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Localized IP₃-Evoked Ca²⁺ Release Activates a K⁺ Current in Primary Vagal Sensory Neurons

¹Medical Biotechnology Center, University of Maryland Biotechnology Institute; and Departments of ²Physiology and ³Pharmacology and Experimental Therapeutics, University of Maryland School of Medicine, Baltimore, Maryland 21201

Submitted 20 October 2003; accepted in final form 4 December 2003

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Electrophysiological and microfluorimetric techniques were used to determine whether intracellular photorelease of caged IP₃, and the consequent release of Ca²⁺, could trigger a Ca²⁺-activated K⁺ current (I_IP3). Photorelease of caged IP₃ evoked an I_IP3 that averaged 2.36 ± 0.35 (SE) pA/pF in 24 of 28 rabbit primary vagal sensory neurons (nodose ganglion neurons, NGNs) voltage-clamped at –50 mV. I_IP3 was abolished by intracellular BAPTA (2 mM), a Ca²⁺ chelator. Changing the K⁺ equilibrium potential by increasing extracellular K⁺ ion concentration caused a predicted Nernstian shift in the reversal potential of I_IP3. These results indicated that I_IP3 was a Ca²⁺-dependent K⁺ current. I_IP3 was unaffected by three common antagonists of Ca²⁺-activated K⁺ currents: bath-applied iberiotoxin (50 nM) or apamin (100 nM), and intracellular 8-Br-cAMP (100 µM) included in the patch pipette. We have previously demonstrated that both IP₃-evoked Ca²⁺ release and Ca²⁺-induced Ca²⁺ release (CICR) are co-expressed in NGNs and that CICR can trigger a Ca²⁺-activated K⁺ current. In the present study, using caffeine, a CICR agonist, to selectively attenuate intracellular Ca²⁺ stores, we showed that IP₃-evoked Ca²⁺ release occurs independently of CICR, but interestingly, that a component of I_IP3 requires CICR. These data suggest that IP₃-evoked Ca²⁺ release activates a K⁺ current that is pharmacologically distinct from other Ca²⁺-activated K⁺ currents in NGNs. We describe several models that explain our results based on Ca²⁺ signaling microdomains in NGNs.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cytosolic Ca²⁺ ions are ubiquitous second messengers that regulate a broad spectrum of cellular phenomena, including membrane excitability, ion channel gating (Hille 2001), gene expression (Bito et al. 1996), neurotransmitter release (Katz and Miledi 1968), muscle contraction (Fabiato and Fabiato 1975), and secretion of hormones (Curry et al. 1968) and digestive juices (Petersen 1992). Increases in cytosolic-free Ca²⁺ ion concentration ([Ca²⁺]_i) can result from Ca²⁺ influx through the plasma membrane, Ca²⁺ release from intracellular Ca²⁺ stores through intracellular Ca²⁺ release channels, or alterations in Ca²⁺ ATPase activity or Na⁺/Ca²⁺ exchange.

Two types of intracellular Ca²⁺ release channels exist: ryanodine receptor (RyR) channels, and d-myo-inositol 1,4,5-trisphosphate receptor (IP₃R) channels (Berridge 1998). While these channels both mediate Ca²⁺ release from intracellular stores, they differ in their mechanisms of activation. Ca²⁺ release via RyRs is activated by increases in [Ca²⁺]_i (Ca²⁺-induced Ca²⁺ release, CICR). Typically, Ca²⁺ ions that trigger CICR arise from Ca²⁺ influx through plasma membrane voltage- or ligand-gated channels (for review of CICR in neurons, see Verkhratsky and Shmigol 1996). In contrast, Ca²⁺ release through IP₃Rs is activated by increases in [IP₃] (IP₃-induced Ca²⁺ release). IP₃ is normally generated through cleavage of phosphoinositide lipids by phospholipase C (PLC) coupled to cell-surface receptors (Berridge 1993).

One important role of intracellular Ca²⁺ ions is the regulation of Ca²⁺-activated K⁺ currents (I_K(Ca)s) that can influence both action potential shape and the pattern of action potential firing. Such I_K(Ca)s, which are triggered as a consequence of action potentials, fall into at least three temporally distinct classes: I_C, a fast current lasting 5–20 ms; I_AHP, a current with intermediate kinetics, lasting hundreds of milliseconds; and I_sAHP, a slow current lasting many hundreds to thousands of milliseconds. These currents can be further distinguished based on their disparate physiological, pharmacological, and biophysical properties (Sah 1996). I_C underlies the fast afterhyperpolarization (fAHP) following an action potential, is activated by both membrane depolarizations and increases in [Ca²⁺]_i, and is inhibited by tetraethylammonium (TEA) ions (<1 mM), iberiotoxin, and charybdotoxin (Adams et al. 1982; Shao et al. 1999). I_C is believed to be mediated by large-conductance (100–200 pS) Ca²⁺-activated K⁺ channels (BK channels) (Sah and Louise Faber 2002; Shao et al. 1999). I_AHP underlies the medium AHP (mAHP), is activated by increases in [Ca²⁺]_i, is voltage independent, and is inhibited by TEA ions (10–20 mM) and apamin (50–200 nM) (Pennefather et al. 1985). Small-conductance (5–15 pS) Ca²⁺-activated K⁺ channels (SK channels), which are voltage insensitive and some of which are apamin-sensitive, are believed to mediate I_AHP (Sah and Louise Faber 2002). I_sAHP underlies the slow afterhyperpolarization evoked by action potentials in primary vagal afferents. It is activated by increases in [Ca²⁺]_i, is voltage independent, and is inhibited by elevations in intracellular [cAMP] (Cordoba-Rodriguez et al. 1999). The molecular identity of the channels that mediate I_sAHP remains undefined (Sah and Louise Faber 2002).

In a population of primary vagal sensory neurons (nodose ganglion neurons, NGNs), action potential-triggered CICR activates an I_sAHP that controls spike frequency adaptation (Moore et al. 1998; Weinreich and Wonderlin 1987). We recently reported that photoreleased IP₃ evokes intracellular Ca²⁺ release in NGNs (Hoesch et al. 2002). In the present study, we ask whether IP₃-induced Ca²⁺ release, like CICR, could also activate membrane currents in NGNs. Our results show that IP₃-evoked intracellular Ca²⁺ release can activate a K⁺ current, I_IP3, with properties distinct from those of the I_sAHP.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Cell dissociation

Male New Zealand White rabbits, weighing 3–4 kg, were purchased from Robinson Services (Clemmons, NC) and killed by pentobarbital sodium overdose (100 mg/kg) as approved by the Institutional Animal Care and Use Committee of the University of Maryland Biotechnology Institute. Dissociated NGNs were prepared as described previously (Leal-Cardoso et al. 1993) with the exception that sterile technique was used and the final neuronal pellet was resuspended in Leibovitz L-15 medium (Gibco-BRL, Grand Island, NY) containing 10% fetal bovine serum (FBS, JRH Bioscience, Lenexa, KS), and 0.1% by volume penicillin-streptomycin (10,000 U penicillin G and 10,000 µg/ml streptomycin sulfate; Gibco-BRL, Grand Island, NY). The resulting cell suspension was then plated in 0.2-ml aliquots onto 25-mm round No. 1 glass coverslips (Fisher Scientific, Newark, DE) coated with poly-D-lysine (0.1 mg/ml; molecular wt: 30–70 kDa, Sigma, St. Louis, MO). NGNs were incubated at 37°C for 12 h, then maintained at room temperature to prevent neurite outgrowth, and used for experiments for 48 h.

[Ca²⁺]_i measurements and calibration

Fluo-3 indicator was used to measure [Ca²⁺]_i. NGNs were loaded with 1 µM fluo-3/AM (Teflabs, Austin, TX) in L-15 medium for 1–3 h. Single-cell microfluorimetry was performed as previously described (Cohen et al. 1997), except that excitation was at 490 nm and that the fluorescence emission was passed through a 530-nm band-pass filter before photometric quantitation. Fluo-3 fluorescence intensity records were corrected by subtracting the background fluorescence intensity, measured after cell lysis with digitonin (20 µM). Ca²⁺ transient amplitudes are reported as changes in fluorescence intensity (F) or changes in fluorescence intensity normalized to baseline fluorescence intensity immediately preceding the transient (F/F₀).

Extracellular solutions

Neurons were superfused with physiological saline solution (20–24°C) that contained (in mM) 120 NaCl, 3.0 KCl, 1.0 NaH₂PO₄, 25.0 NaHCO₃, 1.5 MgCl₂, 2.2 CaCl₂, and 10.0 dextrose, equilibrated with 95% O₂-5% CO₂; pH adjusted to 7.4. For photorelease experiments where it was important to eliminate any potential Ca²⁺ influx, nominally Ca²⁺-free medium, from which CaCl₂ was omitted, was used. For experiments that examined the effects of shifting E_K, NGNs were superfused with saline solution with elevated [K⁺] (high-K⁺ medium) that contained (in mM) 100 NaCl, 23.0 KCl, 1.0 NaH₂PO₄, 25.0 NaHCO₃, 1.5 MgCl₂, 2.2 CaCl₂, and 10.0 dextrose, equilibrated with 95% O₂-5% CO₂; pH adjusted to 7.4. In high-K⁺ medium, [Cl^-]_o and E_Cl are unchanged.

A recording chamber with a narrow rectangular flow path allowed superfusion of NGNs on a glass coverslip at 7 ml/min via a gravity flow system. The chamber was mounted on an inverted microscope (Diaphot, Nikon, Melville, NY) equipped with a x40 phase-contrast oil-immersion objective (Fluor, N.A. 1.3, Nikon) to allow fluorescence measurements and direct visualization of NGNs for positioning patch pipettes. In experiments where Ca²⁺-free solution was used, nominally Ca²⁺-free physiological saline was superfused for 30 s before and after drug application. Solution changes were complete in 14 s as determined with fluorescent tracers.

Electrophysiological measurements

INTRACELLULAR SOLUTIONS. Control patch pipette solutions contained (in mM) 152 KCH₃SO₃, 10.0 HEPES, 2.0 MgCl₂, 1.0 Na₃ATP, 1.0 Na₃GTP, and 1.0 KCl; pH adjusted with KOH to 7.2. KCH₃SO₃ was used to avoid excess intracellular Cl^-, which is known to inhibit G proteins (Lenz et al. 1997). Aliquots of stock pipette solution were stored frozen at -20°C. Each aliquot of pipette solution was thawed, stored on ice, and used for only one day. K₅Fluo-3 was added to the pipette solution to a final concentration of 50 µM; sufficient CaCl₂ was added to set free [Ca²⁺] = 100 nM (taking the Ca²⁺ dissociation constant of fluo-3 under physiological conditions to be 400 nM (Minta et al. 1989)). For photolysis experiments, 0.5 mM of the trisodium salt of d-myo-inositol 1,4,5-trisphosphate P⁴⁽⁵⁾-1-(2-nitrophenyl)ethyl ester (caged IP₃) was included in the pipette solution, which was loaded only into the tip of the pipette. The shaft of the pipette was filled with pipette solution containing no caged IP₃. For BAPTA experiments, NGNs were loaded with 20 µM BAPTA/AM (Molecular Probes, Eugene, OR) in L-15 medium containing 10% vol/vol FBS and 0.0075% wt/vol of the surfactant, Pluronic F-127 (BASF Wyandotte, Wandotte, WI) for 1–2 h. The patch-pipette solutions contained (in mM) 146.4 KCH₃SO₃, 10.0 HEPES, 2.0 MgCl₂, 1.0 Na₃ATP, 1.0 Na₃GTP, 1.0 KCl, and 2.0 K₄BAPTA; pH adjusted with KOH to 7.2. Sufficient CaCl₂ was added to set free [Ca²⁺] 75 nM (taking the Ca²⁺ dissociation constant of BAPTA under physiological conditions to be 190 nM (Tsien 1980)). Caged IP₃, 8-Br-cAMP, K₄BAPTA, and K₅Fluo-3 were delivered via patch pipette and allowed to equilibrate for 5 min before start of experiments.

PATCH-CLAMP RECORDING. The whole cell configuration of the patch clamp technique (Hamill et al. 1981) was used to measure membrane currents. Patch pipettes (2–3 M), fabricated from boro-silicate glass stock (1.5 mm OD, 1.12 mM ID, World Precision Instruments, Sarasota, FL) on a Flaming-Brown P97 micropipette puller (Sutter Instruments, Novato, CA) were connected to an Axopatch 200B amplifier (Axon Instruments, Union City, CA). Data acquisition through the Digidata 1200 interface was controlled with pClamp 8 software (Axon Instruments). NGNs were first loaded with fluo-3/AM to allow fluorimetric measurement of [Ca²⁺]_i in parallel with electrophysiological recording. After a gigaohm seal (>1.0 G) was formed, the whole cell configuration was established, with neurons voltage-clamped to -50 mV. Membrane input resistance and capacitance were determined from current transients elicited by 5-mV depolarizing voltage steps from the holding potential. NGNs were considered suitable for study if membrane input resistance was >150 M and holding current was <200 pA.

RAMP PROTOCOLS. To determine I-V relations, the following voltage command protocol was applied to voltage-clamped NGNs: 1) from a holding potential of –50 mV, the membrane potential was stepped to +50 mV for 100 ms to inactivate voltage-gated Na⁺ channels; 2) an I-V relation was then generated by a voltage ramp that decreased from +50 to –110 mV at 1 mV/ms; and 3) at the end of the ramp, the cell was returned to a holding potential of –50 mV. This protocol was applied twice to each NGN tested: first immediately before IP₃ photorelease and again 2 s after IP₃ photorelease, when the IP₃-evoked current had developed substantially (a representative time course of the current is shown in Fig. 1). Taking the difference between these two I-V relations yielded the I-V relation for the IP₃-evoked current.

View larger version (15K): [in this window] [in a new window]   FIG. 1. Inositol 1,4,5-trisphosphate (IP3) photorelease evokes a Ca2+ transient and an outward current simultaneously in the same nodose ganglion neuron (NGN). Trace 1: Ca2+ transient evoked by IP3 photorelease. Trace 2: outward current recorded simultaneously in the same NGN. The NGN was loaded with 500 µM caged IP3 and 50 µM fluo-3 via whole cell patch pipette and voltage-clamped at –50 mV. , the start of the 500-ms UV laser pulse. · · ·, the interval during which the UV flash precluded photometry. Changes in fluo-3 fluorescence intensity relative to baseline (F/F0) are plotted. Fluorimetric and electrophysiological data were acquired at 4 Hz.

To photolyze caged IP₃, we delivered 500-ms flashes of ultraviolet (UV) light to NGNs loaded with 0.5 mM caged IP₃. The multiline UV output (333.6–363.8 nm) of an argon ion laser (BeamLok 2065–7S, Spectra-Physics, Mountain View, CA) was used for photolysis. The output beam of the laser was directed through an objective lens (U-27X, Newport, Irvine, CA) and focused onto the 50-µm-diam silica core of a step-index multimode optical fiber (CeramOptec, East Longmeadow, MA) that was cleaved orthogonally to expose an optically flat surface. For adjusting alignment, the objective lens and the chuck holding the optical fiber were both mounted on a multimode fiber coupler assembly (F-915T, Newport). The output end of the optical fiber was ensheathed in a glass pipette, which was mounted on a hydraulic micromanipulator to allow the output of the fiber to be directed onto cells being viewed under the microscope. The duration of UV flashes was regulated by a laser shutter (LS200F, NM Laser Products, Sunnyvale, CA) interposed between the laser head and the UV objective lens of the coupler. Shutter gating was controlled by TTL signals through pClamp 8 software (Axon Instruments, Union City, CA).

Data analysis

Unless otherwise stated, the following conventions apply: numerical results are reported as a mean ± SE; when multiple responses were elicited from a NGN, the response amplitude under a given experimental condition was normalized to the control response amplitude; and Student's t-test (2-tailed) was used to assess significant differences between calculated means and P < 0.05 was considered significant. Origin software (Microcal Software, Northampton, MA) was used for all data analysis and least-squares curve fitting.

Reagents

Reagents were obtained from the following sources: caged IP₃, 8-Br-cAMP, heparin sulfate (13.5–15 kDa), and ryanodine from Calbiochem (La Jolla, CA); acetoxymethyl ester and pentapotassium salt of fluo-3 (fluo-3/AM and K₅Fluo-3) from Teflabs; caffeine and forskolin from Sigma; iberiotoxin and apamin from Tocris (Ellisville, MO); acetoxymethyl ester and tetrapotassium salt of BAPTA (BAPTA/AM and K₄BAPTA) from Molecular Probes. Inorganic salts were from VWR (Piscataway, NJ).

Reagent solutions were prepared daily from concentrated stock solutions in dimethylsulfoxide (Fisher Biotech, Fair Lawn, NJ) or water that were stored frozen. Unless otherwise noted, drugs were delivered via the superfusate by switching a three-way valve to a reservoir containing a known concentration of the drug in the extracellular solution.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We have previously reported (Hoesch et al. 2002) that intracellular photorelease of IP₃ evokes Ca²⁺ release from intracellular stores in primary vagal sensory neurons (NGNs). Because these neurons are known to express a variety of Ca²⁺-activated currents (Christian et al. 1994; Fowler et al. 1985; Hay and Kunze 1994b; Higashi et al. 1984; Lancaster et al. 2002; Schild et al. 1994; Weinreich and Wonderlin 1987), we asked whether IP₃-evoked Ca²⁺ release could also activate Ca²⁺-dependent membrane currents. To address this question, we loaded NGNs with caged IP₃ and fluo-3 via patch pipette and measured whole cell current and [Ca²⁺]_i simultaneously at a holding potential of -50 mV. As shown in Fig. 1, IP₃ photorelease triggered both a transient rise in [Ca²⁺]_i, i.e., a Ca²⁺ transient (trace 1), and an outward current (trace 2). During photorelease episodes, NGNs were superfused with nominally Ca²⁺-free medium to prevent Ca²⁺ influx. In 24 of 28 NGNs (86%) tested, IP₃ photorelease evoked an outward current (I_IP3; Fig. 1, trace 2) that averaged 108.1 ± 16.2 pA, or 2.36 ± 0.35 pA/pF when normalized to membrane capacitance. I_IP3 decayed with a t_1/2 of 13.9 ± 3.0 s (n = 14). In four control NGNs loaded via patch pipette with fluo-3, but no caged IP₃, UV pulses did not evoke detectable currents or Ca²⁺ transients. Similarly, in 10 control NGNs that were loaded with fluo-3/AM only and not patched, UV pulses never evoked Ca²⁺ transients. These control experiments indicate that the photorelease-evoked Ca²⁺ transients and currents were activated by IP₃ and not by UV light alone. Intracellular application of heparin (13.5–15 kDa, 1 mg/ml), a selective IP₃R antagonist (Ehrlich et al. 1994), completely inhibited both I_IP3 and the Ca²⁺ transient evoked by IP₃ photorelease (n = 3; data not shown). Thus functional IP₃Rs are required for both I_IP3 and the IP₃-evoked Ca²⁺ transient (see also Hoesch et al. 2002).

Because IP₃ photorelease activated a Ca²⁺ transient and an outward I_IP3 in parallel, I_IP3 could be a current (IK(Ca)). To determine if I_IP3 Ca²⁺-activated K⁺ was a K⁺ current, we tested whether its reversal potential (E_rev) showed a Nernstian dependence on extracellular [K⁺]. Ramp voltage commands were used to generate I-V relations, as described in METHODS. Trace A in Fig. 2 is the averaged I-V relation of I_IP3 determined in 24 NGNs in normal physiological saline containing 3 mM K⁺. The I-V relation showed slight outward rectification and E_rev = –90.4 ± 2.3 mV. The proximity of E_rev to E_K (–100.0 mV) suggested that I_IP3 may be principally a K⁺ current. To further test the role of K⁺ ions as the predominant charge carrier of I_IP3, we examined the effects of changing E_K on the E_rev of I_IP3. Superfusing NGNs with high-K⁺ medium containing 23 mM K⁺ shifted E_K to –48.5 mV without changing E_Cl (see METHODS). Trace B in Fig. 2 is the averaged I-V relation for I_IP3 in high-K⁺ medium for four NGNs with E_rev = –40.8 ± 1.6 mV. Thus raising [K⁺]_o from 3 to 23 mM shifted the E_rev of I_IP3 by 49.6 mV, close to the value expected for ideal Nernstian behavior (51.5 mV). Under the ionic conditions used, Cl^- was the only other ion that might have carried an outward current at the holding potential of –50 mV (E_Cl = -82.5 mV). This possibility was ruled out because while E_Cl was kept constant, a large positive shift in E_K caused a corresponding shift in E_rev (Fig. 2, trace B). Therefore our results indicate that I_IP3 is principally a K⁺ current. Most ion channels have imperfect selectivity, and K⁺ channels typically exhibit detectable Na⁺ permeability. From the values of E_K, E_Na, and E_rev in normal and high-K⁺ media, the relative permeability of Na⁺ to K⁺ for I_IP3 was estimated, using the GHK equation, to be P_Na/P_K = 0.037 ± 0.028.

View larger version (17K): [in this window] [in a new window]   FIG. 2. I-V relations for IIP3. Trace A: averaged, control I-V relation for IP3-evoked current (IIP3) recorded from 24 NGNs in normal medium ([K+]o = 3 mM); the trace shows slight outward rectification. Mean reversal potential, Erev = -90.4 ± 2.3 mV. Trace B: averaged I-V relation for IIP3 recorded in high-K+ medium ([K+]o = 23 mM), with Erev = -40.8 ± 1.6 mV (n = 4). Compared with control, Erev for IIP3 in high-K+ medium was more positive by 49.6 mV, close to the 51.5 mV expected for ideal Nernstian behavior. Trace C: averaged I-V relation for IIP3 recorded in the presence of intracellular BAPTA. Four NGNs were loaded via patch pipette with 2 mM BAPTA to buffer [Ca2+]i. The flat I-V relation indicates that IIP3 was blocked by BAPTA and thus dependent on an elevation in [Ca2+]i. NGNs were loaded with 500 µM caged IP3 and 50 µM fluo-3 via patch pipette for >5 min. Photorelease experiments were performed in nominally Ca2+-free external medium. The horizontal dotted line marks the 0-current level. Vertical dotted lines mark the reversal potentials of IIP3 in control (Erev) and in high-K+ medium (Erev, Hi-K+). I-V relations were generated by ramp protocols as described in METHODS. For clarity, I-V relations are shown from -30 to -100 mV.

Because I_IP3 is dependent on an elevation of [Ca²⁺]_i, we asked if there was a correlation between the amplitudes of I_IP3 and the activating Ca²⁺ transient. Figure 3 is a plot of I_IP3 (normalized to membrane capacitance) versus peak Ca²⁺ transient amplitude (normalized to baseline fluorescence intensity measured immediately preceding photorelease), for 24 NGNs that expressed I_IP3. Linear regression of the data yielded a correlation coefficient of R = 0.0267, indicating essentially no correlation between the amplitude of the IP₃-evoked Ca²⁺ release and the amplitude of I_IP3. Thus I_IP3 appears insensitive to the magnitude of the global, whole cell Ca²⁺ signal. One possible interpretation of this result is that whereas all IP₃Rs can contribute to the global Ca²⁺ transient, only relatively few IP₃Rs are closely apposed to the plasma membrane and only Ca²⁺ release from such closely apposed IP₃Rs can trigger I_IP3. Such a spatial arrangement constitutes a Ca²⁺ signaling "microdomain" (e.g., Delmas et al. 2002). Because of this arrangement, the K⁺ channels mediating I_IP3 are optimally positioned for activation by localized Ca²⁺ release within the microdomain, even though such localized release may not contribute significantly to the measured global (whole cell) Ca²⁺ transients.

View larger version (9K): [in this window] [in a new window]   FIG. 3. IIP3 and Ca2+ transient amplitudes are poorly correlated. For each NGN, the amplitude of IIP3 was normalized to membrane capacitance, and then plotted against the amplitude of the IP3-evoked Ca2+ transient. Linear regression (—) yielded a correlation coefficient of R = 0.027, indicating that the 2 variables are poorly correlated.

View larger version (15K): [in this window] [in a new window]   FIG. 4. Time course of IIP3 onset. A: averaged IIP3 for 12 NGNs evoked by a 500-ms UV flash. UV flash duration is indicated by the bar below the trace. The part of the trace enclosed in the rectangular box corresponds to the start of photorelease and is shown at higher time resolution in B. B: expanded portion of the trace in A corresponding to the start of photorelease. Solid curve is an exponential fit to the data points. The latency between IP3 photorelease and onset of IIP3 is <50 ms. The horizontal dashed line marks the 0-current level. All NGNs were loaded with 500 µM caged IP3 and 50 µM fluo-3 via patch pipette for >5 min. Data were acquired at 4 kHz.

We have previously reported that NGNs exhibit robust CICR (Cohen et al. 1997; Cordoba-Rodriguez et al. 1999; Hoesch et al. 2001). Because IP₃-evoked Ca²⁺ release may, in turn, trigger CICR, we asked if CICR contributes to the activation of I_IP3. To address this question, caffeine (Caf), a CICR agonist, was used to selectively deplete Caf-releasable intracellular Ca²⁺ stores on which CICR depends (Trafford et al. 1998, 2001). The trace in Fig. 5A shows a [Ca²⁺]_i record from a NGN, to which six 10-s pulses of 10 mM Caf were applied at 15-s intervals. This "Caf-pulse series" was followed by a 10-s "test" Caf pulse, which was delivered 75 s after completion of the Caf-pulse series. The six Caf pulses evoked progressively smaller Ca²⁺ responses, and the test Caf pulse was markedly diminished relative to the first Caf-evoked response. Similar profiles of responses to caffeine were observed in four other NGNs. The results from all five NGNs are summarized in Fig. 5B, which shows the average response to each Caf pulse. In each NGN, all responses were normalized to the first response. Each point in Fig. 5B is the average of responses evoked in two intact NGNs and in three NGNs patch-clamped at –50 mV; the responses from these two groups were not significantly different. These results show that the Caf-pulse series effectively diminishes the CICR store, and thus attenuates subsequent CICR to 49 ± 5.5% of control.

View larger version (11K): [in this window] [in a new window]   FIG. 5. Repetitive caffeine pulses diminish caffeine-releasable intracellular Ca2+ stores. A: 6 10-s pulses of 10 mM caffeine (Caf) were delivered at 15-s intervals (, the start of a Caf pulse). After 75 s, a 10-s "test" pulse of 10 mM Caf was applied. Compared with the response to the 1st Caf pulse, the response to the "test" Caf pulse was significantly attenuated. Fluo-3 was used to monitor changes in [Ca2+]i. B: average attenuation of the caffeine-releasable Ca2+ store by a series of Caf pulses (following the protocol shown in A). For each of 5 NGNs, every response to Caf was normalized to the 1st response. The normalized responses from individual cells to each Caf pulse were then averaged. These data show that the Caf pulse series attenuates the test Caf response by 50% relative to the 1st (control) response. Data recorded from intact (n = 2) and patch-clamped (n = 3) NGNs were not significantly different and were grouped in this study. Error bars are ± SE.

View larger version (22K): [in this window] [in a new window]   FIG. 6. Depletion of CICR Ca2+ stores by repetitive caffeine pulses does not attenuate Ca2+ transients evoked by IP3 photorelease, but does attenuate IIP3. A: 2 pairs of Ca2+ transients evoked by photoreleasing IP3 300 s apart in 2 separate NGNs. In the 1st NGN (left), there was no pharmacological manipulation during the 300-s interval between the 2 photorelease events (labeled 1 and 2; , time of photorelease). In the 2nd NGN (right), 90 s after the 1st IP3 photorelease (labeled 1), a "Caf-pulse series" was used to attenuate the CICR Ca2 stores before the 2nd IP3 photorelease. A Caf-pulse series consisted of 6 10-s pulses of 10 mM caffeine delivered at 15-s intervals. Seventy-five seconds after the last Caf pulse, a 2nd Ca2+ transient (labeled 2) was evoked by photoreleasing IP3. In these 2 experiments, the ratio of Ca2+ transient amplitude 2 to amplitude 1 was 1.02 in the untreated NGN and 1.16 in the NGN that received the Caf-pulse series. Therefore diminishing the CICR Ca2+ stores does not attenuate the IP3-evoked Ca2+ transients. NGNs were loaded with 500 µM caged IP3 and 50 µM fluo-3 via whole cell patch pipette >5 min before the 1st photorelease and voltage-clamped at –50 mV. , the start of a 500-ms UV laser pulse. - - -, the interval during which the UV flash precluded photometry. IP3 photorelease was performed during superfusion of nominally Ca2+-free medium. Changes in fluo-3 fluorescence intensity (F) are plotted. B: 2 pairs of photorelease-evoked IIP3s recorded simultaneously with the Ca2+ transients shown in A. Left: 2 IIP3s evoked 300 s apart (labeled 1 and 2) in the untreated NGN; right: IIP3s triggered in the 2nd NGN before (1) and after (2) the Caf-pulse series to reduce CICR Ca2+ stores. In these 2 experiments, the ratio of IIP3 amplitude 2 to amplitude 1 was 1.09 in the untreated NGN and 0.70 in the NGN that received Caf-pulse series. Therefore diminishing the CICR Ca2+ stores by applying the Caf-pulse series markedly attenuated the 2nd IP3-evoked current response relative to the 1st.

Although CICR did not contribute significantly to global IP₃-evoked Ca²⁺ transients, it was conceivable that Ca²⁺ release via IP₃Rs could trigger CICR via nearby RyRs within a Ca²⁺ signaling microdomain. Such localized CICR would not contribute significantly to global Ca²⁺ transients, but it could participate in the activation of I_IP3. To test this possibility, we examined the effects of a Caf-pulse series (see Fig. 5) on the magnitude of I_IP3. Figure 6B shows the two pairs of photorelease-evoked I_IP3s recorded simultaneously with the Ca²⁺ transients shown in Fig. 6A. Figure 6B, left, shows two I_IP3s evoked 300 s apart (labeled 1 and 2) in the untreated NGN, while the right panel shows I_IP3s triggered in the second NGN before (1) and after (2) the Caf-pulse series to reduce CICR Ca²⁺ stores. Comparing the amplitudes of the second (test) I_IP3 to the first (control) I_IP3 shows that the Caf-pulse series markedly attenuated the second IP₃-evoked current response relative to the first. In these two neurons, the test-to-control I_IP3 amplitude ratios were 1.09 and 0.70, respectively. In six NGNs treated with the Caf-pulse series, the ratio of the test to control I_IP3 amplitudes averaged 0.77 ± 0.09, which was significantly smaller (by 35%) than the average ratio (1.18 ± 0.13) recorded in 10 control NGNs that were not subjected to the Caf-pulse series. These results suggest that CICR can contribute to I_IP3 activation. We further tested whether random fluctuation might have accounted for the difference in the test-to-control ratios for the IP₃-evoked Ca²⁺ and current responses (1.21 ± 0.06 and 0.77 ± 0.09, respectively). The difference proved statistically significant (P = 0.00152) and therefore not attributable to random fluctuation. The observation that CICR participates in activating I_IP3, and yet does not contribute significantly to the global IP₃-evoked Ca²⁺ transient (Fig. 6), further suggests the existence of Ca²⁺ signaling microdomains. That is, Ca²⁺ release via IP₃Rs could trigger CICR via nearby RyRs; together, Ca²⁺ released from neighboring IP₃Rs and RyRs could activate K⁺ channels residing within the same Ca²⁺ signaling microdomain.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
Our major findings in rabbit primary vagal sensory neurons (NGNs) are the following: IP₃-evoked Ca²⁺ release activates a K⁺ current (I_IP3); I_IP3 is insensitive to apamin, iberiotoxin and 8-Br-cAMP, three common antagonists of I_K(Ca)s; and Ca²⁺-induced Ca²⁺ release (CICR) consequent to IP₃-evoked Ca²⁺ release makes a small, but significant, contribution to the activation of I_IP3.

In the present study, we found that I_IP3 is activated by IP₃-evoked Ca²⁺ release in 86% of rabbit NGNs, develops with little or no latency following IP₃ photorelease (<50 ms), has a minor dependence on CICR, and is insensitive to apamin, iberiotoxin and 8-Br-cAMP. In our previous studies, we have found that a subpopulation of rabbit NGNs (35%) express a Ca²⁺-activated K⁺ current that underlies the slow afterhyperpolarization following action potentials (I_sAHP) (Cohen et al. 1997; Cordoba-Rodriguez et al. 1999; Moore et al. 1998). Activation of the I_sAHP is absolutely dependent on CICR consequent to action-potential-induced Ca²⁺ influx through N-type channels and exhibits postspike latency to onset of 100 ms (Cohen et al. 1997; Cordoba-Rodriguez et al. 1999; Moore et al. 1998). We have shown that I_sAHP is insensitive to apamin and iberiotoxin but is completely blocked by treatments that elevate intracellular cAMP (Cordoba-Rodriguez et al. 1999). Contrasting the preceding observations suggests that I_IP3 is distinct from I_sAHP. We have no evidence that different channels underlie these two macroscopic currents. Therefore it is possible that a single-channel protein mediates both currents and that observed differences in pharmacology and kinetics reflect differences in regulation. Interestingly, Ca²⁺-induced Ca²⁺ release and IP₃-induced Ca²⁺ release both give rise to global (whole cell) Ca²⁺ transients in NGNs and yet activate functionally distinguishable Ca²⁺-dependent K⁺ currents. This suggests the existence of Ca²⁺ signaling microdomains in NGNs (see also Cordoba-Rodriguez et al. 1999).

Our data suggest several possible models for the activation of I_IP3, some of which are illustrated in Fig. 7. In the first model (Fig. 7A), Ca²⁺ is released via IP₃Rs and RyRs from separate intracellular Ca²⁺ stores. Ca²⁺ released via IP₃Rs induces consequent CICR via closely neighboring RyRs. Ca²⁺ released through both of these channels activates closely apposed I_IP3 channels in the plasma membrane. Such a tight spatial organization of channels would constitute a Ca²⁺ signaling microdomain. This model fits well with two key observations. First, that global IP₃-evoked Ca²⁺ release was not measurably affected by depletion of CICR stores (Fig. 6) implies that IP₃Rs and RyRs likely mediate Ca²⁺ release from separate intracellular Ca²⁺ stores. Second, that depletion of CICR stores attenuated I_IP3 without affecting global IP₃-evoked Ca²⁺ release suggests that IP₃-evoked Ca²⁺ release can trigger localized CICR to augment I_IP3 activation.

View larger version (17K): [in this window] [in a new window]   FIG. 7. Microdomain models of IIP3 activation by intracellular Ca2+ release. A: microdomain containing IIP3 channels, and IP3Rs and RyRs on separate Ca2+ stores. In microdomains (delimited by - - -) near the plasma membrane, IP3-evoked Ca2+ release can trigger CICR via closely neighboring RyRs. IP3Rs and RyRs mediate Ca2+ release from separate Ca2+ stores. Ca2+ release through both IP3Rs and RyRs in the microdomain activates IIP3 channels in the plasma membrane. B: microdomain containing IIP3 channels and IP3Rs and RyRs on a common Ca2+ store. In microdomains (delimited by - - -), IP3-evoked Ca2+ release triggers CICR via closely-neighboring RyRs, and Ca2+ release through both IP3Rs and RyRs activates IIP3 channels in the plasma membrane. IP3Rs and RyRs mediate Ca2+ release from a common (shared) store in microdomains, but from separate stores elsewhere in the cell. C: microdomain containing IIP3channels and IP3Rs. Only Ca2+ released from IP3Rs in the microdomain (delimited by - - -) can activate IIP3 channels in the plasma membrane. Because RyRs are not contained within the microdomain, Ca2+ release through IP3Rs cannot trigger CICR through RyRs; nor can Ca2+ release via RyRs activate IIP3 channels. IP3Rs and RyRs share a common Ca2+ store near the plasma membrane but mediate Ca2+ release from separate stores elsewhere in the cell.

Intracellular Ca²⁺ release evoked by photoreleased IP₃ has also been reported to activate K⁺ currents in cerebellar Purkinje neurons (Khodakhah and Armstrong 1997) and in mid-brain dopamine neurons (Morikawa et al. 2000). The expression of I_IP3 by central and peripheral neurons raises the question of the nature of the physiological stimuli that trigger I_IP3. ATP, through P2Y receptor activation, can trigger IP₃ signaling in NGNs (Hoesch et al. 2002) as well as many other cell types (Dubyak and el-Moatassim 1993). Therefore ATP is a probable physiological stimulus to trigger I_IP3 in NGNs. Other signaling molecules are also likely to employ IP₃ signaling in NGNs and thus could activate I_IP3. In midbrain dopamine neurons, activation of both metabotropic glutamate and muscarinic acetylcholine receptors, which are known to act through IP₃-evoked Ca²⁺ signaling, both evoked outward currents (Fiorillo and Williams 1998, 2000), suggesting physiological roles for IP₃-evoked outward currents in those neurons (Morikawa et al. 2000). Metabotropic glutamate receptors in NGNs have been physiologically characterized (Hay and Kunze 1994a) and recently cloned (Hoang and Hay 2001). Therefore if activation of metabotropic glutamate receptors could trigger IP₃-evoked Ca²⁺ signaling, then glutamate might evoke I_IP3 in NGNs. The prevalence of metabotropic receptors and the ubiquity of the IP₃ signaling pathway favor the hypothesis that I_IP3 is a common and robust mechanism through which metabotropic receptor activation could control membrane excitability.

	ACKNOWLEDGMENTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION ACKNOWLEDGMENTS REFERENCES

 
We thank Dr. Michael Gold for helpful discussions and for reading an earlier draft of this manuscript.

GRANTS

This work was supported by National Institutes of Health Grants GM-56481 to J.P.Y. Kao and NS-22069 to D. Weinreich.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ The time interval from the start of photolysis to the release of Ca²⁺ via IP₃Rs was reported to be of the order of 8–14 ms (Carter and Ogden 1997). Therefore in our experiments, the minimum expected latency for I_IP3 onset would be 8–14 ms.

² In all NGNs, the second IP₃-evoked Ca²⁺ response was always somewhat larger than the first. The cause of this increase is not clear.

Address for reprint requests and other correspondence: J.P.Y. Kao, Medical Biotechnology Center, University of Maryland, 725 W. Lombard St., Baltimore, MD 21201 (E-mail: jkao{at}umaryland.edu).

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