible for this difference in cGMP exchange rates. Removal of the ␦ and/or ␥ subunits yields a catalytic ␣␤ dimer with identical catalytic and binding properties for both PDE-M and PDE-S as follows: high affinity cGMP binding is abolished at one site (K D >1 M); cGMP binding affinity at the second site (K D ϳ60 nM) is reduced 3-4-fold compared with the nonactivated enzyme; the kinetics of cGMP exchange to activated PDE-M and PDE-S are accelerated to similar extents. The properties of nonactivated PDE can be restored upon addition of ␥ subunit. Occupancy of the noncatalytic sites by cGMP may modulate the interaction of the ␥ subunit with the ␣␤ dimer and thereby regulate cytoplasmic cGMP concentration and the lifetime of activated PDE during visual transduction in photoreceptor cells. The cGMP phosphodiesterase (PDE)1 present in rod and cone retinal photoreceptors is the central effector enzyme for vertebrate visual excitation. Photoactivation of the visual pigment rhodopsin leads to G-protein (transducin) activation, which then proceeds to the activation of PDE. The activated ␣ subunit of transducin (␣ t -GTP) is believed to activate the membraneassociated rod PDE holoenzyme (subunit stoichiometry, ␣␤␥ 2 ) by displacing the inhibitory ␥ subunits (P␥) from the active sites of the catalytic heterodimer (P␣␤). The enhanced hydrolytic activity of the activated PDE rapidly reduces cytoplasmic cGMP levels and then leads to dissociation of bound cGMP from the cGMP-gated ion channel, closure of the ion channel, and finally a hyperpolarization of the membrane. These steps constitute the excitation pathway for vertebrate visual transduction (for reviews see Refs. 1-3).The photoreceptor PDE is a member of a family of phosphodiesterases that all share the ability to hydrolyze cyclic nucleotides. The PDE in rods and cones (classified as PDE6 (4)) is most closely related to the cGMP-specific PDE (PDE5) based on several criteria, including overall amino acid sequence similarity, substrate preference for cGMP over cAMP, inhibition of catalysis by isozyme-selective drugs, and the presence of a consensus sequence in the N-terminal half of the protein that represents noncatalytic cGMP binding domains. In the case of PDE5, binding of cGMP to the noncatalytic cGMP-binding sites may regulate activity indirectly via protein phosphorylation of the enzyme (reviewed in Ref. 5). The cGMP-stimulated PDE (PDE2) also contains noncatalytic cGMP-binding sites that allosterically regulate catalysis at the active site (6).For the photoreceptor PDE, most...

PDE6 (type 6 phosphodiesterase) from rod outer segments consists of two types of catalytic subunits, alpha and beta; two inhibitory gamma subunits; and one or more delta subunits found only on the soluble form of the enzyme. About 70% of the phosphodiesterase activity found in rod outer segments is membrane-bound, and is thought to be anchored to the membrane through C-terminal prenyl groups. The recombinant delta subunit has been shown to solubilize the membrane-bound form of the enzyme. This paper describes the site and mechanism of this interaction in more detail. In isolated rod outer segments, the delta subunit was found exclusively in the soluble fraction, and about 30% of it did not coimmunoprecipitate with the catalytic subunits. The delta subunit that was bound to the catalytic subunits dissociated slowly, with a half-life of about 3.5 h. To determine whether the site of this strong binding was the C-termini of the phosphodiesterase catalytic subunits, peptides corresponding to the C-terminal ends of the alpha and beta subunits were synthesized. Micromolar concentrations of these peptides blocked the phosphodiesterase/delta subunit interaction. Interestingly, this blockade only occurred if the peptides were both prenylated and methylated. These results suggested that a major site of interaction of the delta subunit is the methylated, prenylated C-terminus of the phosphodiesterase catalytic subunits. To determine whether the catalytic subunits of the full-length enzyme are methylated in situ when bound to the delta subunit, we labeled rod outer segments with a tritiated methyl donor. Soluble phosphodiesterase from these rod outer segments was more highly methylated (4.5 +/- 0.3-fold) than the membrane-bound phosphodiesterase, suggesting that the delta subunit bound preferentially to the methylated enzyme in the outer segment. Together these results suggest that the delta subunit/phosphodiesterase catalytic subunit interaction may be regulated by the C-terminal methylation of the catalytic subunits.

The ␦ subunit of the rod photoreceptor PDE has previously been shown to copurify with the soluble form of the enzyme and to solubilize the membrane-bound form (1). To determine the physiological effect of the ␦ subunit on the light response of bovine rod outer segments, we measured the real time accumulation of the products of cGMP hydrolysis in a preparation of permeablized rod outer segments. The addition of ␦ subunit GST fusion protein (␦-GST) to this preparation caused a reduction in the maximal rate of cGMP hydrolysis in response to light. The maximal reduction of the light response was about 80%, and the half-maximal effect occurred at 385 nM ␦ subunit. Several experiments suggest that this effect was not due to the effects of ␦-GST on transducin or rhodopsin kinase. Immunoblots demonstrated that exogenous ␦-GST solubilized the majority of the PDE in ROS but did not affect the solubility of transducin. Therefore, changes in the solubility of transducin cannot account for the effects of ␦-GST in the pH assay. The reduction in cGMP hydrolysis was independent of ATP, which indicates that it was not due to effects of ␦-GST on rhodopsin kinase. In addition to the effect on cGMP hydrolysis, the ␦-GST fusion protein slowed the turn-off of the system. This is probably due, at least in part, to an observed reduction in the GTPase rate of transducin in the presence of ␦-GST. These results demonstrate that ␦-GST can modify the activity of the phototransduction cascade in preparations of broken rod outer segments, probably due to a functional uncoupling of the transducin to PDE step of the signal transduction cascade and suggest that the ␦ subunit may play a similar role in the intact outer segment.Retinal photoreceptor cells, rods and cones, can directly sense and respond to photons of light. In these cells, light induces a highly regulated and well studied cascade of events that leads to changes in photoreceptor membrane potential and neurotransmitter release. PDE61 is an integral part of this cascade. When a photon of light hits the chromophore in the transmembrane protein rhodopsin, a conformational change occurs that allows the G protein, transducin, to be activated. Transducin then activates PDE6 by binding to its inhibitory ␥ subunits. When active, PDE6 rapidly hydrolyzes cGMP, causing the closure of cGMP-gated ion channels in the plasma membrane of the photoreceptor, reduced influx of sodium and calcium, and hyperpolarization of the photoreceptor cell. The light signal ends when transducin becomes inactive by hydrolyzing its GTP, and rhodopsin kinase phosphorylates rhodopsin, blocking further interaction between transducin and rhodopsin. GDP-bound transducin releases the ␥ subunits of PDE6, allowing the catalytic subunits of PDE6 to become reinhibited after transducin has hydrolyzed its GTP. Reductions in calcium caused by light stimulate guanylate cyclase to replace the hydrolyzed cGMP.Rhodopsin is a transmembrane protein that is found in the internal membranous disks of photoreceptor cells. Therefore, it is ...