Calcium currents from jellyfish striated muscle cells: preservation of phenotype, characterisation of currents and channel localisation
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 and Bamfield Marine Station, Bamfield, British Columbia, Canada V1R 1B0
*Author for correspondence (e-mail: aspencer{at}bms.bc.ca)
Accepted July 31, 2001
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: LVA calcium channel, striated muscle, jellyfish, voltage-clamp, dihydropyridine, Cnidaria, jellyfish, Polyorchis penicillatus.
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Voltage-gated calcium channels can be categorised into high-voltage-activated (HVA) and low-voltage-activated (LVA) channels. In general, HVA calcium channels activate above 40 mV. The activation and inactivation kinetics of HVA calcium channels are quite variable but they can be identified by their sensitivity to various pharmacological agents. They are categorised into at least five classes, namely L, N, P, Q and R types, based on their pharmacological properties. LVA channels activate at around 70 to 40 mV, inactivate rapidly, and are sensitive to nickel and mibefradil (Bean, 1985; Akaike et al., 1989; Huguenard, 1996; Ertel et al., 1997). To date, only one type of LVA channel has been identified, the T-type calcium channel.
Voltage-gated calcium channels are found in all muscle types; however, their roles in muscle contraction differ depending on the muscle type. Influx of external calcium ions is required to initiate contraction in both smooth and cardiac muscle but not in skeletal striated muscle of vertebrates (Bers, 1991), where depolarisation of the membrane triggers release of calcium from the sarcoplasmic reticulum (Endo, 1977). All cardiac myocytes possess L-type calcium channels, while T-type channels are also present in cardiac cells that display primary or secondary pacemaker activity such as at the sinoatrial node, sinus venosus and Purkinje fibres (Hagiwara et al., 1988; Hirano et al., 1989; Tseng and Boyden, 1989; Bois and Lenfant, 1991). However, T-type channels are absent or present at low densities in ventricular myocytes (Bers, 1991; Campbell and Strauss, 1995). Thus, the primary role of L-type calcium channels appears to be the control and modulation of the electromechanical coupling process, while T-type channels are involved in pacemaker activity.
Several voltage-gated calcium currents have been recorded from neurones in jellyfish (Mackie and Meech, 1985; Anderson, 1987; Przysiezniak and Spencer, 1992), smooth muscles in ctenophores (Bilbaut et al., 1988; Dubas et al., 1988), and fertilised eggs in ctenophores (Barish, 1984). Calcium currents are present in the striated swimming muscle of the medusa Polyorchis penicillatus (Spencer and Satterlie, 1981). More recently, using isolated striated muscle strips from this jellyfish, we have shown that contraction depends on extracellular calcium and can be inhibited by the calcium channel blocker nitrendipine (Lin et al., 2000). Using both electrophysiological techniques and specific labelling, we report the presence of low-voltage-activated calcium currents in striated myocytes of the hydrozoan jellyfish Polyorchis penicillatus. Retention of functional channels in these cells required dissociation at 30°C, with much of the current being lost at room temperature. A primary function of calcium currents through this channel population is to initiate muscle contraction. We have determined that the muscle feet have a far higher density of these channels than the somata.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell dissociation and electrophysiology
Subumbrellar sections were excised from the whole jellyfish and the radial and ring canals trimmed away. The muscle layer was stripped off the endodermal lamella using a pair of forceps. One medium-sized jellyfish (about 1520 mm in bell diameter) yielded approx. 200 µl of striated muscle cells with some mesoglea attached. The stripped tissues were put into a 1.5 ml Eppendorf tube and a digestion solution of 1 mg ml1 Pronase (Boehringer Mannheim) dissolved in artificial sea water (ASW), either prewarmed to 30°C or at room temperature (2022°C), was added to the Eppendorf tube to a final volume of 1 ml. Test tubes were left undisturbed at either room temperature (2022°C) or at 30°C for 1520 min. After digestion, a few gentle taps on the test tube was all that was needed to separate the tissue into single muscle cells. Muscle cells were dissociated at 30°C because the contractile phenotype appeared to be fixed by heat. Muscle cells dissociated at 30°C were easily identified by their long contractile processes, whereas muscle cells dissociated at room temperature could be identified by the lack of muscle feet and the retracted myofibres within the cytoplasm. Any remaining endodermal cells could be identified by their cuboidal or columnar shape. Before recording, cells were washed with the appropriate bath solution three times and resuspended in bath solution to a final volume of 500 µl.
Recordings were made immediately after dissociation. Each batch of cell suspension was used for about 34 h. One or two drops of cell suspension were added into a stagnant perfusion chamber and then cells were allowed to settle (some remained suspended) before recording.
Electrophysiological recording and data analysis followed the technique of Grigoriev et al. (Grigoriev et al., 1996). Briefly, whole-cell, tight-seal recordings were made using borosilicate glass pipettes (TW-150-4, World Precision Instruments), which were made on a Sutter automated puller, with resistances of 23 M when filled with the pipette solution. Recordings were made with an Axopatch-1D amplifier (Axon Instruments), low-pass filtered at 3 kHz using a 4-pole Bessel filter, and digitised using a Labmaster TL-125 acquisition board (Axon Instruments). Individual cells were viewed under phase contrast with a Nikon Diaphot inverted microscope. A piezo-electric driver (Burleigh) was used to manipulate the pipette onto the cell surface. Perfusion with the relevant external recording solution started after successful seal formation. No contraction was observed during recording. Stimulus control, data acquisition and analyses were performed with pCLAMP 6.0 software (Axon Instruments) on a Dell personal computer. Leakage and capacitative currents were subtracted, prior to test pulses, using P/4 or P/5 protocols from a holding potential of 80 mV (pCLAMP 6.0). Series resistance (Rs) was compensated optimally to minimise voltage errors, and was usually set to values of
80 %. Cm (membrane capacitance) and Rs were obtained by minimising the capacitative transient in response to a hyperpolarising voltage step. Mean Rs was 4.39±0.81 M
(N=20) before compensation, and mean Cm was 5.49±1.07 pF (N=20) before compensation. All experiments were carried out at room temperature (2022°C).
Solutions
Before use all recording solutions were filtered through cellulose acetate membrane cartridges with a 0.2 µm pore size. ASW at pH 7.5 contained: NaCl 376 mmol l1, Na2(SO4) 26 mmol l1, MgCl2 41.4 mmol l1, CaCl2 10 mmol l1, KCl 8.5 mmol l1 and N-2-hydroxy-ethylpiperazine-N'-2-ethanesulphonic acid (Hepes) hemisodium salt 10 mmol l1. The bath solution at pH 7.5 contained: MgCl2 40 mmol l1, CaCl2 (or BaCl2 or SrCl2) 10 mmol l1, KCl 15 mmol l1, N-methyl glucamine-HCl (NMG-Cl) 429 mmol l1, Hepes (free acid) 10 mmol l1. The pipette solution (pH 7.5) used for recording total membrane currents contained: NaCl 50 mmol l1, MgCl2 2 mmol l1, CaCl2 1 mmol l1, ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) 11 mmol l1, KCl 350 mmol l1, KOH 30 mmol l1, Hepes (free acid) 10 mmol l1. The pipette solution (pH 7.5) used for isolating calcium currents contained: NaCl 50 mmol l1, MgCl2 2 mmol l1, CaCl2 1 mmol l1, EGTA 11 mmol l1, CsCl 350 mmol l1, CsOH 30 mmol l1, Hepes (free acid) 10 mmol l1, TEA-Cl 20 mmol l1. Using the latter pipette solution all outward currents were blocked totally within the first minute after break-through.
Pharmacology of muscle contraction
The considerable thickness and elasticity of the mesoglea in the bell region made muscle tension recordings from this region extremely variable. A preliminary study indicated that muscle strips from bell regions and vela (the shelf of tissue at the opening of the bell) had similar pharmacologies. Therefore we used muscle strips from vela, which had a thin lamina of mesoglea, to measure tension development. The vela of medusae, anaesthetised in 1:1 isotonic MgCl2 (0.33 mol l1) and ASW, were excised so as to provide continuous strips of maximal width. Care was taken not to include portions of the innervating motor-neurone network in the strips by bisecting the strip lengthwise and using only the half closest to the free margin. The free ends of each velar strip were pinned to the Sylgard base of a 35 mm Petri dish, which also contained a pair of embedded Ag/AgCl2 stimulating electrodes connected to a Grass S44 stimulator. The velar strip ran between the two stimulating electrodes and around a small hook attached to a capacitative force transducer (Kent Scientific Corporation). Tension on the strip was adjusted using a micromanipulator so as to remove any slack in the muscle strip. Repetitive square pulses of 30 ms duration and 0.2 Hz were given to produce a train of twitch contractions that did not show fatigue. The stimulation voltage was determined by increasing the voltage until no increase in the amplitude of contraction at the stimulation frequency of 0.1 Hz occurred. The voltages used usually were between 30 and 40 V. Perifusion was by a peristaltic pump at 1.5 ml min1 and the perifusate was removed by vacuum. All perifusates were kept at 1214°C during the experiment by running part of the perfusion tubing through an ice bucket. Transduced tension was recorded on a digital, Dash-IV pen-recorder (Astro-Med Inc.). The amplitude of contractile force for each condition (control, drug effect and wash) was calculated by averaging 30 individual twitch contractions.
Localisation of calcium channels
The method used to localise calcium channels using dihydropyridine-BODIPY (fDHP) followed the protocol of Schild et al. (Schild et al., 1995), with some modification. Dissociated cells were incubated with both fDHP and the styryl dye, RH414 (Molecular Probes, Inc.) at final concentrations of 10 µmol l1 and 5 µmol l1, respectively, for 15 min at room temperature before examination. A laser-scanning, confocal microscope (Molecular Dynamic, Inc.) was used to examine the spatial distribution of calcium channels. The wavelength of the excitation beam was set to 488 and 568 nm (argon ion laser, 4.0 mW). The emitted fluorescence from labelled cells was split by a dichroic mirror (565 nm) into two wavelength bands. The intensities of the green (fDHP) and red (RH414) fluorescent labelling were recorded by two photomultipliers with cut-off filters at 530 and 590 nm, respectively.
To calculate the ratio of fluorescence intensity between green (fDHP) and red (RH414), each confocal image was split into two grey-scale images registering green and red fluorescence intensity. Images were then generated by determining the ratio of the intensity of green fluorescence (fDHP) to that of the red fluorescence (RH414) using the image math function in NIH image software (National Institute of Health, USA at http://rsb.info.nih.gov/nih-image/about.html). Only optical sections, 1 µm thick, with sharp and definitive membrane labelling by RH414 (red images) were used for data analysis as this indicated a perpendicular section through the cell membrane. Data were collected and measured using the tool function in NIH image software by drawing lines along the membrane where RH414 showed sharp and definitive labelling. Unless otherwise stated, results are expressed as mean ± S.E.M.
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
Electrical and kinetic properties
The rate of activation was voltage-dependent (Fig. 3A) with the time-to-peak for maximal current of 1.49±0.23 ms at 30 mV (N=10). The time course for inactivation was also voltage-dependent (Fig. 3B) with the inactivation time constant varying between 1.5 ms at +80 mV and 3.5 ms at 70 mV (N=10).
|
|
|
|
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Channel localisation and fixation
Since only cells with intact feet showed large currents, it is possible that most of the channels are located on the feet. Our fDHP data also support the proposition that DHP-sensitive ion channels are at a higher density on the feet. Several other excitable cells also show a differential distribution pattern of ion channels that are obviously related to the excitability properties of the cell in question. For example, voltage-gated sodium channels are known to be clustered at the nodes of Ranvier in vertebrate myelinated axons and neuromuscular junctions (Ritchie and Rogart, 1977; Ellisman and Levinson, 1982; Lupa and Caldwell, 1994), and L-type calcium channels are known to aggregate in T-tubules (Carl et al., 1995). Jellyfish striated muscle cells are morphologically separated into a somal and a foot region (Spencer, 1979). These muscle feet contain the contractile myofibres that use extracellular calcium during excitationcontraction coupling (Spencer and Satterlie, 1981). Localisation of calcium channels on muscle feet in jellyfish striated muscle is further supported by the presence of membranous calcium stores just beneath the membrane of muscle feet (Lin and Spencer, 2001). A close association between calcium channels and calcium stores would allow for possible direct electrical coupling or calcium-induced calcium release to augment the influx of calcium through voltage-gated channels for regulation of muscle contraction. The precise way ion-channel proteins and the cytoskeleton interact mechanically is not known, especially for jellyfish. Nevertheless, several adapter proteins have been reported to be associated with voltage and ligand-gated ion channels, which link them to the cytoskeleton. For example, ankyrin and spectrin immobilise sodium channels with actin filaments at presynaptic terminals and along axons (Srinivasan et al., 1988), while rapsyn links nicotinic acetylcholine receptors to actin filaments at neuromuscular junctions (Apel et al., 1995) and gephyrin links glycine receptors to microtubules at postsynaptic sites (Kirsch et al., 1993). GABAA-receptor-associated protein (GABARAP) links GABAA receptors to microtubules at synapses (Wang et al., 1999).
Channel kinetics
The electrophysiological characteristics of the calcium currents recorded from isolated jellyfish muscle cells are similar to those of T-type currents: activation occurred at low voltages (70 mV), with both activation and inactivation being very rapid and voltage-dependent. Moreover, this current showed another characteristic of T-type channels (Randall and Tsien, 1997) wherein, with progressively stronger depolarisations, successive currents activated and inactivated faster. A noticeable difference between the current we recorded and T-type currents in other systems was the greater rate of both activation and inactivation in jellyfish muscle cells. Time-to-peak for maximum current at a membrane potential of 30 mV was 1.49±0.23 ms and the inactivation time constant was 2.59±0.38 ms for the calcium currents recorded from jellyfish muscles. In comparison the activation time constants for most other T-type currents are usually greater than 10 ms, with inactivation constants greater than 20 ms (Huguenard, 1996; Randall, 1998; Randall and Tsien, 1997).
Pharmacology
Our experiments examining the effects of various known calcium channel blockers on muscle contraction were an indirect method for evaluating channel properties, but they also provide us with estimates of the doses that might be effective when using whole-cell, patch-clamp recording. Although dihydropyridines are known to have higher inhibitory potency for L-type calcium channels than T-type channels (Hille, 1992), the sensitivity of the jellyfish calcium current to dihydropyridines, with an IC50 in the µmol l1 range, was similar to that reported for T-type calcium channels found in some vertebrate cells, including mouse spermatocytes (Arnoult et al., 1996; Lievano et al., 1996; Santi et al., 1996), hippocampal CA1 neurones (Takahashi and Akaike, 1991), cerebellar Purkinje cells (Kaneda et al., 1990), amygdaloid neurones (Kaneda and Akaike, 1989), rat hypothalamic neurons (Akaike et al., 1989) and mouse sensory neurones (Richard et al., 1991). LVA T-type calcium currents have been reported in some excitable cells of invertebrates (Hagiwara et al., 1975; Deitmer, 1984; Yamoah and Crow, 1994; Ödblom et al., 2000), where the LVA calcium currents in cardiac myocytes of squid are shown to be sensitive to dihydropyridines at micromolar levels (Ödblom et al., 2000). Nickel ions are known to block the T-type calcium channels selectively (Hille, 1992). Jellyfish LVA calcium currents were sensitive to low concentrations of Ni2+ (<100 µmol l1), which is similar to reports for rat Purkinje cells (Regan, 1991) and amygdaloid neurones (Kaneda and Akaike, 1989). Based upon channel kinetics, activation and inactivation ranges and pharmacological properties, these LVA calcium channels in jellyfish striated muscle cells most closely resemble those of T-type channels.
Possible function of LVA calcium channels in jellyfish striated muscle cells
Due to their low activation threshold and fast inactivation, T-type currents are generally believed to function in combination with other currents to produce pacemaker potentials where rhythmical activity is required (Randall, 1998; Tsien, 1998). In addition, T-type currents are implicated in regulation of cell growth and proliferation (Hermsmeyer, 1998; Triggle, 1998). As the LVA calcium current appears to be the only significant inward current in jellyfish striated muscle cells, this is the first case reported of a LVA calcium current being expressed in isolation without other inward currents. The action potentials of these jellyfish muscles, which have been recorded in current-clamp with sharp electrodes (Spencer, 1978; Spencer and Satterlie, 1981), are reminiscent of those in vertebrate cardiac muscle having large amplitudes (>100 mV) and a long plateau phase (up to 170 ms). Although the ionic basis of the action potential was not clearly established by Spencer and Satterlie, it was demonstrated that most of the inward current was carried by calcium. However, some sodium was required externally to support an action potential. Our results contradict this study; nevertheless it is obvious that the rising phase of the action potential is mostly due to calcium influx through LVA channels, with a delayed repolarisation due to a delayed, rectifier-like potassium current and/or a calcium-dependent potassium current. It must be assumed that the resulting increase in cytoplasmic calcium concentration initiates muscle contraction.
Implications for channel evolution
Due to their phylogenetic position at the base of the metazoan radiation, and being one of the first animal groups to possess sodium currents, cnidarians have been a popular taxon in which to look for ancestral types of sodium channels or hybrid calcium/Na+ channels (Anderson, 1987; Spafford et al., 1996). Hille (Hille, 1992) noted that T-type calcium channels could be the ancestor of sodium channels, based on the similarity of their kinetics of activation and inactivation. Furthermore, sequence comparisons between sodium and calcium channels show the relatedness of domains in these two channel types (Spafford et al., 1998). Calcium selectivity of HVA channels is conferred by four negatively charged glutamate residues, which form a high-affinity, EDTA-like calcium-binding site in the pore region (Tsien, 1998). This contrasts with sodium channels where two of the negatively charged residues are replaced by a positively charged residue (lysine) and a neutral residue (alanine) (Heinemann et al., 1992; Ellinor et al., 1995). T-type channels might represent the transition state where these four critical residues are two glutamates and two aspartates (Tsien, 1998). Measurements of sequence similarity between L-type, non-L-type, T-type and sodium channels also suggests that there was an early divergence of these channel types (Tsien, 1998). However, the presence of a functionally undefined, glycosylation-rich, extracellular loop between segments 5 and 6 (located just in front of the pore in domain I and found only in sodium channels and T-type calcium channels) might represent a remnant of their shared ancestry (Spafford et al., 1998).
This jellyfish LVA current activates and inactivates much faster than most vertebrate T-type currents reported to date, with kinetic properties similar to sodium currents. In jellyfish striated muscle cells this calcium current is the major inward current of action potentials, and it is tempting to speculate that this LVA calcium channel is closely related to an ancestral sodium channel.
![]() |
Acknowledgments |
---|
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akaike, N., Kostyuk, P. G. and Osipchuk, Y. V. (1989). Dihydropyridine-sensitive low-threshold calcium channels in isolated rat hypothalamic neurones. J. Physiol. 412, 181195.[Abstract]
Anderson, P. A. V. (1987). Properties and pharmacology of a TTX-insensitive Na+ current in neurones of the jellyfish, Cyanea capillata. J. Exp. Biol. 133, 233248.
Apel, E. D., Roberds, S. L., Campbell, K. P. and Merlie, J. P. (1995). Rapsyn may function as a linker between the acetylcholine receptor and the agrin-binding dystrophin-associated glycoprotein complex. Neuron 5, 115126.
Arnoult, C., Cardullo, R. A., Lemos, J. R. and Florman, H. M. (1996). Activation of mouse sperm T-type Ca2+ channels by adhesion to the egg zona pellucida. Proc. Natl. Acad. Sci. USA 93, 1300413009.
Barish, M. E. (1984). Calcium-sensitive action potential of long duration in the fertilized egg of the ctenophore Mnemiopsis leidyi. Dev. Biol. 105, 2940.[Medline]
Bean, B. P. (1985). Two kinds of calcium channels in canine atrial cells. J. Gen. Physiol. 86, 130.[Abstract]
Berridge, M. J. (1993). Inositol trisphosphate and calcium signalling. Nature 361, 315325.[Medline]
Bers, D. M. (1991). ExcitationContraction Coupling and Cardiac Contractile Force, 258 p. Boston: Kluwer Academic Publishers.
Bilbaut, A., Hernandez-Nicaise, M. L., Leech, C. A. and Meech, R. W. (1988). Membrane currents that govern smooth muscle contraction in a ctenophore. Nature 331, 533535.[Medline]
Bois, P. and Lenfant, J. (1991). Evidence for two types of calcium currents in frog cardiac sinus venosus cells. Pflugers Arch. 417, 591596.[Medline]
Campbell, D. L. and Strauss, H. C. (1995). Regulation of calcium channels in the heart. In Advances in Second Messenger and Phosphoprotein Research, Vol. 30 (ed. R. Means), pp. 2588. New York: Raven Press, Ltd.
Carl, S. L., Felix, K., Caswell, A. H., Brandt, N. R., Brunschwig, J. P., Meissner, G. and Ferguson, D. G. (1995). Immunolocalisation of triadin, DHP receptors, and ryanodine receptors in adult and developing skeletal muscle of rats. Muscle Nerve 18, 12321243.[Medline]
Deitmer, J. (1984). Evidence for two voltage-dependent calcium currents in the membrane of the ciliate Stylonychia. J. Physiol. 355, 137159.[Abstract]
Dubas, F., Stein, P. G. and Anderson, P. A. (1988). Ionic currents of smooth muscle cells isolated from the ctenophore Mnemiopsis. Proc. Roy. Soc. Lond. B 233, 99121.[Medline]
Ellinor, P. T., Yang, J., Sather, W. A., Zhang, J. F. and Tsien, R. W. (1995). Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. Neuron 15, 11211132.[Medline]
Ellisman, M. H. and Levinson, S. R. (1982). Immunocytochemical localisation of sodium channel distribution in the excitable membrane of Electrophorus electricus. Proc. Natl. Acad. Sci. USA 79, 67016711.
Endo, M. (1977). Calcium release from the sarcoplasmic reticulum. Physiol. Rev. 57, 71108.
Ertel, S. I., Ertel, E. A. and Clozel, J. P. (1997). T-type Ca2+ channels and pharmacological blockade: potential pathophysiological relevance. Cardiovasc. Drugs Ther. 11, 723739.[Medline]
Grigoriev, N. G., Spafford, J. D., Przysiezniak, J. and Spencer, A. N. (1996). A cardiac-like sodium current in motor neurons of a jellyfish. J. Neurophysiol. 76, 22402249.
Hagiwara, N., Irisawa, H. and Kameyama, M. (1988). Contribution of two types of calcium currents to the pacemaker potential of rabbit sino-atrial node cells. J. Physiol. 395, 233253.[Abstract]
Hagiwara, S. Ozawa, S. and Sand, O. (1975). Voltage clamp analysis of two inward current mechanisms in the egg cell membranes of a star fish. J. Gen. Physiol. 65, 617644.[Abstract]
Heinemann, S. H., Terlau, H., Imoto, K. and Numa, S. (1992). Calcium channel characteristics conferred on the sodium channel by single mutations. Nature 356, 441443.[Medline]
Hermsmeyer, K. (1998). Role of T channels in cardiovascular function. Cardiology 89, 29.[Medline]
Hille, B. (1992). Ionic Channels of Excitable Membranes, 607 p. Sunderland: Sinauer.
Hirano, Y., Fozzard, H. A. and January, C. T. (1989). Characteristics of L- and T-type Ca2+ currents in canine cardiac Purkinje fibers. Am. J. Physiol. 256, 14781492.
Huguenard, N. J. (1996). Low-threshold calcium currents in central nervous system neurons. Ann. Rev. Physiol. 58, 329348.[Medline]
Kaneda, M. and Akaike, N. (1989). The low-threshold Ca2+ current in isolated amygdaloid neurons in the rat. Brain Res. 497, 187190.[Medline]
Kaneda, M., Ito, C. and Akaiko, N. (1990). Low-threshold calcium current in isolated Purkinje cell bodies of rat cerebellum. J. Neurophysiol. 63, 10461051.
Kirsch, J., Wolters, I., Triller, A. and Betz, H. (1993). Gephyrin antisense oligonucleotide prevents glycine receptor clustering in spinal neurones. Nature 366, 745748.[Medline]
Lievano, A., Santi, C. M., Serrano, C. J., Trevino, C. L., Bellve, A. R., Hernandez-Cruz, A. and Darszon, A. (1996). T-type Ca2+ channels and alpha 1E expression in spermatogenic cells, and their possible relevance to the sperm acrosome reaction. FEBS Lett. 388, 150154.[Medline]
Lin, Y.-C. J., Grigoriev, N. G. and Spencer, A. N. (2000). Wound healing in jellyfish striated muscle involves rapid switching between two modes of cell motility and a change in the source of regulatory calcium. Dev. Biol. 225, 87100.[Medline]
Lin, Y.-C. J. and Spencer, A. N. (2001). Localisation of intracellular calcium stores in the striated muscles of the jellyfish Polyorchis penicillatus: possible involvement in excitationcontraction coupling. J. Exp. Biol. 204, 37273736.
Lupa, M. T. and Caldwell, J. M. (1994). Sodium channels aggregate at former synaptic sites in innervated and denervated regenerating muscles. J. Cell Biol. 124, 139147.[Abstract]
Mackie, G. O. and Meech, R. W. (1985). Separate sodium and calcium spikes in the same axon. Nature 313, 791793.[Medline]
Ödblom, M. P., Williamson, R. and Jones, M. B. (2000). Ionic currents in cardiac myocytes of squid, Alloteuthis subulata. J. Comp. Physiol. B 170, 1120.[Medline]
Plickert, G. and Kroiher, M. (1988). Proliferation kinetics and cell lineages can be studied in whole mounts and macerates by means of BrdU/anti-BrdU technique. Development 103, 791794.[Abstract]
Przysiezniak, J. and Spencer, A. N. (1992). Voltage-activated calcium currents in identified neurons from a hydrozoan jellyfish, Polyorchis penicillatus. J. Neurosci. 12, 20652078.[Abstract]
Randall, A. D. (1998). The molecular basis of voltage-gated Ca2+ channel diversity: is it time for T? J. Membr. Biol. 161, 207213.[Medline]
Randall, A. D. and Tsien, R. W. (1997). Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels. Neuropharmacology 36, 879893.[Medline]
Regan, L. J. (1991). Voltage-dependent calcium currents in Purkinje cells from rat cerebellar vermis. J. Neurosci. 11, 22592269.[Abstract]
Richard, S., Diochot, S., Nargeot, J., Baldy-Moulinier, M. and Valmier, J. (1991). Inhibition of T-type calcium currents by dihydropyridines in mouse embryonic dorsal root ganglion neurons. Neurosci. Lett. 132, 229234.[Medline]
Ritchie, J. M. and Rogart, R. B. (1977). Density of sodium channels in mammalian myelinated nerve fibers and nature of the axonal membrane under the myelin sheath. Proc. Natl. Acad. Sci. USA 74, 211215.[Abstract]
Santi, C. M., Darszon, A. and Hernandez-Cruz, A. (1996). A dihydropyridine-sensitive T-type Ca2+ current is the main Ca2+ current carrier in mouse primary spermatocytes. Am. J. Physiol. 271, 15831593.
Schmid, V., Stidwill, R., Bally, A., Marcum, B. and Tardent, P. (1981). Heat dissociation and maceration of marine cnidarians. Rouxs Arch. Dev. Biol. 190, 143149.
Schild, D., Geiling, H. and Bischofberger, J. (1995). Imaging of L-type Ca2+ channels in olfactory bulb neurones using fluorescent dihydropyridine and a styryl dye. J. Neurosci. Meth. 59, 183190.[Medline]
Spafford, J. D., Grigoriev, N. G. and Spencer, A. N. (1996). Pharmacological properties of voltage-gated sodium currents in motor neurones from a jellyfish Polyorchis penicillatus. J. Exp. Biol. 199, 941948.
Spafford, J. D., Spencer, A. N. and Gallin, W. J. (1998). A putative voltage-gated sodium channel subunit (PpSCN1) from the hydrozoan jellyfish, Polyorchis penicillatus: structural comparisons and evolutionary considerations. Biochem. Biophys. Res. Comm. 244, 772780.[Medline]
Spencer, A. N. (1978). Neurobiology of Polyorchis. I. Function of effector systems. J. Neurobiol. 9, 143157.[Medline]
Spencer, A. N. (1979). Neurobiology of Polyorchis. II. Structure of effector systems. J. Neurobiol. 10, 95117.[Medline]
Spencer, A. N. and Satterlie, R. A. (1981). The action potential and contraction in subumbrellar swimming muscle of Polyorchis penicillatus (Hydromedusae). J. Comp. Physiol. 144, 401407.
Srinivasan, Y., Elmer, L. W., Davis, J. Q., Bennett, V. and Angelides, K. J. (1988). Ankyrin and spectrin associate with voltage-dependent sodium channels in brain. Nature 333, 177180.[Medline]
Takahashi, K. and Akaike, N. (1991). Calcium antagonist effects on low threshold (T-type) calcium current in rat isolated hippocampal CA1 pyramidal neurons. J. Pharm. Exp. Ther. 256, 169175.[Abstract]
Triggle, D. J. (1998). The physiological and pharmacological significance of cardiovascular T-type, voltage-gated calcium channels. Am. J. Hypertns. 11, 8087.
Tseng, G. N. and Boyden, P. A. (1989). Multiple types of Ca2+ currents in single canine Purkinje cells. Circ. Res. 65, 17351750.[Abstract]
Tsien, R. W. (1998). Key clockwork component cloned. Nature 391, 839841.[Medline]
Wang, H., Bedford, F. K., Brandon, N. J., Moss, S. J. and Olsen, R. W. (1999). GABAA-receptor-associated protein links GABAA receptors and the cytoskeleton. Nature 397, 6972.[Medline]
Yamoah, E. N. and Crow, T. (1994). Two components of calcium currents in the soma of photoreceptors of Hermissenda. J. Neurophysiol. 72, 13271336.