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In this study we examine the effects of ionic conditions on the gating charge movement in the fast inactivation–removed wild-type Shaker channel and its W434F mutant. Our results show that various ionic conditions influence the rate at which gating charge returns during repolarization following a depolarizing pulse. These effects are realized through different mechanisms, which include the regulation of channel closing by occupying the cavity, the modulation of transitions into inactivated states, and effects on transitions between closed states via a direct interaction with the channel's gating charges. In generating these effects the cations act from the different binding sites within the pore. Ionic conditions, in which conducting wild-type channels close at different rates, do not significantly affect the rate of charge recovery upon repolarization. In these conditions, channel closing is fast enough not to be rate-limiting in the charge recovery process. In the permanently P-inactivated mutant channel, however, channel closing becomes the rate-limiting step, presumably due to weakened ion–ion interactions inside the pore and a slower intrinsic rate of gate closure. Thus, variations in closing rate induced by different ions are reflected as variations in the rate of charge recovery. In 115 mM internal Tris+ and external K+, Cs+, or Rb+, low inward permeation of these ions can be observed through the mutant channel. In these instances, channel closing becomes slower than in Tris+O//Tris+I solutions showing resemblance to the wild-type channel, where higher inward ionic fluxes also retard channel closing. Our data indicate that cations regulate the transition into the inactivated states from the external lock-in site and possibly the deep site. The direct action of barium on charge movement is probably exerted from the deep site, but this effect is not very significant for monovalent cations.

Time constants of slow inactivation were investigated in NH(2)-terminal deleted Shaker potassium channels using macro-patch recordings from Xenopus oocytes. Slow inactivation is voltage insensitive in physiological solutions or in simple experimental solutions such as K(+)(o)//K(+)(i) or Na(+)(o)//K(+)(i). However, when [Na(+)](i) is increased while [K(+)](i) is reduced, voltage sensitivity appears in the slow inactivation rates at positive potentials. In such solutions, the I-V curves show a region of negative slope conductance between approximately 0 and +60 mV, with strongly increased outward current at more positive voltages, yielding an N-shaped curvature. These changes in peak outward currents are associated with marked changes in the dominant slow inactivation time constant from approximately 1.5 s at potentials less than approximately +60 mV to approximately 30 ms at more than +150 mV. Since slow inactivation in Shaker channels is extremely sensitive to the concentrations and species of permeant ions, more rapid entry into slow inactivated state(s) might indicate decreased K(+) permeation and increased Na(+) permeation at positive potentials. However, the N-shaped I-V curve becomes fully developed before the onset of significant slow inactivation, indicating that this N-shaped I-V does not arise from permeability changes associated with entry into slow inactivated states. Thus, changes in the relative contributions of K(+) and Na(+) ions to outward currents could arise either: (a) from depletions of [K(+)](i) sufficient to permit increased Na(+) permeation, or (b) from voltage-dependent changes in K(+) and Na(+) permeabilities. Our results rule out the first of these mechanisms. Furthermore, effects of changing [K(+)](i) and [K(+)](o) on ramp I-V waveforms suggest that applied potential directly affects relative permeation by K(+) and Na(+) ions. Therefore, we conclude that the voltage sensitivity of slow inactivation rates arises indirectly as a result of voltage-dependent changes in the ion occupancy of these channels, and demonstrate that simple barrier models can predict such voltage-dependent changes in relative permeabilities.

Shaker channel mutants, in which the first (R362), second (R365), and fourth (R371) basic residues in the S4 segment have been neutralized, are found to pass potassium currents with voltage-insensitive kinetics when expressed in Xenopus oocytes. Single channel recordings clarify that these channels continue to open and close from −160 to +80 mV with a constant opening probability ( P o ). Although P o is low (∼0.15) in these mutants, mean open time is voltage independent and similar to that of control Shaker channels. Additionally, these mutant channels retain characteristic Shaker channel selectivity, sensitivity to block by 4-aminopyridine, and are partially blocked by external Ca 2+ ions at very negative potentials. Furthermore, mean open time is approximately doubled, in both mutant channels and control Shaker channels, when Rb + is substituted for K + as the permeant ion species. Such strong similarities between mutant channels and control Shaker channels suggests that the pore region has not been substantially altered by the S4 charge neutralizations. We conclude that single channel kinetics in these mutants may indicate how Shaker channels would behave in the absence of voltage sensor input. Thus, mean open times appear primarily determined by voltage-insensitive transitions close to the open state rather than by voltage sensor movement, even in control, voltage-sensitive Shaker channels. By contrast, the low and voltage-insensitive P o seen in these mutant channels suggests that important determinants of normal channel opening derive from electrostatic coupling between S4 charges and the pore domain.

The kinetic properties of wild-type rat brain IIa sodium channels in excised macropatches were studied using step depolarizations and ramp depolarizations to imitate the slow settling-time of voltage in two-electrode voltage clamp. Ramp depolarizations longer than 1 ms produce an increasing suppression of peak sodium current (I[Na]). Two rates of inactivation can be seen in macroscopic sodium current records from excised patches following both step and ramp depolarizations. During slow ramp depolarizations, reduction in peak I[Na] is associated with selective loss of the fastest rate of test-pulse inactivation. This change can be interpreted as resulting from inactivation of a separate sub-population of 'fast mode' channels. The slow rate of test-pulse inactivation is relatively unaffected by changing ramp durations. These results are sufficient to explain the typically slow inactivation kinetics seen in two-electrode voltage clamp recordings of sodium channels in Xenopus oocytes. Thus, the kinetics of sodium channels expressed in Xenopus oocytes are not readily characterizable by two-electrode clamp because of the large membrane capacitance and resulting slow clamp settling time which artifactually selects for slow mode channels.

C-type inactivation of Shaker potassium channels involves entry into a state (or states) in which the inactivated channels appear nonconducting in physiological solutions. However, when Shaker channels, from which fast N-type inactivation has been removed by NH2-terminal deletions, are expressed in Xenopus oocytes and evaluated in inside-out patches, complete removal of K+ ions from the internal solution exposes conduction of Na+ and Li+ in C-type inactivated conformational states. The present paper uses this observation to investigate the properties of ion conduction through C-type inactivated channel states, and demonstrates that both activation and deactivation can occur in C-type states, although with slower than normal kinetics. Channels in the C-type states appear “inactivated” (i.e., nonconducting) in physiological solutions due to the summation of two separate effects: first, internal K+ ions prevent Na+ ions from permeating through the channel; second, C-type inactivation greatly reduces the permeability of K+ relative to the permeability of Na+, thus altering the ion selectivity of the channel.

Illumination of crayfish giant axons, during internal perfusion with 0.5 mM methylene blue (MB), produces photodynamic effects that include (i) reduction in total sodium conductance, (ii) shifting of the steady-state inactivation curve to the right along the voltage axis, (iii) reduction in the effective valence of steady-state inactivation and, (iv) potentially complete removal of fast inactivation. Additionally, the two kinetic components of fast inactivation in crayfish axons are differentially affected by MB+light. The intercept of the faster component (tau h1) is selectively reduced at shorter MB+light exposure times. Neither tau h1 nor the slower (tau h2) process was protected from MB+light by prior steady-state inactivation of sodium channels. However, carotenoids provide differing degrees of protection against each of the photodynamic actions listed above, suggesting that the four major effects of MB+light are mediated by changes occurring within different regions of the sodium channel molecule.

In the adult mouse, signaling through c-Jun N-terminal kinases (JNKs) links exposure to acute stress to various physiological responses. Inflammatory cytokines, brain injury and ischemic insult, or exposure to psychological acute stressors induce activation of hippocampal JNKs. Here we report that exposure to acute stress caused activation of JNKs in the hippocampal CA1 and CA3 subfields, and impaired contextual fear conditioning. Conversely, intrahippocampal injection of JNKs inhibitors sp600125 (30 μm) ord-JNKI1 (8 μm) reduced activity of hippocampal JNKs and rescued stress-induced deficits in contextual fear. In addition, intrahippocampal administration of anisomycin (100 μg/μl), a potent JNKs activator, mimicked memory-impairing effects of stress on contextual fear. This anisomycin-induced amnesia was abolished after cotreatment with JNKs selective inhibitor sp600125 without affecting anisomycin's ability to effectively inhibit protein synthesis as measured by c-Fos immunoreactivity. We also demonstrated milder and transient activation of the JNKs pathway in the CA1 subfield of the hippocampus during contextual fear conditioning and an enhancement of contextual fear after pharmacological inhibition of JNKs under baseline conditions. Finally, using combined biochemical and transgenic approaches with mutant mice lacking different members of the JNK family (Jnk1, Jnk2, andJnk3), we provided evidence that JNK2 and JNK3 are critically involved in stress-induced deficit of contextual fear, while JNK1 mainly regulates baseline learning in this behavioral task. Together, these results support the possibility that hippocampal JNKs serve as a critical molecular regulator in the formation of contextual fear.

Changes in holding potential (Vh), affect both gating charge (the Q(Vh) curve) and peak ionic current (the F(Vh) curve) seen at positive test potentials. Careful comparison of the Q(Vh) and F(Vh) distributions indicates that these curves are similar, having two slopes (approximately 2.5e for Vh from -115 to -90 mV and approximately 4e for Vh from -90 to -65 mV) and very negative midpoints (approximately -86 mV). Thus, gating charge movement and channel availability appear closely coupled under fully-equilibrated conditions. The time course by which channels approach equilibration was explored using depolarizing prepulses of increasing duration. The high slope component seen in the F(Vh) and Q(Vh) curves is not evident following short depolarizing prepulses in which the prepulse duration approximately corresponds to the settling time for fast inactivation. Increasing the prepulse duration to 10 ms or longer reveals the high slope, and left-shifts the midpoint to more negative voltages, towards the F(Vh) and Q(Vh) distributions. These results indicate that a separate slow-moving voltage sensor affects the channels at prepulse durations greater than 10 ms. Charge movement and channel availability remain closely coupled as equilibrium is approached using depolarizing pulses of increasing durations. Both measures are 50% complete by 50 ms at a prepulse potential of -70 mV, with proportionately faster onset rates when the prepulse potential is more depolarized. By contrast, charge movement and channel availability dissociate during recovery from prolonged depolarizations. Recovery of gating charge is considerably faster than recovery of sodium ionic current after equilibration at depolarized potentials. Recovery of gating charge at -140 mV, is 65% complete within approximately 100 ms, whereas less than 30% of ionic current has recovered by this time. Thus, charge movement and channel availability appear to be uncoupled during recovery, although both rates remain voltage sensitive. These data suggest that channels remain inactivated due to a separate process operating in parallel with the fast gating charge. We demonstrate that this behavior can be simulated by a model in which the fast charge movement associated with channel activation is electrostatically-coupled to a separate slow voltage sensor responsible for the slow inactivation of channel conductance.

We have previously reported that repeated central administration of sub-anxiogenic doses of the corticotropin releasing factor 1 (CRF(1)) agonist Cortagine, termed "priming," elicits a phenotype of increased anxiety-like behaviors in the elevated plus maze (EPM) and open-field test, and enhanced retention of contextual conditioned fear in C57BL/6J mice. Observed behavioral changes were functionally coupled to CRF(1)-mediated elevated central cholecystokinin (CCK) tone in discrete brain regions. However, the changes in gene expression that mediated "priming"-induced behavioral and concurrent molecular changes in specific brain regions remained unknown. In the present study, a complementary DNA microarray analysis was used to investigate gene expression profiles in the hippocampus and prefrontal cortex (PFC) of C57BL/6J mice following the "priming" procedure. Here, we report that chronic stimulation of CRF(1), by i.c.v. administration of 10 ng Cortagine for five days, brought about alterations in the expression of a wide range of hippocampal (31 genes) and PFC (18 genes) genes, implicated in anxiety and aversive memory formation. These expression changes involved genes associated with signal transduction, neurotransmitter secretion, synaptic transmission, myelination, and others involved in the transport, biosynthesis, and binding of proteins. In particular, several genes of the protein kinase A (PKA) and protein kinase C (PKC) signaling cascades, known to be involved in synaptic plasticity, such as neurogranin, calmodulin 3, and the PKA regulatory subunit 1 b were found to be upregulated in the PFC and hippocampus of CRF(1) agonist "primed" mice. Moreover, we show pharmacologically that one of the newly implicated memory regulatory elements, diazepam-binding inhibitor (DBI) is functionally involved in hippocampus-dependent enhancement of contextual fear, a cardinal phenotypic feature of the "primed" mice. Finally, an interaction network mapping of the altered genes and their known interacting partners identified additional molecular candidates responsible for CRF(1)-mediated hypersensitive fear circuitry.

The neuropeptide corticotropin-releasing factor (CRF) plays a critical role in the proper functioning of the stress response system through its actions on its receptors, CRF receptor 1 (CRF1) and CRF receptor 2 (CRF2), located at multiple anatomical sites. Clinical data indicate that stress response dysfunctions, such as excessive CRF activity and hyperstimulation of CRF1, are present in a range of stress-related disorders, including depression and anxiety disorders. Our previous work along with that of other laboratories has demonstrated that mice deficient in CRF2 (CRF2-/-) display increased anxiety and depression-like behaviors. In this study, we found CRF2-/- mice display increased hippocampal levels of activated (phosphorylated) mitogen-activated protein kinase (MAP kinase)/ERK kinase (MEK), extracellular signal-regulated kinases 1 and 2 (ERK1/2), and ribosomal protein S6 kinases 1 (RSK1). These changes can be explained by overactive hippocampal CRF1, in view of the finding that the application of the nonselective CRF receptor antagonist [Glu(11,16)] astressin ([Glu(11,16)]Ast) into the dorsal hippocampus of mutant mice returned the levels of the phosphorylated proteins to baseline. Moreover, inhibition of the hippocampal MEK/ERK pathway with the specific MEK inhibitor U0126, decreased depression-like behaviors in the forced swim test and tail suspension test of CRF2-/- mice. Similarly, treatment with [Glu(11,16)]Ast reversed depression phenotype of CRF2-/- mice without affecting the phenotype of wild-type littermates. Our results support an involvement of CRF receptors in the development of depression, such that elevated hippocampal CRF1 activity, in the absence of CRF2, produces a depression-dominated phenotype through the activation of the MEK/ERK pathway.

C-type inactivation in Shaker potassium channels inhibits K+ permeation. The associated structural changes appear to involve the outer region of the pore. Recently, we have shown that C-type inactivation involves a change in the selectivity of the Shaker channel, such that C-type inactivated channels show maintained voltage-sensitive activation and deactivation of Na+ and Li+ currents in K+-free solutions, although they show no measurable ionic currents in physiological solutions. In addition, it appears that the effective block of ion conduction produced by the mutation W434F in the pore region may be associated with permanent C-type inactivation of W434F channels. These conclusions predict that permanently C-type inactivated W434F channels would also show Na+ and Li+ currents (in K+-free solutions) with kinetics similar to those seen in C-type-inactivated Shaker channels. This paper confirms that prediction and demonstrates that activation and deactivation parameters for this mutant can be obtained from macroscopic ionic current measurements. We also show that the prolonged Na+ tail currents typical of C-type inactivated channels involve an equivalent prolongation of the return of gating charge, thus demonstrating that the kinetics of gating charge return in W434F channels can be markedly altered by changes in ionic conditions.

Macroscopic currents of wild-type rat brain IIA (RBIIA) and mutant Na channels were recorded in excised patches from Xenopus oocytes. A charge deletion (K859Q) and an adjacent conservative mutation (L860F) in the second domain S4 membrane-spanning region differentially altered voltage sensitivity and kinetics. Analysis of voltage dependence was confined to Na currents with fast inactivation kinetics, although RBIIA and K859Q (but not L860F) also showed proportional shifts between at least two gating modes, rendering currents with fast or slow inactivation kinetics, respectively. Compared to RBIIA, the midpoint of the activation curve was shifted in both K859Q and L860F by 22 mV to more positive potentials, yet this shift was not associated with a corresponding change in the voltage dependence of time constants for activation (tau a) or inactivation (tau h1, tau h2). L860F showed faster activation time constants tau a than RBIIA, while K859Q was slower for both the activation (tau a) and the inactivation components (tau h1). Similarly, the steady-state inactivation curve of L860F but not K859Q shifted by 9 mV in the hyperpolarizing direction. Thus, the fourth charge in the IIS4 transmembrane segment exerts control over voltage sensitivity and kinetics of activation and may interact with structure that influence other aspects of channel gating.

Publisher Summary This chapter focuses on axial-wire voltage clamping in crayfish medial giant axons. The crayfish medial giant axon preparation provides substantial advantages for axial-wire voltage-clamp studies on axonal sodium channels in their native membrane. Crayfish are inexpensive and readily available on a year-round basis. The axoplasm is fluid and easily washed out of the axon when the internal perfusate is driven by a very low hydrostatic pressure head to yield a clean membrane preparation without requiring either dialysis, enzymatic, or roller techniques for axoplasm removal. The crayfish Schwann cells permit very rapid re-equilibration of periaxonal ion concentrations. The close spacing of the tubular lattice openings reduces the possibility that microscopic space clamp errors can arise from the lateral resistance of the Frankenhaeuser–Hodgkin space. Current magnitudes are sufficient to permit detailed measurements of ionic and gating current kinetics without some of the problems inherent in the squid axon system. The crayfish voltage-clamp electrode designed by Shrager is constructed as a piggy-back electrode. The piggy-back electrode can be used either for the intact axon or for the perfused axon.

Kinetic effects of osmotic stress on sodium ionic and gating currents have been studied in crayfish giant axons after removal of fast inactivation with chloramine-T. Internal perfusion with media made hyperosmolar by addition of formamide or sucrose, reduces peak sodium current (before and after removal of fast inactivation with chloramine-T), increases the half-time for activation, but has no effect on tail current deactivation rate(s). Kinetics of ON and OFF gating currents are not affected by osmotic stress. These results confirm (and extend to sodium channels) the separation of channel gating mechanisms into voltage-sensitive and solvent-sensitive processes recently proposed by Zimmerberg J., F. Bezanilla, and V. A. Parsegian. (1990. Biophys. J. 57:1049-1064) for potassium delayed rectifier channels. Additionally, the kinetic effects produced by hyperosmolar media seem qualitatively similar to the kinetic effects of heavy water substitution in crayfish axons (Alicata, D. A., M. D. Rayner, and J. G. Starkus. 1990. Biophys. J. 57:745-758). However, our observations are incompatible with models in which voltage-sensitive and solvent-sensitive gating processes are presumed to be either (a) strictly sequential or, (b) parallel and independent. We introduce a variant of the parallel model which includes explicit coupling between voltage-sensitive and solvent-sensitive processes. Simulations of this model, in which the total coupling energy is as small as 1/10th of kT, demonstrate the characteristic kinetic changes noted in our data.

Palytoxin, produced by a stationary marine animal and one of the most toxic substances known, was used as a spear poison in ancient Hawaii to cause death by cardiovascular contracture. We report here that the motility of hamster caudal epididymal (HCE) and other sperm can be inhibited by as little as 10 −13 M palytoxin in a time-dependent manner. This inhibition manifested itself as a loss in flagellar amplitude, often accompanied by an increase in beat frequency, resulting in a loss of forward progression and ultimately cessation of movement. Similar effects were observed in sperm from guinea pigs, rabbit, cattle, sea urchins and man. Preincubation with palytoxin did not prevent the induction of motility from quiescence in HCE sperm when free calcium ion was added. However, regardless of the timing of palytoxin addition this very vigorous motility disappeared shortly after it appeared. These, plus earlier observations showing palytoxin did not cause lysis under similar conditions or inhibit the progressive motility of demembranated sperm axoneme preparations, suggest both that this large molecule acts via the plasma membrane to cause its exceedingly toxic effects and that spermatozoa may be useful for the investigation of the mechanism of action of palytoxin.

Effects of highly purified and partially purified ciguatoxic extracts on the sodium-potassium activated adenosine triphosphatase (NaK ATPase) of human erythrocyte ghosts have been evaluated and compared with the effects of a similarly prepared extract from a nontoxic eel. No inhibition of NaK ATPase was found with the most highly purified extract at an estimated ciguatoxin concentration as high as 10 −4 m and with an erythrocyte membrane protein to toxin ratio of 0.66. In contrast, three of five partially purified extracts studied gave significant inhibition ( p

We report some preliminary studies on ether-soluble and water-soluble residues obtained from the midgut glands of two Hawaiian species of sea hares (Aplysiidae). Sublethal doses of the crude ether-soluble residue produce hypertension when injected intravenously into anesthetized rats, whereas the crude water-soluble residue produces a transient hypotension, bradycardia and apnea. Dose-effect curves for more purified extracts show actions similar to those seen with crude extracts. The hypertension produced by the ether-soluble toxin is resistant to both alpha- and beta-adrenergic blocking agents. The hypotensive effect of the water-soluble extract could not be abolished by vagotomy or pretreatment with either atropine (25 mg/kg) or Benadryl (22 mg/kg). It is concluded that both extracts may have direct effects on the contractility of vascular smooth muscle which are not mediated by alpha-adrenergic or cholinergic mechanisms. The possible relationship of these toxic extracts to the previously described aplysin is discussed.

Although ciguatoxin, prepared from the liver and flesh of the moray eel Gymnothorax javanicus, is an in vitro anticholinesterase, it has not been found to be an in vivo anticholinesterase. Respiratory failure and death due to ciguatoxin is not a function of cholinesterase inhibition as previously speculated, although the toxin may have a widespread action at cholinergic junctions.

The effect of Ciguatoxin (CT) on Na transport across isolated frog skin ( Rana pipiens ) was studied by measuring the electrical potential difference (PD) and the short-circuit current (SCC). When NaCl-Ringer was used, CT (0.25–0.5 mg/100 ml) added to the outside bathing medium induced a substantial reduction in PD and a slight reduction in SCC, indicating a reduction in the skin resistance. The recovery was extremely slow and incomplete. The above reduction in SCC was associated with a significant increase in 22 Na outflux in the face of constant 22 Na influx. The CT effect was much less pronounced and more variable when it was added to the inside bathing medium. When Na 2 SO 4 -Ringer was used, the reduction in PD was smaller than that of SCC. The presence of antidiuretic hormone (ADH) in the inside bathing medium did not alter the response of the skin to CT. Similarly, the presence of CT in the outside bathing medium did not affect the response of the skin to ADH. However, the action of CT was not evident in the presence of 10 m m calcium in the outside bathing medium. CT had no effect on the water permeability of the toad urinary bladder preparation. These results suggest that CT primarily increases the passive permeability of the skin to Na, although an effect on the Na pump cannot be ruled out completely.

Anticholinesterase action of ciguatoxin has been reported. A study of the pharmacology of the action of the toxin on the respiratory system suggests that additional properties may be involved in the respiratory failure-the usual cause of death in ciguatera poisoning.