A diverse family of novel peptide toxins from an unusual cone snail, Conus californicus.

Diversity among Conus toxins mirrors the high species diversity in the Indo-Pacific region, and evolution of both is thought to stem from feeding-niche specialization derived from intra-generic competition. This study focuses on Conus californicus, a phylogenetic outlier endemic to the temperate northeast Pacific. Essentially free of congeneric competitors, it preys on a wider variety of organisms than any other cone snail. Using molecular cloning of cDNAs and mass spectrometry, we examined peptides isolated from venom ducts to elucidate the sequences and post-translational modifications of two eight-cysteine toxins (cal12a and cal12b of type 12 framework) that block voltage-gated Na(+) channels. Based on homology of leader sequence and mode of action, these toxins are related to the O-superfamily, but differ significantly from other members of that group. Six of the eight cysteine residues constitute the canonical framework of O-members, but two additional cysteine residues in the N-terminal region define an O+2 classification within the O-superfamily. Fifteen putative variants of Cal12.1 toxins have been identified by mRNAs that differ primarily in two short hypervariable regions and have been grouped into three subtypes (Cal12.1.1-3). This unique modular variation has not been described for other Conus toxins and suggests recombination as a diversity-generating mechanism. We propose that these toxin isoforms show specificity for similar molecular targets (Na(+) channels) in the many species preyed on by C. californicus and that individualistic utilization of specific toxin isoforms may involve control of gene expression.

Diversity of conotoxin types from Conus californicus reflects a diversity of prey types and a novel evolutionary history.

Most species within the genus Conus are considered to be specialists in their consumption of prey, typically feeding on molluscs, vermiform invertebrates or fish, and employ peptide toxins to immobilize prey. Conus californicus Hinds 1844 atypically utilizes a wide range of food sources from all three groups. Using DNA- and protein-based methods, we analyzed the molecular diversity of C. californicus toxins and detected a correspondingly large number of conotoxin types. We identified cDNAs corresponding to seven known cysteine-frameworks containing over 40 individual inferred peptides. Additionally, we found a new framework (22) with six predicted peptide examples, along with two forms of a new peptide type of unusual length. Analysis of leader sequences allowed assignment to known superfamilies in only half of the cases, and several of these showed a framework that was not in congruence with the identified superfamily. Mass spectrometric examination of chromatographic fractions from whole venom served to identify peptides corresponding to a number of cDNAs, in several cases differing in their degree of posttranslational modification. This suggests differential or incomplete biochemical processing of these peptides. In general, it is difficult to fit conotoxins from C. californicus into established toxin classification schemes. We hypothesize that the novel structural modifications of individual peptides and their encoding genes reflect evolutionary adaptation to prey species of an unusually wide range as well as the large phylogenetic distance between C. californicus and Indo-Pacific species.

Decrease in inflammatory hyperalgesia by herpes vector-mediated knockdown of Nav1.7 sodium channels in primary afferents.

Induction of peripheral inflammation increases the expression of the Nav1.7 sodium channel in sensory neurons, potentially increasing their excitability. Peripheral inflammation also produces hyperalgesia in humans and an increase in nociceptive responsiveness in animals. To test the relationship between these two phenomena we applied a recombinant herpes simplex-based vector to the hindpaw skin of mice, which encoded both green fluorescent protein (GFP) as well as an antisense sequence to the Nav1.7 gene. The hindpaw was subsequently injected with complete Freund's adjuvant to induce robust inflammation. Application of the vector, but not a control vector encoding only GFP, prevented an increase in Nav1.7 expression in GFP-positive neurons and prevented development of hyperalgesia in both C and Adelta thermonociceptive tests. These results provide clear evidence of the involvement of an increased expression of the Nav1.7 channel in nociceptive neurons in the development of inflammatory hyperalgesia.

Identified ion channels in the squid nervous system.

Our modern understanding of channels as discrete voltage-sensitive and ion-selective entities comes largely from a series of classical studies using the squid giant axon. This system has also been critical for understanding how transporters and synaptic transmission operate. This review outlines attempts to assign molecular identities to the extensively studied physiological properties of this system. As it turns out, this is no simple task. Molecular candidates for voltage-gated Na(+), K(+), and Ca(2+) channels, as well as ion transporters have been isolated from the squid nervous system. Both physiological and molecular approaches have been used to equate these cloned gene products with their native counterparts. In the case of the delayed rectifier K(+) conductance, the most thoroughly studied example, two major issues further complicate the equation. First, the ability of K(+) channel monomers to form heteromultimers with unique properties must be considered. Second, squid K(+) channel mRNAs are extensively edited, a process that can generate a wide variety of channel proteins from a common gene. The giant axon system is beginning to play an important role in understanding the biological relevance of this latter process.

Cloning and characterization of an ionotropic glutamate receptor subunit expressed in the squid nervous system.

In this paper we describe the cloning of a putative ionotropic glutamate receptor subunit, SqGluR, and its distribution in the nervous system of the squid. A full-length cDNA was assembled from a cDNA library of the stellate ganglion/giant fibre lobe complex of Loligo opalescens. The deduced amino acid sequence of the mature SqGluR displayed 44-46% amino acid identity with mammalian GluR1-GluR4 and 53% with Lym-eGluR1 from Lymnaea stagnalis. In situ hybridizations in adult squid confirmed that the SqGluR mRNA is abundant in giant fibre lobe neurons, in large, presumptive motor neurons of the stellate ganglion proper and in the supraoesophageal and optic lobes of the central nervous system. In newborn squid, SqGluR mRNA expression was detected throughout the nervous system but not elsewhere. A synthetic peptide corresponding to the last 15 amino acids of the SqGluR C-terminus was used to generate polyclonal antibodies, which were used for immunoblot analysis to demonstrate widespread expression in the squid central and peripheral nervous systems. Injection of the synthetic peptide into the postsynaptic side of the giant synapse inhibited synaptic transmission.

Selective open-channel block of Shaker (Kv1) potassium channels by s-nitrosodithiothreitol (SNDTT).

Large quaternary ammonium (QA) ions block voltage-gated K(+) (Kv) channels by binding with a 1:1 stoichiometry in an aqueous cavity that is exposed to the cytoplasm only when channels are open. S-nitrosodithiothreitol (SNDTT; ONSCH(2)CH(OH)CH(OH)CH(2)SNO) produces qualitatively similar "open-channel block" in Kv channels despite a radically different structure. SNDTT is small, electrically neutral, and not very hydrophobic. In whole-cell voltage-clamped squid giant fiber lobe neurons, bath-applied SNDTT causes reversible time-dependent block of Kv channels, but not Na(+) or Ca(2)+ channels. Inactivation-removed ShakerB (ShBDelta) Kv1 channels expressed in HEK 293 cells are similarly blocked and were used to study further the action of SNDTT. Dose-response data are consistent with a scheme in which two SNDTT molecules bind sequentially to a single channel, with binding of the first being sufficient to produce block. The dissociation constant for the binding of the second SNDTT molecule (K(d2) = 0.14 mM) is lower than that of the first molecule (K(d1) = 0.67 mM), indicating cooperativity. The half-blocking concentration (K(1/2)) is approximately 0.2 mM. Steady-state block by this electrically neutral compound has a voltage dependence (about -0.3 e(0)) similar in magnitude but opposite in directionality to that reported for QA ions. Both nitrosyl groups on SNDTT (one on each sulfur atom) are required for block, but transfer of these reactive groups to channel cysteine residues is not involved. SNDTT undergoes a slow intramolecular reaction (tau approximately 770 s) in which these NO groups are liberated, leading to spontaneous reversal of the SNDTT effect. Competition with internal tetraethylammonium indicates that bath-applied SNDTT crosses the cell membrane to act at an internal site, most likely within the channel cavity. Finally, SNDTT is remarkably selective for Kv1 channels. When individually expressed in HEK 293 cells, rat Kv1.1-1.6 display profound time-dependent block by SNDTT, an effect not seen for Kv2.1, 3.1b, or 4.2.

Interaction of a toxin from the scorpion Tityus serrulatus with a cloned K+ channel from squid (sqKv1A).

A toxin from the scorpion Tityus serrulatus (TsTX-Kalpha) blocks native squid K(+) channels and their cloned counterpart, sqKv1A, at pH 8 ((native)K(d) approximately 20 nM; (sqKv1A)K(d) approximately 10 nM). In both cases, decreasing the pH below 7.0 significantly diminishes the TsTX-Kalpha effect (pK = 6.6). In the cloned squid channel, the pH dependence of the block is abolished by a single point mutation (H351G), and no change in toxin affinity was observed at higher pH values (pH > or =8.0). To further investigate the TsTX-Kalpha-sqKv1A interaction, the three-dimensional structure of TsTX-Kalpha was determined in solution by NMR spectroscopy, and a model of the TsTX-Kalpha-sqKv1A complex was generated. As found for other alpha-K toxins such as charybdotoxin (CTX), site-directed mutagenesis at toxin residue K27 (K27A, K27R, and K27E) significantly reduced the toxin's affinity for sqKv1A channels. This is consistent with the TsTX-Kalpha-sqKv1A model reported here, which has K27 of the toxin inserted into the ion conduction pathway of the K(+) channel. This toxin-channel model also illustrates a possible mechanism for the pH-dependent block whereby lysine residues from TsTX-Kalpha (K6 and K23) are repelled by protonated H351 on sqKv1A at low pH.

Temperature-dependent expression of a squid Kv1 channel in Sf9 cells and functional comparison with the native delayed rectifier.

SqKv1A is a cDNA that encodes a Kv1 (Shaker-type) alpha-subunit expressed only in the giant axon and the parental giant fiber lobe (GFL) neurons of the squid stellate ganglion. We incorporated SqKv1A into a recombinant baculovirus for expression in the insect Sf9 cell line. Whole-cell patch-clamp recordings reveal that very few cells display functional potassium current (IK) if cultured at the standard postinfection temperature of 27 degrees C. At 18 degrees C, less SqKv1A protein is produced than at 27 degrees C, but cells with IK currents are much more numerous and can survive for at least 20 days postinfection (vs. approximately 5 days at 27 degrees C). Activation and deactivation kinetics of SqKv1A in Sf9 cells are slower (approximately 3- and 10-fold, respectively) than those of native channels in GFL neurons, but have similar voltage dependencies. The two cell types show only subtle differences in steady-state voltage-dependence of conductance and inactivation. Rates of IK inactivation in 20 mM external K are identical in the two cell types, but the sensitivity of inactivation to external tetraethylammonium (TEA) and K ions differ: inactivation of SqKv1A in Sf9 cells is slowed by external TEA and K ions, whereas inactivation of GFL IK is largely insensitive. Functional differences are discussed in terms of factors that may be specific to cell-type, including the presence of presently unidentified Kv1 subunits in GFL neurons that might form heteromultimers with SqKv1A.

Natural substitutions at highly conserved T1-domain residues perturb processing and functional expression of squid Kv1 channels.

Shaker-type K-channel alpha-subunits (SqKv1A, B, D) expressed in neurons of the squid stellate ganglion differ in the length of their N-termini and in the species of amino acid present at several points in the T1 domain, an intracellular region involved in the tetramerization process during channel assembly. Heterologous expression of wild-type SqKv1A, B, and D in Xenopus oocytes reveals large differences in the level of both functional channels (assayed by whole-oocyte voltage clamp) and total channel protein (assayed by immunoblotting). Functional expression is poorest with SqKv1A and by far the best with SqKv1D. Biophysical properties of the three SqKv1 channels are essentially identical (assayed by cell-attached patch clamp). Site-directed mutagenesis was used to determine whether the observed differences in expression level are impacted by two residues in the T1 domain at which SqKv1A and B (but not D) differ from the consensus sequences found in many other taxa. In SqKv1A, glycine is substituted for arginine in an otherwise universally conserved sequence (FFDR in the T1(B) subdomain). In SqKv1B, glycine replaces serine in a sequence that is conserved within the Kv1 subfamily (SGLR in the T1(A) subdomain). Restoration of the consensus amino acid at these positions largely accounts for the observed differences in expression level. Analysis of the glycosylation state of aberrant versus restored alpha-subunits suggests that the anomalous amino acids in SqKv1A and B exert their influence during early steps in channel processing and assembly which take place in the endoplasmic reticulum (ER).

Effects of temperature on escape jetting in the squid Loligo opalescens.

In Loligo opalescens, a sudden visual stimulus (flash) elicits a stereotyped, short-latency escape response that is controlled primarily by the giant axon system at 15 C. We used this startle response as an assay to examine the effects of acute temperature changes down to 6 C on behavioral and physiological aspects of escape jetting. In free-swimming squid, latency, distance traveled and peak velocity for single escape jets all increased as temperature decreased. In restrained squid, intra-mantle pressure transients during escape jets increased in latency, duration and amplitude at low temperature. Recordings of stellar nerve activity revealed repetitive firing of the giant motor axon accompanied by increased activity in the non-giant motor axons that run in parallel. Selective stimulation of giant and non-giant motor axons in isolated nerve-muscle preparations failed to show the effects seen in vivo, i.e. increased peak force and increased neural activity at low temperature. Taken together, these results suggest that L. opalescens is able to compensate escape jetting performance for the effects of acute temperature reduction. A major portion of this compensation appears to occur in the central nervous system and involves alterations in the recruitment pattern of both the giant and non-giant axon systems.

Role of prey-capture experience in the development of the escape response in the squid Loligo opalescens: a physiological correlate in an identified neuron.

Although extensively used for biophysical studies, the squid giant axon system remains largely unexplored in regard to in vivo function and modulation in any biologically relevant context. Here we show that successful establishment of the recruitment pattern for the giant axon in the escape response elicited by a brief electrical stimulus depends on prey-capture experience early in life. Juvenile squid fed only slow-moving, easy-to-capture prey items (Artemia salina) develop deficits in coordinating activity in the giant axon system with that of a parallel set of non-giant motor axons during escape responses. These deficits are absent in cohorts fed fast-moving, challenging prey items (copepods). These results suggest that the acquisition of inhibitory control over the giant axon system is experience-dependent and that both prey-capture and escape behavior depend on this control.

State-dependent nickel block of a high-voltage-activated neuronal calcium channel.

Effect of nickel ions (Ni2+) on noninactivating calcium channels in squid giant fiber lobe (GFL) neurons were investigated with whole cell voltage clamp. Three different effects of Ni2+ were observed to be associated with distinct Ca2+ channel activation states. 1) Nickel ions appear to stabilize closed channel states and, as a result, slow activation kinetics. 2) Nickel ions block open channels with little voltage dependence over a wide range of potentials. 3) Block of open channels by Ni2+ becomes more effective during an extended strong depolarization, and this effect is voltage dependent. Recovery from this additional inhibition occurs at intermediate voltages, consistent with the presence of two distinct types of Ni2+ block that we propose correspond to two previously identified open states of the calcium channel. These results, taken together with earlier evidence of state-dependent block by omega-agatoxin IVA, suggest that Ni2+ generates these unique effects in part by interacting differently with the external surface of the GFL calcium channel complex in ways that depend on channel activation state.

A family of delayed rectifier Kv1 cDNAs showing cell type-specific expression in the squid stellate ganglion/giant fiber lobe complex.

Squid giant axons are formed by giant fiber lobe (GFL) neurons of the stellate ganglion (SG). Other large motoneurons in the SG form a parallel system. A small family of cDNAs (SqKv1A-D) encoding Kv1 alpha-subunits was identified in a squid (Loligo opalescens) SG/GFL library. Members have distinct 5' untranslated regions (UTRs) and initial coding regions, but beyond a certain point (nucleotide 34 of SqKv1A) only nine differences exist. 3' UTRs are identical. Predicted alpha-subunits are nearly identical, and only the N termini differ significantly, primarily in length. RNase protection assays that use RNA isolated from specific SG regions show that SqKv1A mRNA is expressed prominently in the GFL but not in the SG proper. SqKv1B yields the opposite pattern. SqKv1D also is expressed only in the SG. SqKv1C expression was not detectable. In situ hybridizations confirm these results and reveal that SqKv1B mRNA is abundant in many large neurons of the SG, whereas SqKv1D expression is limited to small isolated clusters of neurons. SqKv1A and B are thus the predominant Kv1 mRNAs in the SG/GFL complex. Activation properties of SqKv1A and B channels expressed in oocytes are very similar to one another and compare favorably with properties of native delayed rectifier channels in GFL neurons and large SG neurons. The Kv1 complement in these squid neurons thus seems to be relatively simple. Several differences exist between cloned and native channels, however, and may reflect differences in the cellular environments of oocytes and neurons.

Post-hatching development of circular mantle muscles in the squid Loligo opalescens.

Post-hatching development of the circular muscles in the mantle of squid was studied morphometrically to identify structural changes and to quantify hyperplasia and hypertrophy of the muscle fibers. Superficial, mitochondria-rich (SMR) fibers and central, mitochondria-poor (CMP) fibers are present at hatching. Although both fiber types increase in size and, even more so, in number during post-hatching development, CMP fibers increase at a much higher rate than do SMR fibers. As a result, the relative proportion of SMR to CMP fibers shifts from about 1:1 in a hatchling to about 1:6 in an 8-week-old animal; it then apparently remains constant to adulthood. These structural changes are consistent with developmental changes in muscular activity. During slow, jet-propelled swimming, 1-week-old animals show mantle contractions that have twice the relative amplitude and frequency of those in adults. The presence of Na-channel protein in mantle muscle was detected bio-chemically by using site-directed antibodies; the protein was found to be preferentially expressed in CMP fibers. These results suggest that SMR fibers are an important source of locomotory power at hatching, but become progressively less important during the first 8 weeks of development as CMP fibers assume the dominant role in jet locomotion.

Fast and slow activation kinetics of voltage-gated sodium channels in molluscan neurons.

Whole cell patch-clamp recordings of Na current (I(Na)) were made under identical experimental conditions from isolated neurons from cephalopod (Loligo, Octopus) and gastropod (Aplysia, Pleurobranchaea, Doriopsilla) species to compare properties of activation gating. Voltage dependence of peak Na conductance (gNa) is very similar in all cases, but activation kinetics in the gastropod neurons studied are markedly slower. Kinetic differences are very pronounced only over the voltage range spanned by the gNa-voltage relation. At positive and negative extremes of voltage, activation and deactivation kinetics of I(Na) are practically indistinguishable in all species studied. Voltage-dependent rate constants underlying activation of the slow type of Na channel found in gastropods thus appear to be much more voltage dependent than are the equivalent rates in the universally fast type of channel that predominates in cephalopods. Voltage dependence of inactivation kinetics shows a similar pattern and is representative of activation kinetics for the two types of Na channels. Neurons with fast Na channels can thus make much more rapid adjustments in the number of open Na channels at physiologically relevant voltages than would be possible with only slow Na channels. This capability appears to be an adaptation that is highly evolved in cephalopods, which are well known for their high-speed swimming behaviors. Similarities in slow and fast Na channel subtypes in molluscan and mammalian neurons are discussed.

Fast inactivation of delayed rectifier K conductance in squid giant axon and its cell bodies.

Inactivation of delayed rectifier K conductance (gk) was studied in squid giant axons and in the somata of giant fiber lobe (GFL) neurons. Axon measurements were made with an axial wire voltage clamp by pulsing to VK (approximately -10 mV in 50-70 mM external K) for a variable time and then assaying available gK with a strong, brief test pulse. GFL cells were studied with whole-cell patch clamp using the same prepulse procedure as well as with long depolarizations. Under our experimental conditions (12-18 degrees C, 4 mM internal MgATP) a large fraction of gK inactivates within 250 ms at -10 mV in both cell bodies and axons, although inactivation tends to be more complete in cell bodies. Inactivation in both preparations shows two kinetic components. The faster component is more temperature-sensitive and becomes very prominent above 12 degrees C. Contribution of the fast component to inactivation shows a similar voltage dependence to that of gK, suggesting a strong coupling of this inactivation path to the open state. Omission of internal MgATP or application of internal protease reduces the amount of fast inactivation. High external K decreases the amount of rapidly inactivating IK but does not greatly alter inactivation kinetics. Neither external nor internal tetraethylammonium has a marked effect on inactivation kinetics. Squid delayed rectifier K channels in GFL cell bodies and giant axons thus share complex fast inactivation properties that do not closely resemble those associated with either C-type or N-type inactivation of cloned Kvl channels studied in heterologous expression systems.

All-or-none contraction and sodium channels in a subset of circular muscle fibers of squid mantle.

Motor function in squid (Loligo) mantle reflects the highly coordinated activity of two motor pathways associated with giant and non-giant motor axons that respectively produce all-or-none and graded contractions in mantle muscle. Whereas both types of axons innervate circular mantle muscle fibers, precise nerve-muscle relationships remain unclear. Are squid like most invertebrates, in which single muscle fibers receive dual innervation from giant and non-giant motor axons, or is squid mantle configured more like vertebrates, in which parallel motor axon systems innervate distinct fast and slow muscle fibers? In this report, we describe giant and nongiant motor pathways that appear to control different pools of circular muscle fibers in squid. A subset of circular muscle fibers possesses large Na currents, and these fibers are proposed to employ Na-dependent action potentials to produce fast, all-or-none muscle twitches associated with giant axon stimulation.

Molecular identification of SqKv1A. A candidate for the delayed rectifier K channel in squid giant axon.

We have cloned the cDNA for a squid Kvl potassium channel (SqKv1A). SqKv1A mRNA is selectively expressed in giant fiber lobe (GFL) neurons, the somata of the giant axons. Western blots detect two forms of SqKv1A in both GFL neuron and giant axon samples. Functional properties of SqKv1A currents expressed in Xenopus oocytes are very similar to macroscopic currents in GFL neurons and giant axons. Macroscopic K currents in GFL neuron cell bodies, giant axons, and in Xenopus oocytes expressing SqKv1A, activate rapidly and inactivate incompletely over a time course of several hundred ms. Oocytes injected with SqKv1A cRNA express channels of two conductance classes, estimated to be 13 and 20 pS in an internal solution containing 470 mM K. SqKv1A is thus a good candidate for the "20 pS" K channel that accounts for the majority of rapidly activating K conductance in both GFL neuron cell bodies and the giant axon.

Spatial localization of calcium channels in giant fiber lobe neurons of the squid (Loligo opalescens).

Whole-cell voltage clamp was used to investigate the properties and spatial distribution of fast-deactivating (FD) Ca channels in squid giant fiber lobe (GFL) neurons. Squid FD Ca channels are reversibly blocked by the spider toxin omega-Agatoxin IVA with an IC50 of 240-420 nM with no effect on the kinetics of Ca channel gating. Channels with very similar properties are expressed in both somatic and axonal domains of cultured GFL neurons, but FD Ca channel conductance density is higher in axonal bulbs than in cell bodies at all times in culture. Channels presumably synthesized during culture are preferentially expressed in the growing bulbs, but bulbar Ca conductance density remains constant while Na conductance density increases, suggesting that processes determining the densities of Ca and Na channels in this extrasomatic domain are largely independent. These observations suggest that growing axonal bulbs in cultured GFL neurons are not composed entirely of "axonal" membranes because FD Ca channels are absent from the giant axon in situ but, rather, suggest a potential role for FD Ca channels in mediating neurotransmitter release at the motor terminals of the giant axon.

Methadone block of K+ current in squid giant fiber lobe neurons.

Voltage-dependent ionic currents were recorded from squid giant fiber lobe neurons using the whole-cell patch-clamp technique. When applied to the bathing solution, methadone was found to block IK, I Na and I Ca. Both I Na and I Ca were reduced without apparent change in kinetics and exhibited IC(50)'s of 50-100 and 250-500 mu M, respectively, at +10 mV. In contrast, IK was reduced in a time-dependent manner that is well fit by a simple model of open channel block (K(D)= 32+/- or 2 mu M, +60 mV, 10 degrees Celsius). The mechanism of I(K) block was examined in detail and involves a direct action of methadone, a tertiary amine, on K channels rather than an opioid receptor-mediated pathway. The kinetics of I(K) block resemble those reported for internally applied long chain quaternary ammonium (QA) compounds; and recovery from I(K) block is QA-like in its slow time course and strong dependence on holding potential. A quaternary derivative of methadone (N-methyl-methadone) only reproduced the effects of methadone on I(K) when included in the pipette solution; this compound was without effect when applied externally. I(K) block thus appears to involve diffusion of methadone into the cytoplasm and occlusion of the open K channel at the internal QA blocking site by the protonated form of the drug. This proposed mode of action is supported by the pH and voltage dependence of block as well as by the observation that high external K+ speeds the rate of drug dissociation. In addition, the effect of methadone on I(K) evoked during prolonged (300 ms) depolarizations suggests that methadone block may interfere with endogenous K+ channel inactivation. The effects of temperature, methadone stereoisomers, and the methadone-like drugs propoxyphene and nor-propoxyphene on IK block were examined. Methadone was also found to block I(K) in GH3 cells and in chick myoblasts.