Size of vesicle pools, rates of mobilization, and recycling at neuromuscular synapses of a Drosophila mutant, shibire.

Two vesicle pools, readily releasable (RRP) and reserve (RP) pools, are present at Drosophila neuromuscular junctions. Using a temperature-sensitive mutant, shibire(ts), we studied pool sizes and vesicle mobilization rates. In shibire(ts), due to lack of endocytosis at nonpermissive temperatures, synaptic currents continuously declined during tetanic stimulation until they ceased as the result of vesicle depletion. By then, approximately 84,000 quanta were released. Vesicles were mobilized from RP at a rate 1/7-1/10 of RRP. Cytochalasin D inhibited mobilization of vesicles from RP, allowing us to estimate the size of RRP as 14%-19% of all vesicles. Vesicle recycling supports synaptic transmission during prolonged tetanic stimulation and the maximum recycling rate was 1000 vesicles/s.

Synaptic defects and compensatory regulation of inositol metabolism in inositol polyphosphate 1-phosphatase mutants.

Phosphoinositides function as important second messengers in a wide range of cellular processes. Inositol polyphosphate 1-phosphatase (IPP) is an enzyme essential for the hydrolysis of the 1-phosphate from either Ins(1,4)P2 or Ins(1,3,4)P3. This enzyme is Li+ sensitive, and is one of the proposed targets of Li+ therapy in manic-depressive illness. Drosophila ipp mutants accumulate IP2 in their system and are incapable of metabolizing exogenous Ins(1,4)P2. Notably, ipp mutants demonstrate compensatory upregulation of an alternative branch in the inositol-phosphate metabolism tree, thus providing a means of ensuring continued availability of inositol. We demonstrate that ipp mutants have a defect in synaptic transmission resulting from a dramatic increase in the probability of vesicle release at larval neuromuscular junctions. We also show that Li+ phenocopies this effect in wild-type synapses. Together, these results support a role for phosphoinositides in synaptic vesicle function in vivo and mechanistically question the "lithium hypothesis."

Thermodynamic and kinetic studies of the gating behavior of a K+-selective channel from the sarcoplasmic reticulum membrane.

A voltage-dependent, K+-selective ionic channel from sarcoplasmic reticulum of rabbit skeletal muscle has been studied in a planar phospholipid bilayer membrane. The purpose [corrected] of this work is to study the mechanism by which the channel undergoes transitions between its conducting and nonconducting states. Thermodynamic studies show that the "open" and "closed" states of the channel exist in a voltage-dependent equilibrium, and that the channel displays only a single open state; the channel conductance is 120 pmho in 0.1 M K+. The channel's gating process follows single exponential kinetics at all voltages tested, and the individual opening and closing rate constants are exponentially dependent on voltage. The individual rate constants may also be determined from a stochastic analysis of channel fluctuations among multiple conductance levels. Neither the thermodynamic nor the kinetic parameters of gating depend on the absolute concentration of channels in the bilayer. The results are taken as evidence that the channel gates by an unusually simple two-state conformational mechanism in which the equivalent of 1.1 net charges are moved across the membrane during the formation of the open channel.

Acetylcholine receptor in planar lipid bilayers. Characterization of the channel properties of the purified nicotinic acetylcholine receptor from Torpedo californica reconstituted in planar lipid bilayers.

The properties of the channel of the purified acetylcholine receptor (AChR) were investigated after reconstitution in planar lipid bilayers. The time course of the agonist-induced conductance exhibits a transient peak that relaxes to a steady state value. The macroscopic steady state membrane conductance increases with agonist concentration, reaching saturation at 10(-5) M for carbamylcholine (CCh). The agonist-induced membrane conductance was inhibited by d-tubocurarine (50% inhibition, IC50, at approximately 10(-6) M) and hexamethonium (IC50 approximately 10(-5) M). The single channel conductance, gamma, is ohmic and independent of the agonist. At 0.3 M monovalent salt concentrations, gamma = 28 pS for Na+, 30 pS for Rb+, 38 pS for Cs+, and 50 pS for NH+4. The distribution of channel open times was fit by a sum of two exponentials, reflecting the existence of two distinct open states. tau o1 and tau o2, the fast and slow components of the distribution of open times, are independent of the agonist concentration: for CCh this was verified in the range of 10(-6) M less than C less than 10(-3)M. tau 01 and tau o2 are approximately three times longer for suberyldicholine ( SubCh ) than for CCh. tau o1 and tau o2 are moderately voltage dependent, increasing as the applied voltage in the compartment containing agonist is made more positive with respect to the other. At desensitizing concentrations of agonist, the AChR channel openings occurred in a characteristic pattern of sudden paroxysms of channel activity followed by quiescent periods. A local anesthetic derivative of lidocaine ( QX -222) reduced both tau o1 and tau o2. This effect was dependent on both the concentration of QX -222 and the applied voltage. Thus, the AChR purified from Torpedo electric organ and reconstituted in planar lipid bilayers exhibits ion conduction and kinetic and pharmacological properties similar to AChR in intact muscle postsynaptic membranes.

Sea urchin sperm cation-selective channels directly modulated by cAMP.

Components of the sea urchin outer egg jelly layer such as speract drastically change second messenger levels and membrane permeability in sperm. Ion channels are deeply involved in the sperm-egg dialogue in sea urchin and other species. Yet, due to the small size of sperm, studies of ion channels and their modulation by second messengers in sperm are scarce. In this report we offer the first direct evidence that cation-selective channels upwardly regulated by cAMP operate in sea urchin sperm. Due to their poor selectivity among monovalent cations, channel activation in seawater could contribute to sperm membrane repolarization during the speract response.

A ciliary K+ conductance sensitive to charibdotoxin underlies inhibitory responses in toad olfactory receptor neurons.

In olfactory neurons from Caudiverbera caudiverbera, a mixture of putrid odorants trigger an inhibitory, K(+)-selective current and a hyperpolarizing receptor potential. The current-voltage relation resembles that of a Ca(2+)-activated K+ conductance; their amplitude depends on extracellular Ca2+. 10 nM charibdotoxin, a blocker of K(+)-selective channels, including Ca(2+)-activated ones, reversibly abolished inhibitory currents and receptor potentials. Focal stimulation demonstrates that the underlying transduction mechanism is confined to the cilia. This represents the first evidence for inhibitory responses in vertebrate olfactory cells mediated by a ciliary CTX-sensitive K+ conductance, most likely a Ca(2+)-activated one.

Selectivity and gating properties of a cAMP-modulated, K(+)-selective channel from Drosophila larval muscle.

The selectivity and gating properties of cAMP-modulated, voltage-independent, K(+)-selective channel from Drosophila larval muscle were investigated using the patch-clamp technique. In symmetrical 115 mM K+ the channel displayed a linear current-voltage relation with slope conductance of 43 pS. Under biionic conditions (115 mM K+ pipette/115 mM X+ cytoplasmic) the permeability sequence was K+ > Rb+ > NH4+ >> Cs+,Na+. The channel was impermeable to Ca2+ (PCa/PK < 0.02). Under steady-state conditions and regardless [cAMP], open dwell times showed a double exponential distribution. [cAMP] did not affect the time constants of the two components of open times, or their relative amplitudes. Moreover, successive openings were correlated in open time. Closed dwell times were made of at least three exponential components. Fast application of cAMP to the cytoplasmic side of the channel induced a transient increase in open probability that relaxed to a lower value within seconds. This last result suggests that cAMP can activate and desensitize this cAMP-modulated, K(+)-selective channel.

L-glutamate activates excitatory and inhibitory channels in Drosophila larval muscle.

Muscle fibers from Drosophila larvae show an L-glutamate-sensitive membrane potential. Bath-applied L-glutamate depolarizes the muscle in the range from 0.5 to 20 microM. Greater concentrations of the agonist repolarize the fibers. The repolarizing effect disappears if chloride is replaced by sulfate in the external medium. Intracellular recordings show the occurrence of depolarizing and hyperpolarizing spontaneous miniature postsynaptic potentials (smpp). Patch-clamp studies indicate the presence of two types of receptor channels: (i) an anion-selective channel activated by both L-glutamate and GABA. In outside out-patches, bathed in symmetrical 140 mM Cl- and 200 microM GABA, the channel displays conductance substates of 40, 80 and 110 pS. In the presence of 200 microM L-glutamate only the 40 and 80 pS substates are observed; (ii) a cation-selective channel activated only by L-glutamate that has a conductance of 104 pS in cell-attached patches (128 mM Na+ outside). The presence of these two types of receptor channels in Drosophila muscle may explain the effect of bath-applied L-glutamate on membrane potential and the presence of inhibitory and excitatory smpp.

Native and chemically modified porin channels from Salmonella typhi Ty2 in planar lipid bilayers.

Native porins, from Salmonella typhi Ty2 outer membrane, and porins alkylated with pyridoxal phosphate (Plp) were studied in planar lipid bilayers. The conductance of bilayers exposed to native or chemically modified porins increases in discrete jumps. Conductance histograms for native porins displayed two major peaks at 1.7 and 6.7 nS (in 0.5 M KCl). On the other hand, Plp-treated porins exhibited a single major peak at 1 nS. The relation between bilayer conductance and native porin concentration was linear. However, this relation became logarithmic in the presence of modified porins. The results support the notion that alkaline reduction of S. typhi Ty2 porins with Plp dissociates porin channel trimers in a reversible fashion.

A high-conductance voltage-dependent multistate Ca2+ channel found in sea urchin and mouse spermatozoa.

Ion fluxes through poorly understood channel-mediated mechanisms participate in the interaction between spermatozoa and egg. Previously, we reported the characterization in planar bilayers of a high conductance Ca(2+)-selective, voltage-dependent multistate channel from S. purpuratus sea urchin sperm plasma membranes. Here we show that this ion channel can be directly transferred to planar lipid bilayers upon sperm addition, from sea urchin (S. purpuratus and L. pictus) and from mouse. We found that spermatozoa from these species possess a conspicuous Ca(2+)-selective, high conductance, multi-state, voltage-dependent channel, which displays similar voltage dependence and equal PBa2+/PK+ approximately 4 in the three species. The presence of this Ca2+ channel in such diverse species suggests it plays a relevant role in sperm physiology. The high sensitivity of planar bilayers to detect single ion channels can now be used to study ion channel regulation and gamete interaction.

Pore accessibility during C-type inactivation in Shaker K+ channels.

Shaker K+ channels inactivate through two distinct molecular mechanisms: N-type, which involves the N-terminal domain and C-type that appears to involve structural modifications at the external mouth of the channel. We have tested pore accessibility of the Shaker K+ channel during C-type inactivation using Ba2+ as a probe. We determined that external Ba2+ binds to C-type inactivated channels forming an extremely stable complex; i.e. there is Ba2+ trapping by C-type inactivated channels. The structural changes Shaker channels undergo during C-type inactivation create high energy barriers that hinder Ba2+ exit to either the extracellular solution or to the intracellular solution.

Mouse sperm patch-clamp recordings reveal single Cl- channels sensitive to niflumic acid, a blocker of the sperm acrosome reaction.

Ion channels lie at the heart of gamete signaling. Understanding their regulation will improve our knowledge of sperm physiology, and may lead to novel contraceptive strategies. Sperm are tiny (approximately 3 microm diameter) and, until now, direct evidence of ion channel activity in these cells was lacking. Using patch-clamp recording we document here, for the first time, the presence of cationic and anionic channels in mouse sperm. Anion selective channels were blocked by niflumic acid (NA) (IC50 = 11 microM). The blocker was effective also in inhibiting the acrosome reaction induced by the zona pellucida, GABA or progesterone. These observations suggest that Cl- channels participate in the sperm acrosome reaction in mammals.

K(+)-channel blockers restore synaptic plasticity in the neuromuscular junction of dunce, a Drosophila learning and memory mutant.

The effects of K(+)-channel blockers on synaptic transmission in dunce (dnc), a Drosophila learning and memory mutant, were investigated. Larvae dnc mutants lack facilitation and post-tetanic potentiation (PTP) at their motor end-plates; dnc mutants are also deficient in a form of phosphodiesterase, and exhibit abnormally high levels of cyclic adenosine 3',5'-monophosphate (cAMP). A two-microelectrode voltage-clamp was used to record end-plate currents and spontaneous end-plate currents from longitudinal ventrolateral third-instar larval muscle. The K(+)-channel blockers 3,4-diaminopyridine (3,4-DAP) and tetraethylammonium (TEA), at micromolar concentrations, caused a reversible decrease in end-plate current amplitudes both in wild-type and mutant end-plates. In the presence of blockers, a period of high-frequency stimulation (tetanus) of the nerve gave way to a transient increase in the end-plate currents of dnc mutants resembling facilitation and PTP in normal end-plates; 3,4-DAP and TEA also restored facilitation and PTP in normal end-plates after incubation with a non-hydrolysable analogue of cAMP (8Br-cAMP). It is suggested that a specific K+ conductance might be relevant to the lack of synaptic plasticity at the dnc neuromuscular synapses.

Inhibitory K+ current activated by odorants in toad olfactory neurons.

Odorant responses of isolated olfactory neurons from the toad Caudiverbera caudiverbera were monitored by using patch-clamp techniques. Depending on the stimulus, the same neuron responded with an increase or a decrease in action potential firing. Odorants that activate the cAMP cascade in olfactory cilia increased electrical activity, caused membrane depolarization, and triggered inward currents. In contrast, odorants that do not activate the cAMP cascade inhibited electrical activity, produced membrane hyperpolarization, and activated outward currents in a dose-dependent fashion. Such currents were carried by K+ and blocked by tetraethylammonium. Similar currents were recorded from Xenopus laevis. Our results suggest that this K+ current is responsible for odorant-induced inhibition of action potential firing in olfactory neurons.

Ion channels from chemosensory olfactory neurons.

The olfactory epithelium has the ability to respond to a large number of volatile compounds of small molecular weight. Ultimately, such a property lies on a specialized type of neuron, the olfactory receptor cell. In the presence of odorants, the olfactory receptor neuron responds with action potentials whose frequency depends on odorant concentration. The primary events in the process of olfactory transduction are thought to occur at the cilia of olfactory receptor neurons and involve the binding of odorants to receptor molecules followed by the opening of ion channels. A crucial step in understanding olfactory transduction requires identifying the mechanisms that regulate the electrical activity of olfactory cells. In the last couple of years, patch-clamp recording from isolated olfactory cells and reconstitution of olfactory membranes in planar lipid bilayers have begun to shed light on some of these mechanisms. Although the information emerging from such studies is still preliminary, there are already well-defined hypotheses on the molecular events that might underlie the primary events in olfactory transduction. Currently, attention is being focused on the notions that second messengers might be involved in the activation of ion channels in olfactory cilia, and that odorant binding to a receptor molecule might lead directly to the gating of ion channels in chemosensory olfactory membranes. The coming years promise to be exciting ones in the field of olfactory transduction. We have now the necessary tools to be able to confront hypotheses and experimental facts.

Properties of whole cell currents in isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera.

Isolated olfactory neurons from the chilean toad Caudiverbera caudiverbera were found to possess a same set of currents. Outward currents, made of a delayed rectifier and a Ca(2+)-dependent component, were blocked by replacing K+ by Cs+ in the patch pipette, in the presence of millimolar concentrations of tetraethylammonium and 4-aminopyridine in the external solution. Inward currents were made of a transient and a maintained component. The transient was abolished in the absence of external Na+ and was blocked by tetrodotoxin, with an apparent dissociation constant (KDapp) of 25.4 +/- 0.3 nM. The maintained inward currents were suppressed on removing external Ca2+, could be carried also by Ba2+, and were selectively blocked by Cd2+ (KDapp = 3.2 +/- 1.3 microM). A variety of agents found to block the maintained Ca2+ inward currents, including Co2+ and Ni2+, at millimolar concentrations, and nifedipine, verapamil, amiloride, and the amiloride analogue benzamil, at micromolar concentrations, were also effective in either modifying the gating of, or in blocking, the transient inward currents.

Shaker mutants lack post-tetanic potentiation at motor end-plates.

The two-electrode voltage clamp technique was employed to measure end-plate currents in larval neuromuscular junctions of wild-type (Canton-S) and of three different Drosophila Shaker mutants: ShakerKS133, Shaker102 and f5Shaker5. In the Shaker mutants, nerve-evoked end-plate currents (neepc) were 4-5-fold larger than those measured in Canton-S. Shaker motor end-plates were found to lack post-tetanic potentiation (PTP), but could undergo facilitation. Moreover, PTP but not facilitation was lost in wild-type larvae if the neuromuscular junction was exposed to 4-aminopyridine (4-AP), a blocker of Shaker A-type K+ currents. End-plate currents were depressed by Ca2+ channel blockers like Mg2+, at millimolar concentrations, and Co2+ and Cd2+, at micromolar concentrations, but not by nifedipine (100 nM) and verapamil (100 nM). After exposure to Ca2+ channel blockers, Shaker end-plates exhibited PTP. In particular, Cd2+ was most effective in depressing neepcs and in restoring PTP in all Shaker mutants. The results obtained indicate the abnormal function of Shaker K+ channels at motor nerves specifically abolishes PTP in Drosophila larval neuromuscular junctions.

Ion channel classes in purified olfactory cilia membranes: planar lipid bilayer studies.

Ciliary membrane fragment fusion to planar lipid bilayers resulted in the insertion of four ion channel types. cAMP-activated, cation-selective channels could be detected only in the absence of Ca2+ and had a conductance of 23 pS. They exhibited an apparent dissociation constant (Kd) for the cyclic nucleotide of approximately 30 microM and an estimated permeability ratio (PNa/PK) of 2.4. The cAMP cation-selective channel coinserted with a K(+)-selective channel refractory to cAMP, Ca2+, and D-myo-inositol 1,4,5-trisphosphate. This K+ channel was voltage independent and exhibited open-conductance substates of 60 and 112 pS. cAMP was also found to modulate a novel K+ channel with a Kd = 140 microM. It displayed three nearly equally spaced open substates with conductances of 34, 80, and 130 pS. In the absence and in the presence of cAMP the probability of occurrence of the open substates was binomially distributed. A fourth channel type was a Ca(2+)-activated K+ channel with a conductance of 240 pS. It was blocked by charybdotoxin at nanomolar concentrations (Kd = 3 nM). These results add support to the idea that, besides cAMP-activated cation-selective channels, vertebrate chemosensory olfactory membranes possess an arrangement of ion channels.