Effects of delta-conotoxins PVIA and SVIE on sodium channels in the amphibian sympathetic nervous system

delta-Conotoxins are a family of small, disulfide-rich peptides found in the venoms of predatory cone snails (Conus). We examined in detail the effects of delta-conotoxin PVIA from the fish hunting cone snail Conus purpurascens on sodium currents in dissociated sympathetic neurons from the leopard frog Rana pipiens. We also compared this toxin's effects with those of delta-conotoxin SVIE from Conus striatus, another piscivorous cone snail. d-PVIA slowed the time-course of inactivation of delta sodium currents and shifted the voltage-dependence of activation and steady-state inactivation to more hyperpolarized potentials. Similar, albeit more pronounced, effects were seen with d-SVIE. While the effects of d-PVIA were reversed by washing, those of d-SVIE were largely irreversible over the time-course of these experiments. The effects of d-PVIA could be suppressed by conditioning depolarizations in a voltage- and time-dependent manner, whereas the effects of d-SVIE were largely resistant to conditioning depolarizations. Last, in intact sympathetic nervous system preparations, d-PVIA inhibited evoked trains of compound action potentials. Many of these effects of d-PVIA and d-SVIE are remarkably similar to those of toxins that bind to site 3 on voltage-gated sodium channels.     

The number of transmitter molecules in a quantum: an estimate from iontophoretic application of acetylcholine at the neuromuscular synapse.

1. The sensitivity of the subsynaptic membrane of twitch muscles of the frog and snake to iontophoretically applied acetylcholine (ACh) was determined. Optimal placement of ACh micropipettes on to the postsynaptic membrane resulted in potentials that were similar, though not identical, to the miniature excitatory post-synaptic potentials (min e.p.s.p.s). A sensitive bio-assay was developed to measure the output of ACh from micropipettes; this allowed an estimate to be made of the upper limit of the number of ACh molecules in a quantum of transmitter that is released from the nerve to produce a min e.p.s.p. 2. The assay to calibrate the output of ACh from micropipettes used the end-plate of the snake muscle as an ACh concentration detector. The end-plate was situated within a few mum of an oil-water interface, and a 0-6 nl. droplet of Ringer solution containing a known concentration of ACh (1 muM or less) was formed in the oil phase. The droplet was brought to the interface and, upon touching it, discharged its contents into the Ringer phase immediately above the end-plate. This resulted in a membrane depolarization that was recorded with an intracellular microelectrode. By applying droplets containing various known ACh concentrations a standard curve was constructed. To measure the ACh output of micropipettes a 0-6 nl. droplet of Ringer solution was suspended in the oil. The ACh pipette tip was inserted into the droplet and several thousand pulses of ACh were then delivered. The ACh content of the test droplet was measured by comparing its effectiveness in depolarizing the end-plate with the standard curve. In this manner the number of ACh molecules released in a single pulse was determined as a function of charge passed through the pipette. The output of ACh was linear and an average of 30,000 molecules of ACh were released per pC. 3. The sensitivity of the subsynaptic membrane to iontophoretically applied ACh, using the linear slopes of dose-response curves, in preparations from frog and snake treated with anticholinesterases was usually about 5 mV/pC. It follows that 6000 molecules of ACh are sufficient to produce a depolarization of 1 mV in the subsynaptic membrane. 4. The mean min e.p.s.p.s of muscle fibres treated with anticholinesterase range from 1 to 3 mV. Since the ACh released from an iontophoretic pipette is less effective than the same amount released from the nerve, it is concluded that a quantum of transmitter consists of less than 10,000 molecules of ACh. 5. It is calculated that for each molecule of ACh released in a quantum there results a minimum net flow of 3000 univalent ions across the synaptic membrane.     

The rhodopsin-porphyropsin system in freshwater fishes—1. Effects of age and photic environment

The freshwater fish Scardinius erythrophthalmus L. has a mixture of rhodopsin (λ_max 507 nm) and porphyropsin (λ_max, 535 nm) in its retina. Fish kept in continuous light for several weeks convert nearly all their porphyropsin into rhodopsin. In darkness this rhodopsin is reconverted to porphyropsin. If, in the light, each fish is fitted with an opaque cap over one eye, then this eye increases its proportion of porphyropsin while there is no change in the exposed contralateral eye. “Dark fish”, with porphyropsin-dominated retinas, converted this pigment into rhodopsin in the light. Conversion was markedly retarded in capped eyes but not in contralateral uncapped ones. Thus light converted prophyropsin into rhodopsin by acting locally within the ocular tissues and not by centrally-controlled endocrine or neuro-endocrine mechanisms. This does not rule out the possibility that endocrine factors also determine visual pigment composition under some circumstances. Thus there is a sharp increase in the proportion of porphyropsin in fish between 5 and 8 yr, although this might reflect a change in the pattern of ocular dehydrogenases converting retinol into 3-dehydroretinol. In any event, it appears that the seasonal visual pigment variation, manifested by a rhodopsin increase in the summer, is absent or much reduced in older fish. The effects of monocular thyroxine injections and exposure to intermittent photic conditions are also investigated and discussed.