Functional consequences of divergence and convergence in physiological signaling pathways.
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Animals↗
Biomedical subjects
K R Weiss
Publications and source records attributed to K R Weiss.
Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia.
Ion currents and mechanisms of modulation in the radula opener muscles of Aplysia. J. Neurophysiol. 78: 2372-2387, 1997. Numerous studies of plasticity in the feeding behavior of Aplysia have shown that substantial plasticity is due to peripheral neuromodulation of the feeding musculature. Extensive previous work focusing on the accessory radula closer (ARC) muscle has led to the realization that a major function of the modulation in that muscle may be to ensure efficient coordination between its contractions and those of its antagonist muscles. For a more complete understanding, therefore, we must study these muscles also. Here we have studied the radula opener muscles I7-I10. Using single isolated muscle fibers under voltage clamp, we have characterized ion currents gated by voltage and by the physiological contraction-inducing neurotransmitter acetylcholine (ACh) and the effects of the physiological modulators serotonin, myomodulins A and B, and FMRFamide. Our results explain significant aspects of the electrophysiological behavior of the whole opener muscles, as well as why the opener and ARC muscles behave similarly in many ways yet differently in some key respects. Opener muscles express four types of K currents: inward rectifier, A-type [IK(A)], delayed rectifier [IK(V)], and Ca2+-activated [IK(Ca)]. They also express an L-type Ca current [ICa] and a leakage current. ACh activates a positive-reversing cationic current [IACh(cat)] and a negative-reversing Cl current [IACh(Cl)]. The opener muscles differ from the ARC in that, in the openers, activation of IK(A) occurs approximately 9 mV more positive and there is much less IACh(Cl). In both muscles, IACh(cat) most likely serves to depolarize the muscle until ICa activates to supply Ca2+ for contraction, but further depolarization and spiking is opposed by coactivation of IK(A), IK(V), IK(Ca), and IACh(Cl). Thus the differences in IK(A) and IACh(Cl) may well be key factors that prevent spikes in the ARC but often allow them in the opener muscles. As in the ARC, the modulators enhance ICa and so potentiate contractions. They also activate a modulator-specific K current, which causes hyperpolarization and depression of contractions. Finally, in the opener muscles but not in the ARC, the modulators activate a depolarizing cationic current that may help phase-advance the contractions. Each modulator exerts these effects to different degrees and thus has a distinct effect on voltage and contraction size and shape. The overall effect then will depend on the specific combinations of modulators released in different behaviors. By understanding the modulation in the opener muscles, as well as in the ARC, we are now in a position to understand how the behavior of the two muscles is coordinated under a variety of circumstances.
4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone↗
Giant protein kinases: domain interactions and structural basis of autoregulation.
The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules. We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain. The structure of the longer fragment shows that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues. Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.
Amino Acid Sequence↗
Substrate specificity and inhibitor sensitivity of Ca2+/S100-dependent twitchin kinases.
Myosin-associated giant protein kinases of the titin/witchin-like superfamily have previously been implicated in the regulation of muscle function, based on genetic and physiological studies. We find that recombinant constitutively active Caenorhabditis elegans and Aplysia twitchin kinase fragments differ in their catalytic activities and peptide-substrate specificities, as well as in their sensitivities to the naphthalene sulfonamide inhibitors 1-(5-chloronaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-7) and 1-(5-iodonaphthalenesulfonyl)-1H-hexahydro-1,4-diazepine (ML-9). The constitutively active Aplysia twitchin kinase fragment has a remarkably high activity (Vmax > 100 mumol.min-1.mg-1) towards some substrate peptides. The autoinhibited forms of these twitchin kinases can be activated in a Ca(2+)-dependent manner by the dimeric form of the S100A1 protein (S100A1(2)). The twitchin kinase S100A1(2)-binding site can also bind Ca2+/calmodulin but neither kinase is activated by calmodulin. The data provide a functional basis for the ongoing crystallographic study of twitchin kinase fragments.
Amino Acid Sequence↗
Costorage and corelease of modulatory peptide cotransmitters with partially antagonistic actions on the accessory radula closer muscle of Aplysia californica.
Many neurons that contain a classical neurotransmitter also contain modulatory peptides, but it has been difficult to establish unequivocally that these peptides are functional cotransmitters. Here, we provide evidence for functional cotransmission in a neuromuscular system of Aplysia. Using immunocytochemical techniques, we localize members of two peptide families, the small cardioactive peptides (SCPs) and the buccalins (BUCs), to a single subset of dense-core vesicles in the terminals of the cholinergic motorneuron B15. We describe a new preparation and method for the direct detection of released peptides and show that the SCPs and BUCs are released when neuron B15 is intracellularly stimulated. Consistent with their subcellular localization, the SCPs and BUCs are released in a stoichiometric ratio that is constant across conditions that change the absolute amount of peptides released. Peptide release is calcium-dependent but does not require muscle contractions. Thus, the release cannot be attributed to a displacement of peptides that may be present in the extracellular space. In previous studies, we characterized the physiological firing patterns of neuron B15. Here, we simulate these firing patterns and show that peptide release occurs. Additionally, we find that significant quantities of material are released under behaviorally relevant conditions. We find that concentrations of released peptides in the muscle are in the concentration range in which exogenously applied peptides exert characterized modulatory actions on muscle contractions. Together, our findings provide strong support for the hypothesis that peptides contained in neuron B15 are functional cotransmitters.
Animals↗
Release of peptide cotransmitters in Aplysia: regulation and functional implications.
To gain insights into the physiological role of cotransmission, we measured peptide release from cell B15, a motorneuron that utilizes ACh as its primary transmitter but also contains putative peptide cotransmitters, the small cardioactive peptides (SCPs) and the buccalins (BUCs). All stimulation parameters used were in the range in which B15 fires in freely moving animals. We stimulated neuron B15 in bursts and systematically varied the interburst interval, the intraburst frequency, and burst duration. Both peptides were preferentially released when B15 was stimulated at higher intra- or interburst frequencies or with longer burst durations. Across stimulation patterns, the amount of peptide released depended on the mean frequency of stimulation and was independent of the specific pattern of stimulation. The parameters of stimulation that produce a larger release of peptides correspond to those that evoke larger contractions. Large and frequent contractions are likely to fuse or summate, thus disrupting the rhythmic behavior mediated by the muscle innervated by motorneuron B15. Because the combined effect of the SCPs and BUCs is to accelerate the relaxation and shorten the duration of muscle contractions, these peptides reduce the probability of the disruptive fusion or summation of muscle contractions. Because these cotransmitters regulate an aspect of muscle contractions that is not controlled by acetylcholine (ACh), the primary transmitter of B15, we suggest that peptides and ACh form parallel but functionally distinct lines of transmission at the neuromuscular junction. Both types of transmission may be necessary to ensure that behavior remains efficient over a wide range of conditions.
Animals↗
A cerebral central pattern generator in Aplysia and its connections with buccal feeding circuitry.
Different feeding-related behaviors in Aplysia require substantial variations in the coordination of movements of two separate body parts, the lips and buccal mass. The central pattern generators (CPGs) and motoneurons that control buccal mass movements reside largely in the buccal ganglion. It was previously thought that control of the cerebral neuronal circuitry and motoneurons that generate lip movements was coordinated directly by feedback from buccal interneurons. Here, we describe cerebral lip motoneuron C15, which drives rhythmic activity in the isolated cerebral ganglion. Other lip motoneurons are active during this program, so we define it as a cerebral motor program (CMP). The C15 in each cerebral hemiganglion drives the CMP in ipsilateral neurons only, suggesting there are independent CPGs in each hemiganglion. The cerebral and buccal CPGs interact at several points. For example, cerebral-to-buccal interneurons (CBIs), which can drive the buccal CPG, receive excitatory input when the cerebral CPG is active. Likewise, C15, which can drive the cerebral CPG, is excited when the buccal CPG is active. This excitation is simultaneous in both C15s, coupling the activity in the two hemiganglionic cerebral CPGs. Therefore, there are independent cerebral and buccal CPGs, which can produce distinct rhythms, but which interact at several points. Furthermore, the connections between the cerebral and buccal CPGs alter during different forms of motor program. We suggest that such alterations in the interactions between these CPGs might contribute to the generation of the various forms of coordination of lip and buccal mass movements that are necessary during different feeding-related behaviors.
Animals↗
An identified interneuron contributes to aspects of six different behaviors in Aplysia.
Previous results have indicated that the bilateral cerebral interneuron CC5 mediates the pedal artery shortening that is a component of defensive withdrawal responses involving the head. Current studies suggest that CC5 contributes to aspects of at least six different behaviors: locomotion, head turning, defensive head withdrawal, local tentacular withdrawal, rhythmic feeding, and head lifting. In addition to receiving input from mechanoreceptors in the head, CC5 receives synaptic input during fictive locomotor and feeding programs. Firing of CC5 produces widespread monosynaptic or polysynaptic actions in all ganglia in the animal. CC5 excites presumptive motor neurons for the neck, and its activity can contract neck muscles. The pedal artery shortener motor neuron (PAS), a key excitatory follower cell of CC5, fires during ipsilateral head turning, head withdrawal, tentacle withdrawal, feeding, and locomotion. For all behaviors, except locomotion and biting, responses of PAS were eliminated by cutting the ipsilateral-pleural connective, which interrupts the only direct connection of CC5 to the ipsilateral PAS. The data suggest that CC5 is a multifunctional interneuron that plays different roles during different behaviors. The neuron appears to be involved in producing coordinated movements of the head, involving both somatic and visceral muscles. For some behaviors, or for certain aspects of behaviors, CC5 appears to act as an individual command-like neuron; for other behaviors, CC5 appears to act more as an element of a distributed circuit and is neither necessary nor sufficient for any aspects of the behavior.
Abdomen↗
Functional uncoupling of linked neurotransmitter effects by combinatorial convergence.
Physiological signaling pathways both diverge and converge-a single neurotransmitter can have multiple effects and multiple transmitters can have the same effects-in the same target cell. Divergence couples the effects of a transmitter together in a relatively fixed ratio. Different physiological circumstances may require a different ratio, however; the coupling must be made modifiable. This can be achieved through convergence. If two transmitters couple the effects in different ratios, then combinations of the transmitters can yield all intermediate ratios of the effects, thus functionally uncoupling them. This mechanism is analyzed in a well-understood, simple invertebrate neuromuscular circuit.
Animals↗
A pair of identified interneurons in Aplysia that are involved in multiple behaviors are necessary and sufficient for the arterial-shortening component of a local withdrawal reflex.
A bilateral pair of cerebral interneurons, called CC5, contribute to the generation of a number of different behaviors involving head movements. Each cell sends its axon to the ipsilateral and contralateral pedal and pleural ganglia. A weak tactile stimulus to the head excites the ipsilateral CC5; a strong stimulus excites both the ipsilateral and contralateral cells. Firing of CC5 produces powerful shortening of the ipsilateral pedal artery (PA) by means of monosynaptic excitation of the pedal artery shortener (PAS) neuron, the single motor neuron for the artery. A weak touch to a tentacle excites the ipsilateral PAS and evokes a local withdrawal response accompanied by shortening of the ipsilateral PA. In vivo recording of the pedal artery nerve (PAn) showed that PAS was activated bilaterally during defensive head withdrawal elicited by a strong stimulus and was activated unilaterally by a weak stimulus. The responses were eliminated by cutting the ipsilateral cerebral-pleural connective (C-PLC). Electrical stimulation of the cerebral-pleural connective provided evidence that all of the excitatory input to PAS via this connective is provided by CC5. A variety of experimental results indicates that during a local withdrawal reflex of the tentacle, CC5 is necessary and sufficient for the unilateral PA-shortening component of the response and therefore functions as a command neuron for a component of the behavior. The data suggest that during defensive head withdrawal, the two CC5 neurons may act conjointly as a two-neuron command system that is necessary and sufficient for the bilateral arterial-shortening component of the behavior.
Animals↗
Ca2+/S100 regulation of giant protein kinases.
Protein phosphorylation by protein kinases plays a central regulatory role in cellular processes and these kinases are themselves tightly regulated. One common mechanism of regulation involves Ca2+-binding proteins (CaBP) such as calmodulin (CaM). Here we report a Ca2+-effector mechanism for protein kinase activation by demonstrating the specific and >1,000-fold activation of the myosin-associated giant protein kinase twitchin by Ca2+/S100A1(2). S100A1(2) is a member of a large CaBP family that is implicated in various cellular processes, including cell growth, differentiation and motility, but whose molecular actions are largely unknown. The S100A1(2)-binding site is a part of the autoregulatory sequence positioned in the active site that is responsible for intrasteric autoinhibition of twitchin kinase; the mechanism of autoinhibition based on the crystal structures of two twitchin kinase fragments is described elsewhere. Ca2+/S100 represents a likely physiological activator for the entire family of giant protein kinases involved in muscle contractions and cytoskeletal structure.
Amino Acid Sequence↗
Studies of the calmodulin-binding site of twitchin with synthetic peptides using fluorescence and CD spectroscopy.
The calcium-dependent interaction of two synthetic peptides derived from the putative calmodulin-binding site in the protein kinase autoinhibitory region of twitchin was studied by fluorescence and CD spectroscopy. The peptides interacted with dansylcalmodulin in the presence of Ca2+ as shown by a change in the fluorescence emission spectra. Fluorescence titration of dansylcalmodulin with the peptides was used to quantify this interaction. The peptides appeared to assume a helical conformation in a non-polar environment as seen by CD spectroscopy. The ellipticity of Ca2+ calmodulin was enhanced in the presence of peptides compared with that of Ca2+ calmodulin and peptides alone, indicating that the peptides had formed a complex with calmodulin. These results support the assignment of the twitchin calmodulin-binding site.
Amino Acid Sequence↗
Synaptic mechanisms in invertebrate pattern generation.
Although individual neurons can be intrinsically oscillatory and can be network pacemakers, motor patterns are often generated in a more distributed manner. Synaptic connections with other neurons are important because they either modify the rhythm of the pacemaker cell or are essential for pattern generation in the first place. Computational studies of half-center oscillators have made much progress in describing how neurons make transitions between active and inactive phases in these simple networks. In addition to characterizing phase transitions, recent studies have described the synaptic mechanisms that are important for the initiation and maintenance of activity in half-center oscillators.
Animals↗
Two ion currents activated by acetylcholine in the ARC muscle of Aplysia.
1. This work continues our examination of the electrophysiology and contractions of single fibers dissociated from a widely studied molluscan muscle, the accessory radula closer (ARC) muscle of Aplysia californica, aimed at understanding its excitation-contraction mechanisms and their modulation. 2. Extensive previous work has characterized a number of basal ion currents present in the fibers and effects of transmitters and peptide cotransmitters that modulate ARC-muscle contractions in vivo. Here we use current clamp, voltage clamp, and contraction measurements to examine the actions of acetylcholine (ACh), the transmitter that induces the contractions. 3. As in the whole ARC muscle, ACh depolarizes unclamped fibers maximally to about -25 mV where, no matter how much ACh is applied, the depolarization saturates. 4. The underlying ACh-activated current is in fact the sum of two quite distinct components, IACh,cat and IACh,Cl. 5. IACh,cat is itself a mixed current carried by cations (physiologically mainly by Na+, but to a significant degree also by Ca2+), reverses near +20 mV, rectifies inwardly, exhibits prominent voltage-dependent kinetics of activation with hyperpolarization, and is selectively blocked by hexamethonium. 6. In contrast, IACh,Cl is carried by Cl-, reverses near -60 mV, exhibits little rectification or voltage-dependent kinetics, is activated selectively by suberyldicholine, and is blocked by alpha-bungarotoxin. 7. Both currents activate fast when ACh is applied, desensitize relatively slowly in its presence, then deactivate fast. Both currents are activated at similar ACh concentrations, half-maximally at approximately 10 microM. Both currents also are activated by carbachol and propionylcholine and blocked by d-tubocurarine, bicuculline and paraoxon. Picrotoxin and atropine block IACh,cat better, 4-acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic acid (SITS), 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), and anthracene 9-carboxylic acid IACh,Cl better. 8. The two currents are virtually identical to ACh-activated cationic (Na) and Cl currents that are ubiquitous in molluscan neurons. As has been proposed for the neuronal currents, IACh,cat resembles vertebrate neuronal nicotinic ACh-receptor (nAChR) currents, whereas IACh,Cl resembles vertebrate skeletal muscle nAChR currents. 9. Functionally, we believe that IACh,cat serves primarily to depolarize the ARC muscle to open voltage-activated L-type Ca channels, allow Ca2+ influx, and initiate contraction. Physiologically significant Ca2+ may also enter through the ACh,cat channels themselves. 10. By superimposing on IACh,cat, IACh,Cl brings the reversal potential of the combined current to around -25 mV and thereby sets a relatively negative upper limit to the ACh-induced depolarization. We propose that this is its physiological role. By limiting the depolarization, IACh,Cl limits the degree of activation of the Ca current and Ca2+ influx, and so prevents excessive contraction. More importantly, it moderates the voltage during contraction to a range where small voltage changes can finely grade contraction amplitude in this nonspiking muscle. 11. Indeed, in contraction experiments on the single fibers, there is an inverse correlation between the IACh,Cl/IACh,cat ratio and the magnitude of the ACh-induced depolarization and contraction. Furthermore, increased pharmacological activation of IACh,Cl depresses, and block of IACh,Cl enhances, both the depolarization and contraction. 12. Obligatory simultaneous coactivation of IACh,cat and IACh,Cl in the ARC muscle may be part of a peripheral control mechanism that automatically keeps the size of its contractions within behaviorally optimal limits.
Acetylcholine↗
Characterization of a radula opener neuromuscular system in Aplysia.
1. Several lines of evidence suggest that the I7-I10 muscle group contributes to the radula opening phase of behavior in Aplysia; 1) extracellular stimulation of these muscles in reduced preparations causes the halves of the radula to separate, 2) synaptic activity can be recorded from muscles I7-I10 in intact animals when the radula is opening, and 3) motor neurons innervating I7-I10 are activated out of phase with retractor/closer motor neurons during cycles of buccal activity driven by the cerebral-to-buccal interneuron 2 (CBI-2). 2. All of the opener muscles are innervated by the B48 neurons, a bilaterally symmetrical pair of cholinergic motor neurons. B48 neurons produce excitatory junction potentials (EJPs) in opener muscle fibers that summate to produce muscle contractions. Contraction size is determined by the size of depolarization in muscle fibers and/or by action potentials that are triggered by summation of B48-evoked EJPs. 3. In addition to input from B48 neurons, opener muscles also receive excitatory input from the cholinergic multiaction neurons B4/B5. EJPs evoked by stimulation of neurons B4/B5 are 1/10 the size of B48-evoked EJPs. Consequently, changes in muscle tension produced by B4/B5 activity are relatively small. In contrast to B48 neurons, neurons B4/B5 are likely to be active during the closing/retraction phase of behavior. During cycles of buccal activity driven by neuron CBI-2, neurons B4/B5 fire in phase with closer/retractor motor neurons. Thus opener muscles may develop a modest amount of tension during the closing/retraction phase of behavior as a result of synaptic input from neurons B4/B5. 4. Opener muscles may also develop tension during closing/retraction simply by virtue of the fact that they have been stretched. When isolated opener muscles are lengthened, depolarizations are recorded from individual muscle fibers, and muscle tension increases. With sufficient changes in fiber length, action potentials are elicited. These action potentials produce twitchlike muscle contractions that become rhythmic with maintained stretch. Stretch-activated depolarizations are generally first apparent when muscle length is increased by 1 mm. Length changes of 4-5 mm are generally necessary to elicit twitchlike muscle contractions. Changes of 1-2 mm in muscle length are observed when the opener muscle's antagonist, the accessory radula closer, is activated in reduced preparations. 5. Stretch may also modulate B48-induced contractions of the opener muscles. When muscle length is increased, B48-elicited contractions of the I7 muscle are larger. These increases in contraction amplitude are accompanied by decreases in contraction latency. 6. We conclude that muscles I7-I10 contract vigorously in response to strong excitatory input from neuron B48 and contribute to radula opening. Stretch may potentiate this activity. Thus, if radula closer muscles contract vigorously and pull on the opener muscles, the opener muscles will respond by contracting more vigorously themselves. This may be a mechanism for maintaining amplitude relationships between antagonistic muscles. Additionally, it is likely that the opener muscles will develop at least a modest amount of tension during closure/retraction of the radula. Part of this activation may derive from the weak excitatory input that the muscles receive from neurons B4/B5. Another part may derive from the stretch. One function of this co-contraction may be to act as a brake on closure, bringing this phase of feeding behavior to a smooth halt.
Action Potentials↗
Phosphorylation of myosin regulatory light chains by the molluscan twitchin kinase.
The unusually large (approximately 600 to > 3000 kDa) myosin-associated proteins of the titin/twitchin superfamily are considered to be important cytoskeletal rulers for thick filament assembly in muscle. This function is maintained by approximately 60-240 modular fibronectin-type-III and immunoglobulin-C2 repeats in these proteins which further contain a protein serine/threonine kinase domain of unknown function. In this study, the bacterially expressed kinase domain of Aplysia twitchin was used in order to identify a potential physiological substrate. Addition of the recombinant kinase to Aplysia actomyosin preparations resulted in the specific phosphorylation of the 19-kDa myosin regulatory light chains. The twitchin kinase phosphorylated purified light chains on Thr15 in a region which shared a high degree of similarity with the phosphorylation site for vertebrate smooth muscle myosin light chain kinase. Peptide analogs of the twitchin substrate sequence and the similar sequence in vertebrate smooth muscle myosin light chains were phosphorylated with good kinetic properties. These data reveal the first potential substrate for any of the giant protein kinases and support a dual role of twitchin in molluscan muscle as a cytoskeletal protein as well as a myosin light chain kinase.
Amino Acid Sequence↗
Distribution in the central nervous system of Aplysia of afferent fibers arising from cell bodies located in the periphery.
The present study using autoradiography to determine the location of the projections of presumptive peripheral afferent neurons into the central nervous system of Aplysia. Selected peripheral tissues (with an emphasis on structures involved in feeding behavior) were exposed to radioactive amino acids, and the distribution of macromolecules transported into the nervous system via afferent fibers was determined by autoradiography. Different regions of the body exhibited different patterns of projections, and, within the neuropil of the cerebral ganglion, there was a loose topographical organization of projections from the head. For some regions of the body, the projections was largely limited to the ganglion from which the nerve enters; for other regions, the projection was very widespread. In some cases (e.g., rhinophore to eye), there was evidence of projections from one peripheral structure to another. Experiments with all peripheral tissues that were studied resulted in extensive labeling of central ganglia, indicating that afferents with peripheral cell bodies may provide a major source of sensory input to the central nervous system and suggesting that many or all of the numerous ultrafine axons visualized via electron microscopy in the nerves of Aplysia may originate from first- or second-order sensory afferents whose cell bodies are located in the periphery.
Animals↗
Ca(2+)-activated K current in the ARC muscle of Aplysia.
1. This work continues our examination of the electrophysiology and contractions of single, functionally intact fibers dissociated from a widely studied molluscan muscle, the accessory radula closer (ARC) muscle of Aplysia californica, aimed at understanding its excitation-contraction mechanisms and their modulation. Extensive previous work has characterized a number of ion currents in the fibers. 2. Here we describe an additional major current that could not be studied earlier because, unlike all of the other currents in the ARC muscle fibers, it becomes prominent only during contraction of the fiber. It is a Ca(2+)-activated K current, associated with contraction most likely because both are activated by the same elevation in intracellular free Ca2+. 3. We used several manipulations to elicit the Ca(2+)-activated K current and contraction: depolarizing voltage steps in the presence of extracellular Ca2+, application of caffeine in the presence or absence of extracellular Ca2+ (and thus presumably working by releasing Ca2+ from intracellular stores), application of the Ca(2+)-ionophore A23187, and direct iontophoretic injection of Ca2+ into the fiber. 4. The Ca(2+)-activated K current reversed around -70 mV, not far from EK, and the reversal potential shifted substantially with elevated extracellular K+. Activation of the current was not only Ca2+ dependent, but also quite strongly voltage dependent, promoted by depolarization. The current was well blocked by tetraethylammonium (KD approximately 2 mM), but not blocked by even 10 mM 4-aminopyridine or low concentrations of the K-current blocking toxins charybdotoxin and apamin. 5. After a depolarizing voltage step in Ca(2+)-containing solution, the Ca(2+)-activated K current appeared, often with some delay, as a large peak of current that soon disintegrated into a prolonged period of individual oscillatory transients of Ca(2+)-activated K current, sometimes correlated with transient contractions. Similar transients could be elicited by caffeine or iontophoretic Ca2+ injection. More extensive study of the underlying Ca2+ dynamics will be presented elsewhere, but we interpret these phenomena in terms of our hypothesis that the ARC muscle generates both contraction and the Ca(2+)-activated K current by Ca(2+)-induced Ca2+ release (CICR), in which a small depolarization-induced influx of extracellular Ca2+ releases more Ca2+ from intracellular stores. 6. The Ca(2+)-activated K current is significant in the physiological operating voltage range of the ARC muscle, and its predicted hyperpolarizing action and consequent negative-feedback depression of contractions is likely to be an important part of the integrated set of mechanisms that regulate the muscle's contractility.
Animals↗