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¹ Department of Physiology, Queen's University, Kingston, ON, Canada, K7L 3N6

	Abstract

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Ca²⁺-activated, non-selective cation channels feature prominently in the regulation of neuronal excitability, yet the mechanism of their Ca²⁺ activation is poorly defined. In the bag cell neurones of Aplysia californica, opening of a voltage-gated, non-selective cation channel initiates a long-lasting afterdischarge that induces egg-laying behaviour. The present study used single-channel recording to investigate Ca²⁺ activation in this cation channel. Perfusion of Ca²⁺ onto the cytoplasmic face of channels in excised, inside-out patches yielded a Ca²⁺ activation EC₅₀ of 10 µM with a Hill coefficient of 0.66. Increasing Ca²⁺ from 100 nM to 10 µM caused an apparent hyperpolarizing shift in the open probability (P_o) versus voltage curve. Beyond 10 µM Ca²⁺, additional changes in voltage dependence were not evident. Perfusion of Ba²⁺ onto the cytoplasmic face did not alter P_o; moreover, in outside-out recordings, P_o was decreased by replacing external Ca²⁺ with Ba²⁺ as a charge carrier, suggesting Ca²⁺ influx through the channel may provide positive feedback. The lack of Ba²⁺ sensitivity implicated calmodulin in Ca²⁺ activation. Consistent with this, the application to the cytoplasmic face of calmodulin antagonists, calmidazolium and calmodulin-binding domain, reduced P_o, whereas exogenous calmodulin increased P_o. Overall, the data indicated that the cation channel is activated by Ca²⁺ through closely associated calmodulin. Bag cell neurone intracellular Ca²⁺ rises markedly at the onset of the afterdischarge, which would enhance channel opening and promote bursting to elicit reproduction. Cation channels are essential to nervous system function in many organisms, and closely associated calmodulin may represent a widespread mechanism for their Ca²⁺ sensitivity.

(Received 25 January 2006; accepted after revision 6 June 2006; first published online 8 June 2006)
Corresponding author N. S. Magoski: Department of Physiology, Queen's University, 4th Floor, Botterell Hall, 18 Stuart Street, Kingston, ON, Canada, K7L 3N6. Email: magoski{at}post.queensu.ca

	Introduction

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Found in the nervous systems of all species examined, Ca²⁺-activated, non-selective cation channels set excitability and firing rates (Partridge et al. 1979; Swandulla & Lux, 1985; Morisset & Nagy, 1999; Egorov et al. 2002), tune information processing (Hall & Delaney, 2000; Van den Abbeele et al. 1994), and play roles in neuropathology (Fraser & MacVicar, 1996; Chen et al. 1997; Smith et al. 2003). These channels are non-selective to monovalent cations, may be Ca²⁺ permeable or voltage dependent, and typically show activation at 1 µM intracellular Ca²⁺, but can respond over the nanomolar to millimolar range (Yellen, 1982; Partridge & Swandulla, 1987, 1988; Razani-Boroujerdi & Partridge, 1993; Cho et al. 2003; Liman, 2003; Liu & Liman, 2003; Prawitt et al. 2003; Guinamard et al. 2004).

Particularly in the nervous system, the means by which Ca²⁺-activated cation channels transduce Ca²⁺ is not fully understood. The ubiquitous Ca²⁺-binding protein, calmodulin, mediates the Ca²⁺-dependent activation, inactivation, or facilitation of many ion channel species (Saimi & Kung, 2002; Xia et al. 1998; Levitan, 1999; Michikawa et al. 1999; Zuhlke et al. 1999). In addition, the effect of Ca²⁺ on ligand-gated cation channels, such as cyclic nucleotide-gated (CNG) channels (Liu et al. 1994; Bradley et al. 2004) and NMDA receptors (Krupp et al. 1999; Rycroft & Gibb, 2004), is due to closely associated calmodulin. Moreover, calmodulin is the Ca²⁺ sensor for a non-neuronal, transient receptor potential/melastatin (TRPM) cation channel, expressed in heart, pancreas, kidney, and intestine (Launay et al. 2002; Nilius et al. 2005). However, for native, neuronal, steady-state, Ca²⁺-activated cation channels, the mechanism of Ca²⁺ sensitivity remains unknown.

The present study examines the Ca²⁺-dependent activation and modulation of a non-selective cation channel from the bag cell neurones of the marine snail, Aplysia californica. This channel provides the depolarizing drive for the afterdischarge, a prolonged burst that initiates egg-laying behaviour (Kupfermann, 1967; Kupfermann & Kandel, 1970; Pinsker & Dudek, 1977; Conn & Kaczmarek, 1989). The afterdischarge is characterized by 30 min of action potential firing, with a concomitant release and influx of Ca²⁺ that results in the neurohaemal secretion of egg-laying hormone (Fink et al. 1988; Fisher et al. 1994). Upon termination of the afterdischarge, a lengthy refractory period ensues, during which a second burst cannot be elicited (Conn & Kaczmarek, 1989). Previously, Wilson et al. (1996) found that elevating Ca²⁺, from nanomolar to micromolar levels, at the cytoplasmic face of patches excised from bag cell neurones increased cation channel activity; however, the extent or the mechanisms of Ca²⁺ activation were not examined. We now provide evidence to suggest that closely associated calmodulin serves as the cation channel Ca²⁺ sensor. Linking the cation channel and calmodulin provides a means to translate a change in intracellular Ca²⁺ into a change in excitability, which, in a system like the bag cell neurones, is essential for triggering behaviour. As a whole, calmodulin may act in this capacity for neuronal cation channels throughout the metazoa.

	Methods

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Animals and cell culture

Adult Aplysia californica weighing 150–300 g were obtained from Marinus Inc. (Long Beach, CA, USA). Animals were housed in an 300 l aquarium containing continuously circulating, aerated sea water (Instant Ocean; Aquarium Systems, Mentor, OH, USA or Kent sea salt; Kent Marine, Acworth, GA, USA) at 14–16°C on a 12 : 12 h light : dark cycle, and fed Romaine lettuce five times a week.

For primary cultures of isolated bag cell neurones, animals were anaesthetized by an injection of isotonic MgCl₂ (50% of body weight), the abdominal ganglion removed and treated with neutral protease (13.33 mg ml^–1; Roche Diagnostics, Indianapolis, IN, USA) for 18 h at 20–22°C, dissolved in tissue culture artificial sea water (tcASW) (composition (mM): 460 NaCl, 10.4 KCl, 11 CaCl₂, 55 MgCl₂, 15 Hepes, 1 mg ml^–1 glucose, 100 U ml^–1 penicillin, and 0.1 mg ml^–1 streptomycin, pH 7.8 with NaOH). The ganglion was then transferred to fresh tcASW for 1 h, after which time the bag cell neurone clusters were dissected from their surrounding connective tissue. Using a fire-polished Pasteur pipette and gentle trituration, neurones were dispersed in tcASW onto 35 mm x 10 mm polystyrene tissue culture dishes (Corning, Corning, NY, USA). Cultures were maintained in tcASW in a 14°C incubator, and used for experimentation within 1–3 days. Salts were obtained from Fisher Scientific (Ottawa, ON, Canada), ICN (Aurora, OH, USA), or Sigma-Aldrich (St Louis, MO, USA).

Excised, patch-clamp recording

Single cation channel current was measured using an EPC-8 amplifier (HEKA Electronics, Mahone Bay, NS, Canada), and primarily the excised, inside-out patch-clamp method. Microelectrodes were pulled from 1.5 mm internal diameter, borosilicate glass capillaries (TW 150 F-4; World Precision Instruments, Sarasota, FL, USA) and were fire polished to a resistance of 2–8 M when filled with normal artificial sea water (nASW) (composition as per tcASW but lacking glucose, penicillin, and streptomycin). To lower the root mean squared noise of the current signal, microelectrode capacitance was reduced by coating the shank and half of the shoulder with dental wax (Heraeus Kulzer, South Bend, IN, USA) under a dissecting microscope. Following excision, the cytoplasmic face was bathed with artificial intracellular saline (composition (mM): 500 potassium aspartate, 70 KCl, 1.2 MgCl₂, 10 Hepes, 11 glucose, 5 EGTA, 10 reduced glutathione, pH adjusted to 7.3 with KOH). In the majority of experiments, CaCl₂ was added for a free Ca²⁺ concentration of 1 µM. Some experiments were performed using intracellular saline with Ca²⁺ or Ba²⁺ concentrations ranging from 100 nM to 300 µM. Experiments involving Ba²⁺ required the substitution of CaCl₂ with BaCl₂. In all cases, the added and free Ca²⁺ and Ba²⁺ concentrations were calculated using the WebMaxC program (http://www.stanford.edu/~cpatton/webmaxc/webmaxcE.htm). In one set of experiments, single-channel current was measured using the excised, outside-out patch-clamp technique. Microelectrodes were filled with artificial intracellular saline containing 1 µM or 300 µM free Ca²⁺. The extracellular face of excised, outside-out patches was exposed to nASW containing 11 mM Ca²⁺ or 11 mM Ba²⁺. In all cases, current was low-pass filtered at 1 kHz using the EPC-8 Bessel filter and acquired at a sampling rate of 10 kHz using an IBM-compatible personal computer, a Digidata 1300 analog-to-digital converter (Axon Instruments, Union City, CA, USA), and the Clampex acquisition program of pCLAMP (version 8.0; Axon Instruments). Data were gathered at room temperature (22°C) in 1–3 min intervals, typically while holding the patch at –60 mV.

Patch perfusion array, drug application and reagents

An eight-barrel perfusion array was constructed by tightly aligning borosilicate square tubing (outer diameter: 0.75 mm, internal diameter: 0.5 mm; VitroCom Inc., Mountain Lakes, NJ, USA) attached to one another using superglue. The section of the array that was submerged into the bath did not come in contact with superglue. The barrels at the opposite end of the array were fitted with silicone tubing (outer diameter: 3.3 mm, internal diameter: 0.8 mm; Cole Parmer, Vernon Hills, IL, USA). Each of these perfusion lines was connected to a 5 ml syringe. Prior to experimentation, the entire perfusion system was rinsed with Sigmacote (SL-2; Sigma-Aldrich) and allowed to dry for at least 24 h. Gravitational flow was controlled by an alligator clip over the tubing and setting the level of the syringes to a fixed height. When the clip was released, the result was a flow of 1 ml min^–1. Any greater flow rate disturbed the patch and led to mechanical-based noise or seal failure. The perfusion system allowed patches to be moved from the mouth of one barrel to the next, permitting an almost instantaneous change in solutions at the face of the channel. During perfusion, the culture dish was gently drained as required using a plastic Pasteur pipette.

Drugs were made up as concentrated stock solutions and frozen at –30°C. They were introduced to the patch at the indicated working concentration, either with the perfusion array or by pipetting a small volume of stock solution into the culture dish. In the latter case, care was taken to pipette the stock near the side of the dish and as far away as possible from the patch at the tip of the microelectrode. In experiments examining the interplay of Ca²⁺ concentration and voltage dependence, TEA (Sigma-Aldrich) was added to nASW in the pipette to a final concentration of 20 mM in order to reduce outward currents through Ca²⁺-activated K⁺ channels. At or approaching 0 mV, these currents interfered with resolving inward current through the cation channel. Three calmodulin pharmacological inhibitors were employed to test the role of calmodulin as the cation channel Ca²⁺ sensor. Calmidazolium chloride (Calbiochem, San Diego, CA, USA) was dissolved in 100% ethanol for a stock solution of 14.5 mM. Calmidazolium (10 µM final) or its ethanol control (0.07% final) were perfused onto the cytoplasmic face of excised, inside-out patches. Similarly, N-(6-aminohexyl)-1-napthalenesulphonamide HCl (W-5) (Calbiochem) was dissolved in 100% ethanol for a stock solution of 70 mM. W-5 (100 µM final) or its ethanol control (0.15% final) were also perfused onto the cytoplasmic face of excised, inside-out patches. Calmodulin-binding domain (CBD) (Calbiochem) was dissolved in sterile, double-distilled H₂O for a stock solution of 2.2 mM. For experiments, a small volume of CBD, 17 µl (50 µM final), was pipetted into a centre-well organ culture dish (VWR, Mississauga, ON, Canada) containing 750 µl of intracellular saline. Recording of cation channel current from excised, inside-out patches began following a short diffusion period of 1 min.

Calmodulin purification

Calmodulin was purified using repeated chromatography on phenyl-Sepharose from an Escherichia coli strain (BL21) stably transformed with the expression plasmid (pCAM) coding for wild-type bovine calmodulin (accession number P62157). A sample of frozen E. coli cell stock was a gift from Dr A. S. Mak (Department of Biochemistry, Queen's University, Kingston, Ontario, Canada). The sample was cultured in 80 ml of LB broth with 100 µg ml^–1 ampicillin (Fisher) and grown overnight at 37°C with shaking at 250 r.p.m. The following day, 20 ml of the overnight culture was inoculated into 1 l of LB broth with 100 µg ml^–1 ampicillin and grown at 37°C, shaking at 250 r.p.m. until an OD600 of 0.8–1.1 was reached. The bacteria were then induced with 1 mM isopropyl-ß-D-thiogalactoside (Fisher) at 30°C for 4 h. Cells were then spun down for 25 min at 600g at 4°C, the supernatant discarded, and the pellet frozen at –80°C overnight. On ice, cells were resuspended in 50 ml of buffer A (containing: 25 mM Tris-HCl pH 7.5, 1 mM DTT (Fisher), 0.02% NaN₃, and 0.1 mg ml^–1 phenylmethylsulphonylfluoride (PMSF) (Sigma-Aldrich). Cells were ruptured by sonicating the solution 5–10 times at 30 s intervals separated by 1 min rest periods, until the solution was visibly clearer and less viscous. The solution was then centrifuged at 17 500g (JA-20) for 40 min at 4°C, after which time the pellet was discarded and the supernatant centrifuged at 110 000g (Ti45) for 1 h at 4°C. CaCl₂ was added to a concentration of 2.5 mM to expose the hydrophobic regions of calmodulin, and stored overnight at 4°C. A disposable chromatography column (732 1010; Biorad, Hercules, CA, USA) was packed with 5 ml phenyl-Sepharose beads (Amersham, Piscataway, NJ, USA) and pre-equilibrated with 25 ml of H₂O and 25 ml of buffer B (containing: 25 mM Tris-HCl (pH 7.5) and 2.5 mM CaCl₂). The column was capped with parafilm and stored overnight at 4°C. At room temperature, the column was rinsed with 5 ml of buffer B. Extracts were mixed with phenyl-Sepharose beads in the column and left to sit for 30 min with gentle mixing every 5 min. The column was washed with 50 ml of buffer B, then washed with 100 ml of buffer C (containing: 50% buffer B and 0.5 M NaCl), and eluted with buffer E (containing: 25 mM Tris-HCl (pH 7.5), 0.5 M NaCl, 5 mM EGTA). Three different elutions of 5 ml were collected in separate tubes and tested for protein content using a protein assay kit (500-0006; Biorad). The second elution tube contained protein and was added to a 5 kDa cut-off ultrafree centricon (UFV2BCC10-5K; Millipore, Nepean, ON, Canada) to concentrate the calmodulin. The centricon was centrifuged at 400g at 4°C until 1 ml of buffer E remained, at which time it was rinsed with 5 ml of intracellular saline containing 10 µM free Ca²⁺. This step was repeated, and centrifugation continued until 1 ml of calmodulin in intracellular saline remained in the centricon. The absorbance of calmodulin (at 280 nm) was then tested with spectrophotometry, and combined with the extinction coefficient of calmodulin (peptide property calculator; http://www.basic.northwestern.edu/biotools/proteincalc.html) to calculate calmodulin concentration. Aliquots (260 µM stock, 25 µl) were prepared and kept at –80°C prior to experimentation. During experiments, the stock calmodulin was diluted down to a final concentration of 3 µM by pipetting into a bath containing an excised, inside-out patch bathed in intracellular saline with 10 µM free Ca²⁺. As a control, aliquots of stock calmodulin were boiled for 10 min prior to application.

As a test of calmodulin protein stability, a SDS-PAGE gel was run. From a working stock of 7.36 mg ml^–1, 1.5 µl (11 µg), 7.5 µl (55 µg), or 15 µl (110 µg) was added to a sample buffer (containing: 62.5 mM Tris-HCl pH 6.8 at 25°C, 2% (w/v) SDS, 10% glycerol, 0.01% (w/v) bromophenol blue and 42 mM DTT totalling a final volume of 30 µl. Samples and standards were boiled for about 5 min, and 20 µl of each loaded into a lane and allowed to run for 4 h. By comparison to a broad-range protein marker (7701-S; New England Biolabs, Ipswich, MA, USA), the gel indicated that the purified protein was calmodulin (17 000 kDa) and that no degradation had occurred.

Data analysis

To determine channel open probability (P_o), events lists were made from data files using the half-amplitude threshold criterion (Colquhoun & Sigworth, 1995) of the Fetchan analysis program of pCLAMP. Fetchan was also used to generate all-points histograms for determining channel amplitude. For display in figures, all data were filtered to a final cut-off frequency of 500 Hz using the Fetchan digital Gaussian filter. The Pstat analysis program of pCLAMP was used to read events lists and determine P_o, either automatically or manually, using the formula:

where t = the amount of time that n channels are open, N = the number of channels in the patch, and t_tot = the time interval over which P_o is measured. The number of channels in the patch was determined by counting the number of unitary current levels, particularly at more positive voltages (typically –20 mV). Pstat was also used to determine the mean open- and closed-state current level by fitting all-points histograms with Gaussian functions using the least-squares method and a simplex search. Channel current amplitude was then calculated by subtracting the mean closed current level from the mean open current level at a given voltage.

The concentration–response curve was constructed using P_o values obtained at each concentration of Ca²⁺ or Ba²⁺, which were normalized by dividing by the P_o at 300 µM Ca²⁺, averaged, and plotted versus divalent concentration using Origin (version 7; OriginLab Corporation, Northampton, MA, USA). The Ca²⁺ concentration–response curve was then fitted with a Hill function to yield the EC₅₀ and Hill coefficient. The Ba²⁺ concentration–response curve could only be fitted by linear regression. To make P_o versus voltage relationships, P_o was first normalized to P_o at 0 mV and then plotted against patch holding potential using Origin. This relationship was then fitted with a Boltzmann function to derive the half-maximal voltage (V_0.5) and the slope factor (k), which is the change in voltage required to move the P_o e-fold. Channel current versus voltage relationships were produced in Origin by plotting channel-current amplitude against patch-holding potential, and single-channel conductance was then determined by linear regression. Predicted reversal potential was extrapolated from the Origin linear regression fit.

Statistical analysis

Data are presented as the mean ± S.E.M. throughout. Statistical analysis was performed using Instat (version 3; GraphPad Software, San Diego, CA, USA). Student's t test (two-tailed or one-tailed, and paired or unpaired) was used to test whether the mean differed between two groups. A standard ANOVA with Dunnett's multiple comparisons test was used to test for differences between multiple means. The test for linear trend was used to determine if there was statistically significant linear trend in a series of multiple means. Analysis of outside-out recordings was based on a Ba²⁺-induced percentage change in P_o or current amplitude. The P_o and current amplitude with Ca²⁺ at the extracellular face was considered to have a mean of zero, and the data with Ba²⁺ at the extracellular face were compared for a difference from that mean of zero using a two-tailed, one-sample t test. In all cases, data were considered significantly different if the P value was < 0.05.

	Results

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The bag cell neurone cation channel is activated by Ca²⁺

Cation channels were identified in excised, inside-out patches from cultured bag cell neurones by their conductance (25–30 pS; 2 pA at –60 mV), voltage dependence of opening (an increase in P_o with depolarization), and absence of voltage-dependent inactivation. Many cation channels are regulated by Ca²⁺, yet few Ca²⁺ concentration–response curves for neuronal cation channels are published. We perfused the cytoplasmic face of cation channel-containing patches with intracellular saline that had a free Ca²⁺ concentration ranging from 100 nM to 300 µM (Fig. 1A). Upon excision, the patch was always initially perfused with intracellular saline containing 300 µM Ca²⁺; subsequently, as many concentrations from within the range as possible were then delivered. Individual P_o values from a total of 24 patches exposed to some or all of the concentration range were normalized to 300 µM Ca²⁺ and plotted versus Ca²⁺ concentration (Fig. 1B). Cation channel activity was maximal at 300 µM Ca²⁺ and above (data not shown), and gradually diminished upon exposure to decreasing concentrations of free Ca²⁺ to a minimum, but still detectable level, in 100 nM Ca²⁺. The concentration–response curve was fitted with a Hill function, yielding an EC₅₀ of 10 ± 5 µM Ca²⁺ and a Hill coefficient of 0.66 ± 0.1. The response of the channel to the concentration of Ca²⁺ was independent of the order in which the Ca²⁺ concentrations were presented after the initial 300 µM Ca²⁺. For a given channel, the P_o upon initial exposure to 300 µM Ca²⁺ was very similar to a repeated exposure, even after application of any of the lower concentrations and vice versa (Fig. 1C). Thus, cation channel activity was reversible and highly dependent on cytoplasmic face Ca²⁺ concentration, confirming its Ca²⁺-dependent activation.

View larger version (24K): [in this window] [in a new window]   Figure 1.  Concentration-dependent effects of Ca2+ on the cation channel A, sample traces of cation channel activity recorded from an excised, inside-out patch held at –60 mV. Cation channel activity, seen as unitary inward current deflections of 2 pA, steadily increases as the cytoplasmic face is perfused with intracellular saline containing 100 nM to 300 µM Ca2+. The closed state is at the top of the trace and designated by C, while the open states are at the bottom and designated by O1, O2 and O3. B, concentration–response of cation channel Po exposed to a range of Ca2+ concentrations. All patches were exposed to 300 µM Ca2+ (n = 24) with the remaining concentrations consisting of: 100 nM (n = 6), 300 nM (n = 6), 1 µM (n = 9), 3 µM (n = 9), 10 µM (n = 13), 30 µM (n = 11) and 100 µM (n = 11). In three of these 24 cases, the patch was exposed to the entire concentration range (100 nM to 300 µM). Po was normalized to Po at 300 µM Ca2+. Data points fitted with a Hill function yield an EC50 of 10 ± 5 µM and a Hill coefficient of 0.66 ± 0.1. C, sample traces of cation channel activity recorded from an excised, inside-out patch held at –60 mV. Upon exchange of the intracellular saline perfusing the cytoplasmic face from one containing 300 µM Ca2+ to one with 100 nM, the activity decreases. However, this effect is completely reversible and the Po reverts back to the prior level upon return to 300 µM Ca2+.

The voltage dependence of cation channel P_o is known to be modulated by Ca²⁺ in hamster vomeronasal sensory neurones (Liman, 2003) and membrane vesicles from placenta (Gonzalez-Perrett et al. 2002). Wilson et al. (1996) examined the effect of Ca²⁺ on the voltage dependence of the bag cell neurone cation channel, and suggested that increasing the cytoplasmic face Ca²⁺ from 100 nM to 1 µM caused a hyperpolarizing shift in the P_o versus voltage curve. The present study examined the voltage dependence of bag cell neurone cation channel P_o as a function of 100 nM, 10 µM, or 300 µM cytoplasmic face Ca²⁺. The voltage dependence of the cation channel was maintained at all three Ca²⁺ levels such that P_o increased with depolarizing potentials. Figure 2A shows an example of this effect at 300 µM Ca²⁺. When held at potentials positive to 0 mV, the cation channel current did not reverse, but became unresolvable. This lack of reversal may be due to block by cytoplasmic face Mg²⁺, as found in astrocyte (Chen & Simard, 2001) and TRPC5 (Obukhov & Nowycky, 2005) cation channels. Thus, despite not being an ideal standardization, P_o was normalized to P_o at 0 mV, which in turn revealed an apparent hyperpolarizing shift in voltage dependence without a change in sensitivity at 10 µM (n = 6) or 300 µM Ca²⁺ (n = 9) as compared to 100 nM Ca²⁺ (n = 5) (Fig. 2B). The V_0.5 increased from –12 ± 0.8 mV in 100 nM Ca²⁺ to –30 ± 0.5 mV or –27 ± 0.2 mV in 10 µM or 300 µM Ca²⁺, without an appreciable change in the k (15 ± 0.7 versus 17 ± 0.4 versus 16 ± 0.2 in 100 nM versus 10 µM versus 300 µM Ca²⁺, respectively). Overall, channel activity not only increased upon exposure to increasing Ca²⁺ levels, but as the membrane was depolarized, the increase in channel activity was further enhanced by the presence of 10 µM or 300 µM Ca²⁺. Not surprisingly, linear regression analysis of current–voltage relationships in the three Ca²⁺ concentrations showed no change in the conductance or, as established by extrapolating beyond 0 mV, the reversal potential (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 2.  Interaction between Ca2+ and voltage dependence of the cation channel A, sample traces of cation channel activity recorded from an excised, inside-out patch perfused with intracellular saline containing 300 µM Ca2+ and held at the indicated voltages. As the membrane potential is depolarized, channel activity increases. B, mean cation channel Po normalized to Po at 0 mV for each respective Ca2+ concentration and plotted against voltage. As given in the inset, points fitted with a Boltzmann function yield values for half-maximal activation (V0.5) and slope factor (k). When Ca2+ is elevated from 100 nM to 10 µM, the voltage dependence shifts in the hyperpolarizing direction (V0.5 = –12 ± 0.8 mV versus –30 ± 0.5 mV) without an appreciable alteration in sensitivity (k = 15 ± 0.7 versus 17 ± 0.4); however, a further increase in Ca2+ to 300 µM does not cause any obvious additional change (V0.5 = –27 ± 0.2 mV; k = 16 ± 0.2).

The Ca²⁺ sensitivity of ion channels is mediated by Ca²⁺ binding directly to the channel or a channel-associated Ca²⁺ sensor (Hille, 2001; Levitan & Kaczmarek, 2001). The most versatile and ubiquitous Ca²⁺ sensor is calmodulin (Berridge et al. 2000), and it has been implicated in mediating the Ca²⁺ sensitivity of numerous ion channels (Levitan, 1999). To initially test this, we used a method first carried out on small-conductance Ca²⁺-activated K⁺ channels (Cao & Houamed, 1999); namely, Ba²⁺ was substituted for Ca²⁺ and perfused on the cytoplasmic face of the cation channel. The rationale is based on Ba²⁺ binding/activating calmodulin poorly because it is a weak substitute for Ca²⁺ at the EF-hand motifs (Haiech et al. 1981; Chao et al. 1984; Wang, 1985; Ozawa et al. 1999). Therefore, if calmodulin mediates Ca²⁺ sensitivity of the cation channel, replacing Ca²⁺ with Ba²⁺ would result in a failure to activate the channel in a concentration-dependent fashion. Cation channel-containing patches were initially exposed to intracellular saline containing 300 µM Ca²⁺, and then subsequently perfused with intracellular saline containing Ba²⁺ ranging in concentration from 100 nM to 300 µM (n = 6). Cation channel P_o was dramatically reduced upon exposure to Ba²⁺ and remained relatively unchanged throughout the concentration range (Fig. 3). Overall, the cation channel showed very little, if any, sensitivity to Ba²⁺, suggesting a possible involvement of calmodulin as the sensor mediating Ca²⁺ dependence of channel activity.

View larger version (20K): [in this window] [in a new window]   Figure 3.  Lack of effect of Ba2+ on cation channel activity A, sample traces of cation channel activity recorded at –60 mV from an excised, inside-out patch. The cytoplasmic face was perfused with intracellular saline with 300 µM Ca2+ followed by intracellular saline with Ba2+ substituted for Ca2+ at concentrations ranging from 100 nM to 300 µM. The Ba2+ intracellular saline contained no added Ca2+. Upon exchange to Ba2+, the Po decreases and remains unchanged regardless of the concentration of subsequently applied Ba2+. B, summary of mean cation channel Po after Ba2+ perfusion compared to that of Ca2+. Po was normalized to Po at 300 µM Ca2+ (perfused onto each patch at the start of the experiment prior to the introduction of Ba2+) (, n = 6). The points could only be fitted with a linear regression function. For comparison, the concentration–response curve for Ca2+ from Fig. 1B is replotted here (•).

For voltage-gated Ca²⁺ channels, Ca²⁺ entry in turn inactivates the channel through associated calmodulin, and this can be prevented by substituting Ba²⁺ for Ca²⁺ as a charge carrier (Zuhlke et al. 1999). To examine if Ca²⁺ entry through the cation channel also serves as a regulator, patches were excised from bag cell neurones in the outside-out configuration, and the extracellular face of the channel was perfused with nASW containing 11 mM Ca²⁺ followed by ASW with Ba²⁺ substituted for Ca²⁺. The pipette, which bathed the cytoplasmic face, contained intracellular saline with either 1 µM or 300 µM Ca²⁺ (n = 4 and 4) (Fig. 4A and B). In agreement with Ba²⁺ permeating the cation channel as a superior charge carrier, following perfusion with Ba²⁺ the current amplitude increased by 18% with 1 µM Ca²⁺, and 15% with 300 µM Ca²⁺ in the pipette (Fig. 4C). Wilson et al. (1996) reported that the relative monovalent cation permeability of the channel was K⁺ Na⁺ >> Tris > NMDG; moreover, consistent with Ca²⁺ permeability and our data on enhanced current amplitude under external Ba²⁺, they showed that replacing external Ca²⁺ with Ba²⁺ caused the conductance to increase from 29 pS to 36 pS. However, Wilson et al. (1996) did not report at all regarding the effect of external Ba²⁺ on P_o. We found that Ba²⁺ perfusion produced an 70% and 50% decrease in P_o with 1 µM and 300 µM Ca²⁺ in the pipette, respectively (Fig. 4C). This drop in P_o is probably due to a loss of Ca²⁺ activation. Changes to both current amplitude and P_o with either 1 µM or 300 µM Ca²⁺ in the pipette were statistically significant, and suggest that Ca²⁺ influx through the channel itself may be a source of Ca²⁺ activation.

View larger version (42K): [in this window] [in a new window]   Figure 4.  Extracellular Ba2+ decreases cation channel Po and increases amplitude A, sample traces recorded from an excised, outside-out patch held at –60 mV. The cytoplasmic face is exposed to intracellular saline (in the pipette) containing 300 µM Ca2+, and upon excision the extracellular face is perfused with ASW (in the bath) containing 11 mM Ca2+ or 11 mM Ba2+. Upper panel, replacement of Ca2+ with Ba2+ at the extracellular face results in both an increased channel amplitude and a lowering of Po. The closed and two open current levels are indicated by dotted lines. The 2 s time base applies. Lower panel, 100 ms portion of cation channel activity during both Ca2+ and Ba2+ perfusion taken from the upper panel at times indicated by arrows. The dotted lines again show the increase in current amplitude when Ba2+ replaces Ca2+. The 25 ms time base applies. B, all-points histograms from the outside-out patch shown in A. The closed (C) and two open (O1, O2) current levels are denoted just to the right of their corresponding peaks. An increase in current amplitude with Ba2+versus Ca2+ application is evident by the left shift in both of the open state peaks. C, summary of Ba2+-induced percentage changes in PO and current amplitude from outside-out patches. Ba2+-induced percentage changes are compared to a mean of zero using a two-tailed, one-sample t test. When recorded with intracellular saline containing either 1 µM or 300 µM Ca2+, the Po and current amplitude (amp) are significantly reduced and enhanced, respectively, by the perfusion of Ba2+ (*P < 0.05 or **P < 0.01). For this and all subsequent bar graphs, the n values are given within, just below, or just above a given bar.

In those cases where calmodulin is the sensor mediating Ca²⁺-dependent channel regulation of ion channels, calmodulin antagonists often inhibit the effects of Ca²⁺ on channel function (Krupp et al. 1999; Michikawa et al. 1999; Bobkov & Ache, 2003; Moreau et al. 2005). Calmidazolium chloride is a potent, specific, and widely effective organic antagonist that inhibits many calmodulin-activated proteins (Van Belle, 1981; Gietzen et al. 1982; DeRiemer et al. 1985). When cation channels excised from bag cell neurones were perfused with intracellular saline containing 10 µM calmidazolium and 1 µM Ca²⁺, the P_o decreased (n = 5) (Fig. 5A). The antagonizing effect of calmidazolium on cation channel activity was also apparent at a cytoplasmic face Ca²⁺ concentration of 10 µM or 300 µM (n = 6 and 6) (Fig. 5B and C). When the cytoplasmic face of excised patches was exposed to ethanol, the vehicle for calmidazolium, cation channel activity decreased by no more than 30%, regardless of the Ca²⁺ concentration (n = 5, 5 and 5) (Fig. 5D). Compared to parallel ethanol controls, 10 µM calmidazolium significantly reduced channel P_o by 60% and 70% in 1 µM and 10 µM, respectively (Fig. 5E). The concentration dependence of calmidazolium was demonstrated by applying additional concentrations in the presence of 300 µM Ca²⁺. At 3 µM and 30 µM (n = 4 and 4), calmidazolium caused lower (45%) and higher (90%) reductions in channel P_o than that produced by 10 µM (75%) (Fig. 5E).

View larger version (25K): [in this window] [in a new window]   Figure 5.  A calmodulin inhibitor decreases cation channel Po A–C, sample traces, before and after 10 µM calmidazolium, of cation channel activity in an excised, inside-out patch held at –60 mV with the cytoplasmic face bathed in intracellular saline containing 1 µM, 10 µM, or 300 µM Ca2+. At each Ca2+ concentration, there is a clear and obvious decrease in Po upon addition of calmidazolium. D, sample traces before and after 0.15% ethanol (the vehicle), of cation channel activity in a patch held at –60 mV with the cytoplasmic face bathed in intracellular saline containing 1 µM Ca2+. Ethanol causes only a modest reduction in Po. E, a summary of the mean percentage change in cation channel Po showing the effect of ethanol versus calmidazolium or W-5-treated patches in intracellular saline containing 1 µM, 10 µM, or 300 µM Ca2+. A two-tailed, unpaired t test comparing separate ethanol controls to 10 µM calmidazolium shows that the decrease in Po reaches significance in the presence of either 1 µM or 10 µM Ca2+ (*P < 0.05). An ANOVA followed by Dunnett's multiple comparisons test shows that 10 and 30 µM calmidazolium produce significant and concentration-dependent decreases in Po in the presence of 300 µM Ca2+ (*P < 0.05 or **P < 0.01). A test for linear trend amoung the 300 µM Ca2+ points also shows that there is a significant linear trend, i.e. a negative slope with increasing calmidazolium concentration (P < 0.005). In 300 µM Ca2+, a one-tailed, unpaired t test finds no significant (n.s.) difference between W-5-treated patches and ethanol controls.

A synthetic peptide inhibitor of calmodulin, known as CBD and corresponding to residues 290–309 of the Ca²⁺–calmodulin-dependent kinase regulatory domain, is highly effective at impairing calmodulin-mediated Ca²⁺ processes (Payne et al. 1988). This peptide is a very specific calmodulin antagonist and a reliable indicator of calmodulin being involved in a process. The cytoplasmic face of excised, inside-out patches containing cation channels was exposed to 50 µM CBD added to intracellular saline containing 1 µM, 10 µM and 300 µM Ca²⁺ (n = 7, 6 and 6) (Fig. 6A–C). CBD was manually added to the bath and allowed to diffuse for 1 min prior to a 5 min recording period. For controls, water (the vehicle) was initially employed, and it produced no change in cation channel activity (n = 2, data not shown). Subsequently, timed controls were used for analysis and can be described as the percentage change in cation channel activity between the first and second half of the control period prior to the addition of CBD. Compared to timed controls, 50 µM CBD caused a significant decline in cation channel P_o of 20%, 40%, and 65% in 1 µM, 10 µM, and 300 µM Ca²⁺, respectively (Fig. 6D). Collectively, the antagonizing effects of pharmacological blockers of calmodulin support the role of calmodulin in Ca²⁺-dependent cation channel activation.

View larger version (22K): [in this window] [in a new window]   Figure 6.  Calmodulin-binding domain inhibitor peptide (CBD) decreases cation channel Po A–C, sample traces of cation channel activity, before and after addition of 50 µM CBD, recorded from excised, inside-out patches held at –60 mV and bathed with intracellular saline containing 1 µM, 10 µM, or 300 µM Ca2+. In each case, the delivery of CBD markedly lowers the Po. D, a summary of the mean percentage change in Po between timed controls and CBD-treated preparations. Timed controls are expressed as the percentage change between the first and second half of the control period. Comparisons using a one-tailed, paired t test show that the reduction in Po during the CBD treatment period produces means that are significantly different from timed controls at 1 µM or 10 µM Ca2+ (*P < 0.05) and 300 µM Ca2+ (**P < 0.01).

The application of exogenous calmodulin to Ca²⁺-sensitive ion channels is a widely practised method to assess possible modulation by this protein (Liu et al. 1994; Zhang et al. 1998; Krupp et al. 1999; Zuhlke et al. 1999; Rycroft & Gibb, 2004; Bradley et al. 2004). Excised, inside-out patches containing cation channels were bathed in 10 µM intracellular Ca²⁺ and exposed to 3 µM of either intact or boiled/denatured purified bovine calmodulin. Calmodulin was used in the presence of a Ca²⁺ concentration that led to a 50% activation of the cation channel, i.e. 10 µM. Exogenous calmodulin caused a significant, 60% increase in cation channel P_o (Fig. 7A and C), whereas when the protein was heat-inactivated it induced a moderate, 20% reduction in P_o (Fig. 7B and C). Overall, 10 µM Ca²⁺ was sufficient to activate 3 µM of added calmodulin to cause an increase in bag cell neurone cation channel activity.

View larger version (38K): [in this window] [in a new window]   Figure 7.  Exogenous calmodulin increases cation channel Po A, sample traces of cation channel activity recorded from an excised, inside-out patch held at –60 mV and bathed in intracellular saline containing 10 µM Ca2+, before and after addition of 3 µM purified bovine calmodulin. The introduction of calmodulin robustly elevates cation channel Po. B, sample traces of cation channel activity recorded from an excised, inside-out patch held at –60 mV and bathed in intracellular saline containing 10 µM Ca2+ before and after addition of 3 µM boiled calmodulin. Heat inactivation renders calmodulin modestly inhibitory. C, a comparison of the mean percentage change in Po between excised inside-out patches treated with 3 µM calmodulin versus boiled calmodulin. A two-tailed, unpaired t test shows that the increase in cation channel Po is significantly different following calmodulin administration compared to patches treated with boiled protein (*P < 0.05).

	Discussion

Top Abstract Introduction Methods Results Discussion References

Ca²⁺ activation is a defining characteristic of many cation channels. First described in cardiac Purkinje fibres (Kass et al. 1978), this process has been studied at the single-channel level in various cells, including cardiomyocytes (Colquhoun et al. 1981; Guinamard et al. 2004), epithelia (Miyashita et al. 2001), kidney cells (Gonzalez-Perrett et al. 2002), hair cells (Van den Abbeele et al. 1994) neuroblastoma (Yellen, 1982), sensory neurones (Razani-Boroujerdi & Partridge, 1993; Cho et al. 2003; Liman, 2003) and Helix neurones (Partridge & Swandulla, 1987). Ca²⁺ activation of the bag cell neurone cation channel has an EC₅₀ of 10 µM and peaks at 300 µM. Although cytoplasmic Ca²⁺ rarely exceeds 1 µM in spiking bag cell neurones (Fisher et al. 1994; Magoski et al. 2000), evidence from squid shows that, at the plasma membrane, the concentration in a Ca²⁺ channel microdomain can be 200–300 µM (Llinas et al. 1992). Also, depending on the distance from the pore, the nanodomain for single Ca²⁺ channels is 5–50 µM (Smith & Augustine, 1988; Neher, 1998). Thus, Ca²⁺ channels opened during the afterdischarge would provide a substantial, near-membrane concentration of Ca²⁺ to the cation channel. In addition, release from intracellular stores would be a source of Ca²⁺ for cation channel activation (Fink et al. 1988; Magoski et al. 2000).

Both Wilson et al. (1996) and the present study provide evidence that the bag cell neurone cation channel is Ca²⁺ permeable. We also now show that substituting Ba²⁺ for Ca²⁺ as an extracellular charge carrier decreased channel P_o. Given that Ba²⁺ activates the channel poorly, this suggests that Ca²⁺ influx through the cation channel itself provides a degree of stimulation. In general, for those Ca²⁺-activated, non-selective cation channels that are Ca²⁺ permeable, the intrinsically higher Ca²⁺ concentrations at the channel mouth may act as a regulator (Lan et al. 1996; Zitt et al. 1997; Strubing et al. 2001; Lambers et al. 2004). The result of Ba²⁺ permeation reducing bag cell neurone cation channel activity is, to our knowledge, some of the first evidence supporting such a mechansim. Conversely, a number of additional cation channels, such as TRPM4 and TRPM5, show little or no Ca²⁺ permeability and require other sources of Ca²⁺ influx or release for activation (Yellen, 1982; Chen & Simard, 2001; Miyashita et al. 2001; Launay et al. 2002; Cho et al. 2003; Liman, 2003; Prawitt et al. 2003; Guinamard et al. 2004).

Comparing the EC₅₀ and Hill coefficient values of the bag cell neurone channel (10 µM and 0.66) to other cation channels reveals both similarities and differences, e.g. 460 nM and 0.49 in chick sensory neurones (Razani-Boroujerdi & Partridge, 1993), 510 µM and 2.1 in vomeronasal neurones (Liman, 2003), 774 µM and 0.97 in rat sensory neurones (Cho et al. 2003), or 10 µM in cochlear hair cells (Van den Abbeele et al. 1994). The EC₅₀ of the non-neuronal TRPM4b has been reported as 400 nM (Launay et al. 2002) or 15 µM (Nilius et al. 2005), while TRPM5, which probably plays a role in taste transduction, has an EC₅₀ of either 840 nM or 21 µM, with a Hill coefficient of either 5.0 or 2.4 (Prawitt et al. 2003; Liu & Liman, 2003). A significant proportion of these EC₅₀ values are, like the bag cell neurone cation channel, in the micromolar range. In addition, the Hill coefficient of less than one for either the bag cell or chick sensory neurone channels suggests negative cooperativity. Perhaps initial Ca²⁺ binding lowers the affinity for additional binding, or Ca²⁺ may partially block the channels at higher concentrations. A Hill coefficient of 1.0, such as for rat sensory neurones, indicates competitive Ca²⁺ binding to a single site on the channel. Regarding the affinity of Ca²⁺ for calmodulin alone, in the absence of an effector protein such as a channel, Haiech et al. (1981) reported EC₅₀s of 4 and 7 µM in 100 and 200 mM KCl, respectively, for ⁴⁵Ca²⁺ binding to sheep calmodulin.

Some cation channels are voltage dependent, including those from cardiomyocytes (Guinamard et al. 2004), epithelia (Miyashita et al. 2001), superchiasmatic neurones (Kononenko et al. 2004), pyramidal neurones (Alzheimer, 1994), and vomeronasal neurones (Liman, 2003). For the bag cell neurone cation channel, activity is steadily enhanced at potentials positive to –60 mV; moreover, when Ca²⁺ is increased from 100 nM to 10 µM or 300 µM, the V_0.5 shifts from –12 mV to –30 mV, with little change in k (16 throughout). This is consistent with Wilson et al. (1996), who intimated that the P_o versus voltage curve shifted to the left when Ca²⁺ was increased from 100 nM to 1 µM. An interdependence of Ca²⁺ and voltage activation has also been observed for cation channels from vomeronasal neurones (Liman, 2003), astrocytes (Chen & Simard, 2001), and endothelia (Csanady & Adam-Vizi, 2003), although for TRPM4b the V_0.5 and k (+32 mV and 9) do not change overall when Ca²⁺ is altered (Nilius et al. 2003). In the case of the bag cell neurone cation channel, Ca²⁺ may not only act as a ligand by increasing P_o in a concentration-dependent manner, but could further augment activity by moving voltage dependence to more hyperpolarized potentials. During the afterdischarge, action potential firing occurs from a membrane potential of –20 to –40 mV (Conn & Kaczmarek, 1989), a range that readily accommodates the V_0.5 (–30 mV) of the cation channel in high Ca²⁺. However, such postulations are made knowing that the high-Ca²⁺-induced shift may be ostensible, given that P_o values in the present study were determined only for a limited range of voltages.

If cation channel Ca²⁺ activation is due to a channel-associated Ca²⁺ sensor, rather than Ca²⁺ binding directly to the channel, calmodulin is a good candidate. A ubiquitous Ca²⁺ sensor, calmodulin, mediates the Ca²⁺-dependent properties of numerous ion channels, including gating of small- and intermediate-conductance Ca²⁺-activated K⁺ channels (Xia et al. 1998; Levitan, 1999), activation of ryanodine receptors (Saimi & Kung, 2002), inactivation and facilitation of L-type Ca²⁺ channels (Zuhlke et al. 1999), as well as inhibition of CNG channels (Liu et al. 1994; Bradley et al. 2004), NMDA receptors (Zhang et al. 1998; Rycroft & Gibb, 2004), and IP₃ receptors (Michikawa et al. 1999). For the bag cell neurone cation channel, the P_o shows no concentration response to Ba²⁺, a metal that binds/activates calmodulin poorly (Haiech et al. 1981; Chao et al. 1984; Wang, 1985; Ozawa et al. 1999). Thus, as with Ca²⁺-activated K⁺ channels (Cao & Houamed, 1999) and Ca²⁺ channels (Zuhlke et al. 1999), the failure of Ba²⁺ to mimic Ca²⁺ suggests that calmodulin may be the cation channel Ca²⁺ sensor.

Calmodulin inhibitors have also been used to implicate calmodulin in channel regulation, e.g. 5–100 µM calmidazolium inhibits Ca²⁺-dependent facilitation and inactivation of Ca²⁺ release-activated Ca²⁺ channels (Moreau et al. 2005), as well as the Ca²⁺ sensitivity of a Na⁺-activated cation channel in lobster olfactory neurones (Bobkov & Ache, 2003) and the Drosophila TRPL cation channel expressed in oocytes (Lan et al. 1996). Moreover, 10 µM calmidazolium strongly inhibits both Aplysia CNS Ca²⁺–calmodulin-dependent kinase and the ability of bag cell neurones to fire an afterdischarge (DeRiemer et al. 1984, 1985). Depending on the added calmodulin concentration, calmidazolium maximally inhibits brain phosphodiesterase and blood cell Ca²⁺-ATPase in the range of 100 nM to 3 µM (Van Belle, 1981). For phosphorylase kinase, a protein that incorporates calmodulin as a subunit, maximal inhibition requires more than 10 µM (Van Belle, 1981). Cation channel activity after exposure to 10 µM calmidazolium in 1 µM, 10 µM and 300 µM Ca²⁺, was reminiscent of that in 100 nM Ca²⁺. Regarding CBD, it was first shown to inhibit Ca²⁺–calmodulin-dependent kinase II and phosphodiesterase by preventing calmodulin–enzyme binding (Payne et al. 1988). In addition, 20–25 µM CBD inhibits Ca²⁺-dependent inactivation of NMDA (Krupp et al. 1999) or IP₃ receptors (Michikawa et al. 1999). Accordingly, we found that 50 µM CBD reduced bag cell neurone cation channel activity at 1 µM, 10 µM and 300 µM Ca²⁺. This again indicates that, rather than Ca²⁺ binding directly to the channel, closely associated calmodulin acts as a Ca²⁺ sensor.

A role for calmodulin in cation channel Ca²⁺ sensitivity was further confirmed by our observation that 3 µM bovine calmodulin, in 10 µM Ca²⁺, increased P_o. Bovine calmodulin readily activates various Aplysia proteins, including Ca²⁺–calmodulin-dependent kinase (500 nM calmodulin with 500 µM Ca²⁺) (DeRiemer et al. 1984), adenylate cyclase (3 µM calmodulin with 3 µM Ca²⁺) (Abrams et al. 1991), and twitchin (100 nM calmodulin with 1 mM Ca²⁺) (Heierhorst et al. 1994). The effectiveness of exogenous calmodulin appears to depend on the combined protein and Ca²⁺ concentration. This is also apparent for exogenous calmodulin-mediated inhibition of CNG channels (500 nM calmodulin with 100 nM Ca²⁺) (Bradley et al. 2004), NMDA receptors (100 nM calmodulin with 100 µM Ca²⁺) (Krupp et al. 1999), and IP₃ receptors (20 µM calmodulin with 200 µM Ca²⁺) (Michikawa et al. 1999), as well as enhancement of non-neuronal TRPM4b (10 µM calmodulin with 100 µM Ca²⁺) (Nilius et al. 2005) and TRPM5 (10 µM calmodulin with 5–7 µM Ca²⁺) (Ordaz et al. 2005). Interestingly, when any of the TRPM2, TRPM4b, TRPV5, or TRPV6 cation channels were coexpressed with a calmodulin mutant whose Ca²⁺-binding sites are impaired, the current was decreased (Lambers et al. 2004; Nilius et al. 2005; Tong et al. 2006). For both the bag cell neurone cation channel and others, exogenous calmodulin may displace endogenous calmodulin and/or bind to unoccupied calmodulin-binding sites.

In the bag cell neurones, intracellular Ca²⁺ rises rapidly upon synaptic stimulation, possibly due to release from intracellular stores (Fink et al. 1988). When the afterdischarge begins, there is a second elevation due to activation of voltage-gated Ca²⁺ channels (Fisher et al. 1994). We propose that intracellular Ca²⁺ binds to cation channel-associated calmodulin to promote channel activation and the afterdischarge. Calmodulin appears to be one of several, closely associated regulatory proteins that are in a complex with the cation channel. In addition to Ca²⁺ activation, the P_o of the cation channel is also increased by closely associated protein kinase C (PKC) (Wilson et al. 1998; Magoski et al. 2002) or decreased by closely associated protein kinase A (PKA) (Magoski, 2004). These kinases can reconfigure depending on bag cell neurone excitability or activity levels. During the afterdischarge, stimulatory PKC is channel associated, while inhibitory PKA is present through the refractory period (Magoski & Kaczmarek, 2005). Calmodulin, which presumably is always present, would allow Ca²⁺ to influence cation channel activity in a graded fashion during the afterdischarge, with PKC maintaining spiking, and PKA then contributing to refractoriness (Fig. 8). Overall, this complex of regulatory proteins determines cation channel function and fundamentally affects species propagation.

View larger version (12K): [in this window] [in a new window]   Figure 8.  A complex of interchanging proteins regulates the cation channel during the afterdischarge A, at rest, calmodulin (CaM) is associated with the cation channel, but activity is minimal due in the presence of low intracellular Ca2+. Channel Po is indicated by the width of the ‘pore’ in the illustration. B, following synaptic stimulation, an increase in intracellular Ca2+ activates the cation channel over the short term, as the afterdischarge begins (Wilson et al. 1996; present study). C, later in the afterdischarge, Ca2+ continues to activate the cation channel along with maintained upregulation provided by the simultaneous association of stimulatory PKC (Wilson et al. 1998; Magoski et al. 2002). D, upon termination of the afterdischarge, intracellular Ca2+ falls with the cessation of action potential firing. Either as a consequence of refractoriness, or as a mechanism contributing to refractoriness, inhibitory PKA is exchanged with PKC to downregulate the cation channel (Magoski, 2004; Magoski & Kaczmarek, 2005). During the refractory period, cation channel activity and bag cell neurone excitability are maintained at lowered levels, thus preventing the generation of additional afterdischarges which could disrupt ongoing egg-laying behaviour.

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