Ca2+-activated Clminus current in cultured myenteric neurons from murine proximal colon

Sok Han Kang, Pieter Vanden Berghe, and Terence K. Smith

Department of Physiology and Cell Biology, University of Nevada School of Medicine, Reno, Nevada 89557


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Whole cell patch-clamp recordings were made from cultured myenteric neurons taken from murine proximal colon. The micropipette contained Cs+ to remove K+ currents. Depolarization elicited a slowly activating time-dependent outward current (Itdo), whereas repolarization was followed by a slowly deactivating tail current (Itail). Itdo and Itail were present in ~70% of neurons. We identified these currents as Cl- currents (ICl), because changing the transmembrane Cl- gradient altered the measured reversal potential (Erev) of both Itdo and Itail with that for Itail shifted close to the calculated Cl- equilibrium potential (ECl). ICl are Ca2+-activated Cl- current [ICl(Ca)] because they were Ca2+ dependent. ECl, which was measured from the Erev of ICl(Ca) using a gramicidin perforated patch, was -33 mV. This value is more positive than the resting membrane potential (-56.3 ± 2.7 mV), suggesting myenteric neurons accumulate intracellular Cl-. omega -Conotoxin GIVA [0.3 µM; N-type Ca2+ channel blocker] and niflumic acid [10 µM; known ICl(Ca) blocker], decreased the ICl(Ca). In conclusion, these neurons have ICl(Ca) that are activated by Ca2+ entry through N-type Ca2+ channels. These currents likely regulate postspike frequency adaptation.

myenteric neurons; chloride currents; cell culture; murine large intestine


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

THE PROXIMAL COLON RECEIVES the liquefied waste products of digestion and reabsorbs the remaining water and electrolytes. These functions are largely dependent upon the integrated activities of the enteric nervous system (ENS; see Ref. 46). In small mammals, the ENS consists of two ganglionated neural networks called the myenteric plexus, which is between the longitudinal and circular muscle layers, and the submucous plexus, which lies in the submucosa on the surface of the inner circular muscle layer. The neurons in the myenteric plexus largely regulate motility reflexes, whereas those in the submucous plexus regulate secretomotor reflexes. The myenteric plexus, which is studied here, contains a number of functionally different neurons that include sensory neurons, interneurons, and excitatory and inhibitory motor neurons supplying the longitudinal and circular muscle layers (31, 34, 47).

Intracellular microelectrode recordings from myenteric neurons in guinea pig small intestine have revealed two broad electrophysiological classes of myenteric neurons, S/type I and AH/type II neurons (23, 36). S neurons are uniaxonal, lack a prolonged slow afterhyperpolarization (AHP, up to 20 s), have prominent fast synaptic input (7, 23), and comprise both interneurons and motor neurons (44). AH neurons, which comprise ~25% of all neurons in the small intestine, are generally multipolar and named for their characteristic AHP (4-20 s) that follows action potential firing in these neurons (7, 21, 23, 36, 49, 50). Many AH neurons appear to be intrinsic primary afferent neurons (17). Ca2+-activated K+ channels underlie the AHP (22, 32) that involves the opening of small-to-intermediate conductance channels that are tetraethylammonium (TEA) and apamin resistant (55).

Myenteric neurons in the large intestine of a number of species, such as human (8), guinea pig (30, 31, 33, 43, 49, 58, 59), rat (9), and mouse (18), appear to be electrically more heterogeneous than those in the small intestine since they exhibit more diverse firing patterns in response to current injection. However, like those in the small intestine, AH neurons are characterized by a prolonged AHP (30, 31, 33, 43, 49, 58), are usually multipolar (30, 31, 33, 43, 49), and project to the mucosa (33, 43). Some ascending interneurons in the colon are also rapidly adapting and exhibit an intermediate AHP (30). However, in the mouse colon, the AH neuronal population (~8%) appears to be only about one-third that in the guinea pig small intestine (18).

Patch-clamp techniques have been used to study Na+, Ca2+, and K+ currents in myenteric neurons in guinea pig small intestine (3, 41, 48, 54, 61) and to a lesser extent in the large intestine (56, 57). However, to date, Cl- channels have not been characterized in myenteric neurons using patch-clamp studies. Indirect evidence, however, suggests that Cl- channels participate in slow synaptic transmission (6) and the depolarizing responses to exogenous gamma -aminobutyric acid (GABA; see Ref. 10) and glycine (35). In addition, the presence of afterdepolarizing responses in some tonic S-type neurons (45) and transient inward currents in AH neurons (54) suggests the possible involvement of Ca2+-activated Cl- (ClCa) channels in these events. Robust action potential-dependent increases in Ca2+ are observed in both of these types of neuron (21, 42, 50, 53, 56, 61), and both the after-depolarization (unpublished observations) and the transient inward current (54) are reduced by niflumic acid. However, Cl- channels cannot be identified definitively by pharmacological means, since there are no specific blockers of these channels (16).

ICl(Ca) have been identified in a variety of peripheral and central neurons, including spinal cord (38), dorsal root ganglia (11), pelvic parasympathetic ganglia (37), trigeminal sensory and parasympathetic neurons (2), rod and cone photoreceptors (4), and olfactory receptor neurons (28). The physiological role of ICl(Ca) is variable, depending on the Cl- equilibrium potential in different types of neurons.

Because there is no clear proof for the presence of ClCa channels in enteric neurons, we determined whether ICl(Ca) could be found in cultured myenteric neurons of the murine proximal colon. We also attempted to directly measure the Cl- equilibrium potential, since this might disclose a physiological role for ICl(Ca). We chose to characterize the ICl(Ca) in the murine intestine in view of the advantages offered by future studies using transgenic mouse models.

A preliminary account of our findings has been published in abstract form (26).


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Dissociation of the myenteric plexus. Adult mice (C57BL/6) were killed by isofluorane inhalation and cervical dislocation in compliance with the requirements of the Animal Ethics Committee at the University of Nevada. A 2.5-cm length of proximal colon was removed, opened longitudinally, and pinned out flat in a dish lined with Sylgard containing Krebs solution. The mucosa and submucosa were completely dissected away, the remaining muscle layer preparation was turned upside down, and the longitudinal muscle layer was peeled away. The remaining myenteric plexus-circular muscle preparation (see Ref. 18) was cut into small pieces and transferred to a test tube containing 0.2% collagenase (type II; Worthington) and 0.12% protease (type IX; Sigma, St. Louis, MO) dissolved in Ca2+-free Hanks' solution. After 30 min of incubation at 37°C, the tissues were washed four times with enzyme-free Ca2+-free Hanks' solution and gently triturated through a fire-polished glass Pasteur pipette for 10-15 min. The suspension was then centrifuged at 200 rpm for 5 min, after which the supernatant was discarded, and the pellet was resuspended in 2 ml Ca2+-free Hanks' solution. Aliquots of this solution were added to 35-mm plastic dishes. Each of the dishes contained 2.5 ml cell culture medium consisting of medium-199 (GIBCO) plus 10% FBS. The medium was also supplemented with 10 mM glucose (GIBCO), 20 µM 5-fluoro-2-deoxyuridine (Sigma), and 1.5% antibiotic/antimycotic solution (10,000 U/ml penicillin, 10 mg/ml streptomycin, and 0.5 mg/ml amphotericin B). The dishes were maintained in a humidified incubator (gassed 5% CO2) at 37°C for 2-5 days before use. The culture medium in the dishes was changed every 2 days.

Patch-clamp recording. Whole cell currents were recorded at room temperature (20-22°C) using a perforated patch configuration with a patch-clamp amplifier (EPC-9; HEKA Instruments, Lambrecht, Germany) and Pulse software. Currents were filtered on-line at 3 kHz and digitized at 0.5-20 kHz. Patch pipettes were drawn from thin-walled borosilicate capillary glass (Sutter Instrument, Novato, Canada) to have resistances of 1.5-3.0 MOmega . An Ag-AgCl reference electrode was connected to the bath through an agar bridge saturated with KCl solution. To obtain a perforated patch, the pipette solution contained gramicidin dissolved in DMSO to a final concentration of 10-60 µg/ml. To measure the reversal potential (Erev) of Cl-, we used nystatin (250 µg/ml) perforated patches to equilibrate the intracellular Cl- with that of the pipette solution (20).

Myenteric neurons were bathed in a solution containing (in mM) 125 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, 10 glucose, and 10 TEA adjusted to pH 7.4 with Trizma. In some experiments, Cl- equilibrium potential (ECl) was modified by substituting methanesulfonate (adjusted to neutral pH with NaOH) for 99 mM Cl- in the bathing solution. The pipette solution contained (in mM) 135 CsCl, 1 MgCl2, 1.5 Na2ATP, 0.5 NaGTP, 10 HEPES, 0.1 EGTA, and 10 TEA, set to pH 7.2 with Trizma. This solution was altered by substituting 102 mM cesium aspartate (made by mixing CsOH with aspartate) for 102 mM of the CsCl in some experiments directed at examining the effects of changing ECl. We measured the normal resting membrane potential of these neurons using the following pipette solution (in mM): 145 KCl, 1 MgCl2, 1.5 Na2ATP, 0.5 NaGTP, 10 HEPES, and 0.1 EGTA, set to pH 7.2 with Trizma, and bath solution containing 135 NaCl, 4 KCl, 1 MgCl2, 2 CaCl2, and 10 glucose, adjusted to pH 7.4 with Trizma. The liquid junction potential between the bath solution and pipette solution was calculated (5) and corrected. Niflumic acid (Sigma) was dissolved in DMSO at a stock concentration of 0.1 M and delivered to the bathing solution at a final concentration of 10 µM. Tetrodotoxin (TTX; Sigma) was dissolved in distilled H2O at a stock concentration of 10-3 M and used at a final concentration of 0.7 µM. omega -Conotoxin GIVA (Alomone Lab, Jerusalem, Israel) was dissolved in distilled H2O to give a stock solution of 10-4 M and was used at final concentration of 0.3 µM.

The relative permeability of external anions (X) with respect to Cl-(PX/PCl) was determined using the Goldman-Hodgkin-Katz equation as follows: Erev = (RT/zF)ln{([Cl-]o + PX/PCl[X]o)/([Cl-]i + PX/PCl[X]i)}, where P is permeability, R is the molar gas constant, T is the absolute temperature, z is the charge, F is the Faraday constant, the indexes i and e indicate the intracellular and extracellular ion species, respectively, and brackets denote concentration. The rise and decay of the currents were fitted by an exponential described by the equation It = A[1-exp-(t/tau )] and It = I0exp-(t/tau ) respectively, where It, I0, and A are the amplitudes of the current at times t, 0 ms, and the end, respectively, and t is time. Data are presented as means ± SE. Paired t-test or one-way ANOVA with a Bonferroni test as a post hoc analysis was used to compare the means, and a P value of 0.05 was used as the cut off for statistical significance.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Depolarizing voltage-activated currents in myenteric neurons. Whole cell currents were recorded from murine colonic myenteric neurons using a Cs+-containing pipette solution to block K+ currents (see MATERIALS AND METHODS). Gramicidin (10-60 µg/ml) was used to perforate cell-attached membrane patches with cation-selective channels to preserve the intracellular Cl- concentration (14, 29). During the recording, the series resistance was <10 MOmega and compensated (~80%). Under these conditions, depolarization (+10 mV, 400 ms) evoked an initial transient inward current and the slowly activating time-dependent outward current (Itdo), whereas repolarizing the membrane potential to -80 mV induced a slowly deactivating tail current (Itail; Fig. 1A). This Itdo and Itail was present in ~70% of recorded neurons (65 out of 94 patched neurons). Initial transient inward currents were composed of fast-activating and fast-inactivating current and sustained inward currents (Fig. 1B). The fast-activating and fast-inactivating current was an Na+ current because it was almost blocked by TTX (0.7 µM, n = 7; Fig. 1B). Myenteric neurons were identified as those cells generating fast Na+ current at the onset of the depolarizing voltage step. The sustained inward current was Ca2+ current because it was abolished by Cd2+ (0.4 mM; see Fig. 7A2).


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Fig. 1.   Currents recorded in a myenteric neuron in response to a command step (duration 400 ms) potential from -80 to +10 mV. A: currents were composed of the early inward current and a time-dependent outward current (Itdo). A slowly deactivating tail current (Itail) occurred when the membrane potential was returned to the holding potential of -80 mV. Itdo and Itail were well fitted by single exponential curves. B: early inward current shown on an expanded time scale. The early inward current consisted of a fast-activating and -inactivating current (*) that was tetrodotoxin (TTX; 0.7 µM) sensitive. This current was followed by a sustained inward current. Note that fast-activating and -inactivating current was not completely blocked by TTX.

Itdo showed almost no inactivation during a depolarizing step lasting up to 1.2 s (Fig. 2A). Itdo and Itail were maximal at a depolarizing step voltage of about +30 and +10 mV, respectively (Fig. 2B). We calculated the time constant of these currents by fitting them with exponential functions (Fig. 2C). We have measured the time constant of Itdo 30 ms after the beginning of the pulse to avoid contamination from dynamic changes in the Na+ and Ca2+ current. Also, Itail was measured 20 ms after the end of the pulse to remove influences from the rapidly deactivating Ca2+ current [as measured in neurons without Cl- current (ICl)]. Depolarizing voltage steps to 0 mV from -80 mV maximally decreased the time constant of Itdo, and stepping to +10 mV maximally increased the time constant of Itail, whereas the changes in time constant of Itdo and Itail were opposite for higher voltage steps (e.g., from +10 to +50 mV; see Fig. 2C). We also tested the effect of the duration of the depolarization step on these currents (Fig. 3). Increasing the duration of a +10-mV voltage step from 100 to 900 ms (in increments of 100 ms) caused an increase in the time constant of Itail (Fig. 3, A and C). However, increasing the step duration had no effect on the time constant of Itdo since its activation kinetics were such that it followed the same exponential time course as shown in Fig. 3A. With longer periods of activation, peak current of Itdo and Itail was increased (see Fig. 3, A and C).


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Fig. 2.   A: currents recorded by depolarizing a myenteric neuron by a series of long-duration (1.2-s) command step potentials ranging from -30 to +50 mV (holding potential = -80 mV). B: current-voltage relationship curves for peak Itdo and Itail. Itdo and Itail were maximal when depolarized to +10 and +30 mV, respectively. C: time constant (tau )-voltage relationship for Itdo and Itail. Values are shown as means ± SE (n = 10).



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Fig. 3.   Effect of duration of a depolarizing voltage step on Itdo, Itail, and tau  of Itail. A: currents were recorded from depolarizing the myenteric neuron from a holding potential of -80 to +10 mV for durations ranging from 100 to 900 ms in increments of 100 ms. B: relationship between the duration of a fixed voltage step (10 mV) and peak current for Itdo and Itail. C: relationship between voltage step duration and tau  for Itail. Values are shown as means ± SE (n = 9). Note that, although the magnitude of Itdo becomes saturated, that of Itail continues to grow.

Measurement of resting membrane potential and cell capacitance. The resting membrane potential of these cultured myenteric neurons measured using a K+-rich (without TEA) pipette solution was -56.3 ± 2.7 mV (n = 15).

The size of the neurons with and without ICl(Ca) appeared to be similar, since there was no significant difference in the capacitance between these two groups [capacitance with ICl(Ca) = 11.3 ± 0.4 pF (n = 65) vs. without ICl(Ca) = 10.5 ± 0.5 pF (n = 29); P > 0.2].

Itdo and Itail are carried by Cl-. To test whether Itdo and Itail are ICl, we measured the Erev of Itail before and after changing the transmembrane Cl- gradient (Fig. 4). Whole cell currents were recorded in a nystatin-perforated patch configuration to equilibrate the intracellular Cl- concentration with that of pipette solution (20). The extracellular Cl- concentration was reduced from 145 to 46 mM, thereby shifting the calculated ECl from ~0 mV to approximately +29 mV. These changes shifted the measured Erev of Itail from 1.4 ± 0.6 to 19.2 ± 0.7 mV (n = 5; Fig. 4, A and B). From these measured Erev, we can calculate the relative permeability of methanesulfonic acid to Cl- (0.23 ± 0.12).


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Fig. 4.   Changes in reversal potential (Erev) of Itail after changes of transmembrane Cl- gradient. A: Itail recorded from a +10-mV depolarizing voltage step followed by different voltage steps from -40 to +30 mV in increments of 10 mV. Left, intracellular and extracelluar Cl- concentrations were 147 and 145 mM, respectively. Right, intracellular and extracelluar Cl- concentrations were 147 and 46 mM, respectively, by replacing extracellular Cl- with methanesulfonic acid. B: decreasing the extracellular Cl- concentration shifted the Erev from +0.1 to +17.6 mV. C: currents recorded after changes in intracellular Cl- concentration. Step protocol similar to A, but with voltage steps from -60 to +20 mV. Intracellular and extracelluar Cl- concentration were 45 and 145 mM, respectively [calculated Cl- equilibrium potential (ECl) is approximately -29.6 mV]. Intracellular Cl- was replaced with aspartic acid. D: Itail were reversed at -29.3 mV by decreasing the intracellular Cl- concentration. Note that changing the ECl resulted in the shift of Erev of Itail, verifying that these currents are carried by Cl-.

To confirm that Itdo and Itail were indeed ICl, we also substituted 102 mM intracellular Cl- for aspartic acid. The intracellular Cl- concentration was reduced from 147 to 45 mM to give a calculated ECl of -30 mV. In these conditions, the measured Erev of Itail was -28.2 ± 1.4 mV (n = 6; Fig. 4, C and D). The Cl- substitution data also prove that that Itdo and Itail are carried by Cl-. Methanesulfonic acid was suggested to be a good choice for Cl- substitution experiments because it has a low relative permeability (0.1) in lacrimal gland, does not bind Ca2+, and completely dissociates at physiological pH (15). Our results show higher relative permeability of methanesulfonic acid than that obtained for lacrimal cells. The Erev of Itail was shifted very closely to the calculated ECl when the intracellular Cl- was replaced with aspartate. These data imply that the intracellular Cl- was equilibrated with that of pipette solution but not the aspartate.

Myenteric neurons accumulate intracellular Cl-. Using the gramicidin-perforated patch, we measured the ICl without disturbing the intracellular Cl- homeostasis (14, 29). Itail reversed at -33.4 ± 1.0 mV (n = 17; Fig. 5), which was more positive than the resting membrane potential. Using the Nernst equation, we could approximate the intracellular Cl- concentration to be ~39 mM. This implies that, in myenteric neurons, the intracellular Cl- concentration is maintained above that expected for passive Cl- distribution.


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Fig. 5.   Measurement of ECl without perturbing the intracellular Cl- concentration by using the gramicidin-perforated patch. A: currents recorded from +10-mV depolarizing voltage steps followed by different voltage steps from -60 to 0 mV in an increment of 10 mV. B: Cl- current (ICl) was reversed at -31.1 mV. Note that ECl is more positive than passive distribution of Cl-.

Does ECl change during long depolarizing pulses? Figure 3 shows that Itdo appears to grow after Itail has saturated; we therefore examined whether such a difference may be attributable to changes in ECl in response to Cl- accumulation during long-duration depolarizing pulses. We therefore measured the Erev of Itdo and Itail using a ramp protocol. Increasing the depolarization duration from 300 to 1,100 ms in increments of 200 ms produced incremental shifts in ECl from -32.8 ± 1.0 mV (300 ms) to -27.8 ± 0.9 mV (1,100 ms; n = 7, P < 0.01; Fig 6).


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Fig. 6.   Effects of duration of a depolarizing voltage step on Erev of Itdo and Itail. A: currents were recorded from depolarizing the myenteric neuron from a holding potential of -80 to +10 mV for durations ranging from 300 to 1,100 ms in increments of 200 ms; these voltage steps were followed by a ramp protocol from -80 to -10 mV for 300 ms. B: summarized data showing that incrementing the duration of the depolarizing pulses shifted Erev to a more positive value. Values are shown as means ± SE (n = 7). Significant differences (**P < 0.01) are indicated.

This result implies that longer-duration depolarizing pulses lead to increasing Cl- accumulation, resulting in a decreased driving force for Itdo but an increased driving force for Itail.

ICl are Ca2+ dependent. To explore the Ca2+ dependence of the ICl, we compared ICl recorded before and after exposure to Cd2+ (0.4 mM) in the bath solution and after changes in the extracellular Ca2+ concentration.

Cd2+ (0.4 mM) abolished ICl (Fig. 7A1), indicating that Ca2+ influx is a prerequisite for the activation of this current.


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Fig. 7.   A1 and A2: effects of Cd2+ (0.4 mM) on ICl. B1 and B2: comparison of currents recorded with an extracellular Ca2+ concentration of 0.7, 2, and 6 mM. A2 and B2: recordings shown on an expanded time scale to disclose changes in Ca2+ current. C and D: summarized data showing that peak Itdo and Itail were increased in 6 mM Ca2+ but decreased in 0.7 mM Ca2+. tau  of Itdo was increased in 0.7 mM Ca2+ but decreased in 6 mM Ca2+. tau  of Itail was increased in 6 mM Ca2+ but decreased in 0.7 mM Ca2+. These currents were abolished by 0.4 mM Ca2+, proving that these are Ca2+-activated ICl. Values are shown as means ± SE (n = 6). Significant differences (*P < 0.05 and **P < 0.01) are indicated.

Changing extracellular Ca2+ concentration from the usual 2 mM to a 0.7 and 6 mM Ca2+-containing bath solution significantly decreased and increased the ICl, respectively, the normalized results of which are shown in Fig. 7. In 0.7 mM Ca2+, peak Itdo and Itail were decreased from 850 ± 190 to 720 ± 180 pA (n = 6, P > 0.05) and from -600 ± 140 to -480 ± 110 pA (n = 6, P < 0.01), respectively. In contrast, in 6 mM Ca2+, the peak Itdo and Itail were increased from 870 ± 200 to 1,040 ± 270 pA (n = 6, P > 0.05) and from -650 ± 160 to -720 ± 150 pA (n = 6, P < 0.05; Fig. 7, B and C). The time constant of Itdo was increased from 89 ± 7 to 110 ± 13 ms in 0.7 mM Ca2+ (n = 6, P < 0.01) but decreased from 87 ± 8.5 to 69 ± 6.2 ms in 6 mM Ca2+ (n = 6, P < 0.01; Fig. 7, B and D). The time constant of Itail was decreased from 910 ± 190 to 700 ± 160 ms in 0.7 mM Ca2+ (n = 6, P < 0.01) but increased from 750 ± 150 to 900 ± 170 ms in 6 mM Ca2+(n = 6, P < 0.05; Fig. 7, B and D).

Role of N-type Ca2+ channel on ICl(Ca). In the guinea pig myenteric neurons, Ca2+-activated K+ current, which is responsible for the prolonged AHP in AH neurons and the intermediate AHP in tonic S neurons, is reported to be dependent on Ca2+ entry through N-type Ca2+ channels (42, 54). To test the role of N-type channel on ICl(Ca), we have used the omega -conotoxin GIVA (0.3 µM). Bath application of omega -conotoxin GIVA decreased Itdo from 1,080 ± 180 to 550 ± 66 pA (n = 6, P < 0.01) and decreased Itail from -990 ± 130 to -480 ± 77 pA (n = 6, P < 0.01). Also, it increased (but not significantly) the time constant of Itdo from 90 ± 9.3 to 210 ± 60 ms (n = 6, P > 0.05) and decreased the time constant of Itail from 470 ± 65 to 355 ± 43 ms (n = 6, P < 0.01; Fig. 8).


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Fig. 8.   Effect of omega -conotoxin GIVA (GIVA) on Ca2+-activated Cl- current [ICl(Ca)]. A1 and A2: recordings were made in the presence and absence of GIVA (0.3 µM). A2: expanded time scale showing the decrease in Ca2+ current after GIVA treatment. B and C: summarized data showing that GIVA irreversibly decreased the peak current of Itdo and Itail. GIVA also increased tau  of Itdo but decreased the tau  of Itail. Values are shown as means ± SE (n = 5). Significant differences (**P < 0.01) are indicated.

Blockade of ICl(Ca) by niflumic acid. Bath application of 10 µM niflumic acid, an ICl(Ca) blocker (16), significantly decreased Itdo from 950 ± 170 to 420 ± 91 pA (n = 7, P < 0.01) and Itail from -600 ± 130 to -250 ± 50 pA (n = 7, P < 0.01). Although niflumic acid increased the deactivation time constant of Itail from 560 ± 62 to 970 ± 98 ms (n = 6, P < 0.01), it had no effect on the activation time constant of Itdo [from 100 ± 9.1 to 110 ± 4.0 ms (n = 7, P > 0.05); Fig. 9]. Niflumic acid did not appear to inhibit Ca2+ entry (Fig. 9A2) but decreased the peak of the ICl(Ca). Furthermore, niflumic acid did not affect the activation time constant of Itdo, suggesting it to be an open channel blocker, as reported previously (24). However, the deactivation time constant of Itail was significantly slowed, even though the Ca2+ load to be buffered was the same.


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Fig. 9.   Effect of niflumic acid (10 µM) on ICl(Ca). A1 and A2: recordings compared before and after niflumic acid treatment. Niflumic acid had no effect on the Ca2+ current but reversibly decreased the ICl(Ca). B and C: summarized data showing means ± SE (n = 7). The peak current of Itdo and Itail was decreased by niflumic acid. tau  of Itail was increased, whereas the tau  of Itdo was not affected by niflumic acid. Significant differences (**P < 0.01) are indicated.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We present, for the first time, direct evidence for ICl(Ca) in myenteric neurons cultured from murine proximal colon. We identified these currents by showing that, when the transmembrane Cl- gradient was changed, the Erev of this Ca2+-dependent current was shifted close to the calculated value for ECl. Using a gramicidin perforated patch so as not to perturb the intracellular Cl- concentration, we found ECl to be -33 mV. This Erev, which is more positive than the resting membrane potential of these neurons (-56.3 ± 2.7 mV), suggests that ICl(Ca) regulates postspike excitability.

ICl(Ca) in murine colonic myenteric neurons have similar characteristics to those described in other cells: a slow rate of activation, little inactivation during sustained depolarization, and very long deactivation kinetics (39, 60). The characteristics of ICl(Ca) were closely related to Ca2+ entry in the cell. ICl(Ca), which started to activate around -20 mV, was dependent upon Ca2+ entry through voltage-dependent Ca2+ channels (blocked by Cd2+). A significant amount of this Ca2+ entry is through voltage-gated N-type Ca2+ channels, since ICl(Ca) was reduced by omega -conotoxin GIVA, as are K+ currents underlying the postspike afterhypolarization in both AH neurons (54) and some tonic S neurons (42). Itail of ICl(Ca) were maximal at the activation voltage for peak Ca2+ current (~10 mV; Fig. 2B). The changes in the deactivation time constant were compared at the same voltage (-80 mV), and, in contrast to tau  of Itdo, are unlikely to be contaminated by other underlying currents. When the Itail was maximal, the deactivation time constant was also maximal (Fig. 2, B and C). Increasing the duration of the depolarizing step prolongs the activation of Ca2+ channels, which promotes sustained Ca2+ entry into the cell. Increasing the duration of the depolarizing step caused an increase in ICl(Ca) and prolonged the deactivation time constant (tau  of Itail; Fig. 3C). Moreover, increased Ca2+ entry because of raised extracellular Ca2+ concentration (from 2 to 6 mM) prolonged the deactivation time constant, whereas decreased Ca2+ entry resulting from lowered extracellular Ca2+ concentration (from 2 to 0.7 mM) or N-type Ca2+ channel blocker (omega -conotoxin GIVA) shortened the deactivation time constant. These data suggest that increased Ca2+ entry increases the intracellular Ca2+ load to be buffered, which reflects slower deactivation of ICl(Ca) and vice versa for decreased Ca2+ entry to the cell. Therefore, the deactivation time constant likely reflects how fast cells remove excess Ca2+. However, increased Ca2+ entry resulting from raised extracellular Ca2+ concentration (from 2 to 6 mM) shortened the activation time constant, whereas decreased Ca2+ entry resulting from lowered extracellular Ca2+ concentration (from 2 to 0.7 mM) or N-type Ca2+ channel blocker (omega -conotoxin GIVA) prolonged the activation time constant. This supports the link between Ca2+ influx and activation of the ICl(Ca). Therefore, the activation time constant may reflect the level of available intracellular Ca2+ needed to activate ClCa channels.

So far, specific blockers for pharmacological identification of ClCa channels are not available. Niflumic acid, a nonsteroidal anti-inflammatory agent, has been used widely. However, its actions can be complex, such as blocking outward currents and enhancing inward currents, depending on the intracellular Ca2+ level (see Ref. 40 for results in rat pulmonary artery). Also, niflumic acid has been shown to have both open-channel and voltage-dependent blocking effects (24). Consistent with its open-channel blocking effects, we found that niflumic acid had no effect on the activation time constant but prolonged the deactivation time constant. However, there was no evidence for a voltage-dependent blocking effect by niflumic acid, since it decreased both the outward currents (Itdo; activated by stepping to +10 mV) and the inward currents (Itail; elicited by stepping to -80 mV) by a similar amount (decreased by 55.6 and 58.3%, respectively).

Although the specific functional classes of neurons possessing ICl(Ca) were not identified in our study, we found the majority (~70%) of cultured myenteric neurons in murine proximal colon to have ICl(Ca). Also, we did not find any relation between the cell capacitance (and therefore cell size) and the presence or absence of this current.

The physiological role of ClCa channels in neurons is determined by the ECl and resting membrane potential. Opening ClCa channels hyperpolarizes cultured spinal neurons (mouse; see Ref. 39) and taste cells (Necturus; see Ref. 51). Also, inhibitory postsynaptic potentials generated by activation of GABAA and glycine receptors are also reported to be the result of activation of Cl- conductances in neuronal cells (12, 25). For Cl- conductances to hyperpolarize or stabilize the membrane potential, the ECl must be maintained at a value equal to or higher than the resting membrane potential. In this case, ECl results from passive distribution of Cl- across the membrane (the Donnan equilibrium; ECl would be the same as the resting membrane potential) and active extrusion of Cl- from the cytoplasm (ECl would be higher than the resting membrane potential; see Ref. 52). On the other hand, ICl(Ca) can be responsible for posttetanic afterdepolarizing potentials in rabbit sympathetic ganglia (1). Furthermore, the fact that GABA and glycine can also depolarize neurons through activation of Cl- channels (13, 19, 35) reflects an ECl lower than the resting membrane potential. These depolarization responses resulting from an increase in Cl- conductance result from intracellular Cl- accumulation (27). Hence, our results (ECl = -33 mV) imply that myenteric neurons actively accumulate intracellular Cl-, which are responsible for membrane depolarization. In myenteric neurons, there is some evidence for membrane depolarization resulting from an increase in Cl- conductance, which is consistent with our results. Previous studies have suggested the possibility of ICl(Ca) in the afterdepolarizing response observed in some tonic S neurons (45) and poststimulus transient inward currents in AH neurons (54). However, these studies relied on either the estimates of the Erev (-34 to -40 mV) of these events or the sensitivity of inward currents to high concentrations of niflumic acid, which is not selective for ICl(Ca) (16). Other studies that examined the membrane potential dependence of depolarizing responses to agonists estimated the Cl- reversal potential in AH neurons to be around -18 mV with sharp KCl electrodes (10, 35) and -39 mV when potassium acetate, citrate, or sulfate electrodes were used (10).

In the present study, the ECl in murine colonic myenteric neurons was found to be approximately -33 mV, which is more positive than the resting membrane potential of these neurons (-56.3 ± 2.7 mV); therefore, they would accumulate Cl-. Activation of ICl(Ca) is likely to depolarize the neuron (up to ECl) and decrease the threshold for neuronal firing, thereby regulating both postspike excitability and spike frequency adaptation.


    ACKNOWLEDGEMENTS

We thank Drs. James Kenyon and Sang Don Koh for helpful suggestions.


    FOOTNOTES

This study was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant PO1 DK-41315. P. Vanden Berghe is a Postdoctoral Fellow of the Fund for Scientific Research, Flanders, Belgium.

Address for reprint requests and other correspondence: T. K. Smith, Dept. of Physiology and Cell Biology, Univ. of Nevada School of Medicine, Reno, Nevada 89557 (E-mail: tks{at}physio.unr.edu).

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.

First published November 27, 2002;10.1152/ajpcell.00437.2002

Received 23 September 2002; accepted in final form 25 November 2002.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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