The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca2+. The role of K+ channels in this spontaneous activity was studied by examining the effects of commonly used K+ channel blockers on K+currents, spontaneous action currents, and spontaneous Ca2+signaling. Whole-cell recordings of voltage-gated outward K+ currents in GT1–1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1–1 cells also express inwardly rectifying K+ currents (I K(IR)) that were activated by hyperpolarization in the presence of elevated extracellular K+. These currents were blocked by 100 μM Ba2+ and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba2+ had distinct effects on the pattern of action current bursts and the resulting Ca2+ oscillations. TEA increased action current burst duration and increased the amplitude of Ca2+ oscillations. Ba2+ caused an increase in the frequency of action current bursts and Ca2+oscillations. These results indicate that specific subtypes of K+ channels in GT1–1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca2+signaling. K+ current modulation of electrical activity and Ca2+ signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.
The pulsatile release of gonadotropin releasing hormone (GnRH) by the hypothalamus is the fundamental stimulus for events in the vertebrate reproductive axis. GnRH release causes the pulsatile release of gonadotropins from the anterior pituitary which in turn controls such reproductive functions as the maturation of gametes, the ovulatory cycle, and the onset of puberty (Carmel et al. 1976; Clarke and Cummins 1982; Dierschke et al. 1970;Knobil 1980; Levine et al. 1982;Terasawa et al. 1999). Individual characteristics of the GnRH pulse, including its amplitude, duration, and frequency, may encode specific functions in the reproductive system. GnRH neurons in vivo exhibit spontaneous electrical activity that has been correlated with the pulsatile release of GnRH or gonadotropins [e.g., luteinizing hormone (LH)]. LH pulses measured in the peripheral circulation have been correlated with increases in multiunit activity (MUA volleys) recorded from the medial basal hypothalamus of the rat (Kawakami et al. 1982), rhesus monkey (Knobil 1988;Wilson et al. 1984), and goat (Mori et al. 1991). The mechanisms for spontaneous activity in individual cells and the coordination of activity in the networks of cells that compromise the GnRH pulse generator, however, are not clearly understood.
GT1 cells are a GnRH secreting hypothalamic cell line derived by genetically targeted tumorigenesis in transgenic mice (Mellon et al. 1990; Wetsel 1995). They spontaneously secrete GnRH in a pulsatile manner with a 25–35 min interpulse interval (Martinez de la Escalera et al. 1992;Wetsel et al. 1992) similar to that reported from in vivo studies in rats (Masotto and Negro-Vilar 1988) and mice (Weiner and Martinez de la Escalera 1993). GnRH secretion in GT1 cells is Ca2+-dependent (Martinez de la Escalera et al. 1992) and inhibited by TTX (Mellon et al. 1990). GT1 cells also exhibit spontaneous electrical activity and Ca2+oscillations in individual cells (Charles and Hales 1995; Hales et al. 1994). Ca2+ oscillations may be limited to single cells or propagated as waves of increased [Ca2+]i across fields of hundreds of cells (Charles et al. 1996). Each Ca2+ oscillation is correlated with a burst of TTX-sensitive Na+ action potentials, and the entry of Ca2+ through high-voltage-activated (HVA) Ca2+ channels. A burst of action potentials precedes the Ca2+ oscillation in individual cells, regardless of whether the Ca2+ oscillation is limited to an individual cell or propagated as an intercellular wave (Costantin and Charles 1999). Multiple lines of evidence suggest that intercellular Ca2+ waves in GT1–1 cells are propagated by electrotonic coupling through gap junctions (Charles et al. 1996), although these cells do form synapse-like structures (Wetsel et al. 1992) and a role for synaptic coupling has not been excluded.
The spontaneous signaling in GT1 cells involves the coordinated activity of multiple ion channels. Ion channels present in GT1 cells include voltage-gated Na+, Ca2+, and K+ channels as well as cAMP-activated cation channels (Bosma 1993;Costantin and Charles 1999; Hales et al. 1992,1994; Spergel et al. 1996; Van Goor et al. 1999b; Vitalis et al. 2000). The action potential bursting behavior in these cells suggests a complicated interplay between these channels. This study focuses on the different K+ channels in GT1–1 cells and their role in the generation and modulation of patterns of spontaneous electrical activity and Ca2+ signaling.
Whole-cell patch-clamp recording
Na+ and K+ channel currents were measured using the whole-cell configuration. The bath solution used to measure the I K(DR)and I K(A) channels consisted of (in mM) 137 NaCl, 2.8 mM KCl, 2.0 MgCl2, 1.3 CaCl2, 10 glucose, and 10 HEPES (pH 7.2 with NaOH). Ca2+-free bath solution was identical except the Ca2+ was omitted and 0.5 mM EGTA was added before the pH was adjusted to 7.2. The pipette solution contained (in mM) 140 KCl, 3 MgATP, Ca2+ chelator, and 10 HEPES (pH 7.2 with KOH). The pipette Ca2+chelator was 10 mM EGTA, 0.5 mM EGTA, or 5.5 mM bis-(o-aminophenoxy)-N,N,N′,N′-tetraacetic acid (BAPTA; pH = 7.2). I K(IR)channel currents were measured with a bath solution containing (in mM) 140 KCl, 4 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES (pH 7.2 with KOH), and a pipette solution containing (in mM) 140 KCl, 10 EGTA, 3 MgATP, and 10 HEPES (pH 7.2 with KOH). BaCl2, tetraethylammonium chloride (TEA-Cl), and 4-AP were obtained from Sigma. Apamin was obtained from RBI (Natick, MA) and charybdotoxin (CTx) was obtained from Latoxan (Valence, France). The potency of the CTx we were using was confirmed by testing it on a preparation of cloned BK channels expressed in Xenopus oocytes. Pipettes were pulled from borosilicate glass (World Precision Instruments, Sarasota, FL; catalog no. TW150F-4) using a Kopf model 730 puller. Pipette tips were fire-polished using a heated platinum/iridium filament to a tip resistance of 3–5 MΩ. Patch-clamp recordings were performed using an Axopatch 200A or B amplifier; the data were stored and analyzed using Axon Instruments (Foster City, CA) software (pClamp) on a Pentium PC. Leak subtraction was not used with the exception of the data in Fig. 1, where a protocol of P/−4 was used (Bezanilla and Armstrong 1977). The access resistance was less than 15 MΩ in all experiments, as determined by the compensation circuitry of the amplifier. The membrane capacitance (Cm ) was determined from the compensation circuitry and was 10.6 ± 3.8 pF (mean ± SD) with a range of 4–21 pF (n = 58). In a subset of cells (n = 7), a direct measurement of the access resistance (9.4 ± 2.6 MΩ) andCm (8.6 ± 2.8 pF) were made (Armstrong and Gilly 1992). The access resistance (Ra ) was calculated after 10-mV pulses were delivered subsequent to compensation of pipette capacitance.Cm was measured as the integral of the capacitive current divided by the voltage step. The time constant (τ) of the decay of the capacitive transient was obtained (by exponential fit) for the calculation of access resistance (Ra = τ/Cm ). A low-resistance 100 mM KCl bridge was used to connect the ground Ag/AgCl electrode to the bath. All data were collected at 10 kHz and analog filtered at 5 kHz. Pairedt-tests were used to compare the magnitude of control currents versus current magnitude after the addition of K+ channel blockers or Ca2+chelators to the bath solution (Zar 1974). TheP values used to test statistical significance are indicated in the figure legends. All means are expressed ± SD.
Cell-attached patch-clamp recording
Action currents were measured in the cell-attached configuration (Hamill et al. 1981). The advantage of this recording configuration compared with whole-cell recording of action potentials is that the intracellular contents of a cell are not disturbed while action currents are monitored. Action currents were measured as a capacitive current that charges the membrane (Charles and Hales 1995; Costantin and Charles 1999; Fenwick et al. 1982; Peters et al. 1989). The currents are displayed with the opposite sign convention as the whole-cell currents; i.e., the capacitive outward current spike that was measured when the cell fired an action potential is plotted as a negative current. The pipette was voltage clamped to the bath potential (0 mV). The bath and pipette solution consisted of (in mM) 137 NaCl, 2.8 KCl, 2.0 MgCl2, 1.0 CaCl2, 10 glucose, and 10 HEPES (pH 7.2 with NaOH). pClamp 7 software and a Digidata 1200 (Axon Instruments) interface were used to record the action currents onto a Pentium PC. Data were collected at a sample frequency of 10 kHz and analog filtered at 5 kHz using an Axopatch 200A or B amplifier. Data were later digitally filtered off-line at 2 kHz before analysis.
Video fluorescence imaging
Changes in [Ca2+]iwere measured after loading the cells in the bath solution containing 5 μM fura2 AM (Calbiochem) for 30–40 min. We used a fluorescence imaging system described previously (Charles et al. 1991). Some sequences of fura2 fluorescence were recorded to videotape and subsequently digitized using the Axon Imaging Workbench software and a Digidata 2000 interface (Axon Instruments). The changes in fura2 fluorescence measured at 380-nm excitation (ΔF) are shown in pixel intensity values (0–255 scale). The ΔFvalues are not normalized to the baseline F value for each cell. Changes in the intracellular Ca2+concentration ([Ca2+]i) in these cells during an oscillation are typically 50–300 nM when calculated from dual wavelength measurements (Charles and Hales 1995; Charles et al. 1996). The bath solution used for video fluorescence experiments was the same as that used for cell-attached patch-clamp recording. Raster plots were constructed using the Transform portion of the Noesys software package (Fortner Software, Sterling, VA).
Synchronization of the video imaging and cell-attached patch-clamping recordings
To achieve simultaneous recording, the Digidata 2000 and Digidata 1200a interfaces were coupled by connecting the digital output of the Digidata 2000 to the trigger input of the Digidata 1200a. The Axon Imaging Workbench (AIW) and Clampex 7 software packages were run simultaneously under Windows 98. AIW was used as the master program and triggered Clampex 7 through the interconnected interfaces.
Cells were maintained in DMEM/F12 culture media (Mediatech, catalog no. 10-092) supplemented with 5% fetal bovine serum, 5% horse serum, 100 I.U./ml penicillin, and 100 μg/ml streptomycin in 25 cm2 flasks. Cells were grown to approximately 60–80% confluency and then passaged or transferred onto poly-d-lysine-coated glass coverslips, on which they were grown for 6–12 days to a confluence of approximately 60–80% prior to experimentation. GT1–1 cells were a generous gift of Dr. Richard Weiner (UCSF).
K+ current types in GT1–1 neurons
Voltage-gated currents in GT1–1 cells were recorded using the whole-cell patch-clamp configuration. Inward Na+and outward K+ currents are shown in Fig.1. The currents in Fig. 1 A are shown on an expanded time scale in Fig. 1 B. The inward Na+ currents were completely blocked by 0.5 μM TTX (Fig. 1 C). In the presence of 0.5 μM TTX in the bath and 10 mM EGTA in the pipette, the mean peak current was 1165 ± 620 pA corresponding to a mean current density of 110 ± 62 pA/pF (n = 38). The outward K+ currents could be separated into a rapidly activating transient component and a more slowly activating sustained component. The transient component could be eliminated from the control outward currents (Fig.2 A) by applying a depolarizing prepulse prior to the voltage steps delivered to elicit outward currents, leaving the sustained current component (Fig. 2 B). The transient component is obtained by subtraction (Fig.2 C). The two components are similar to transient A-type (I K(A)) and sustained delayed-rectifier currents (I K(DR)) reported in the literature (Adams and Nonner 1990).
The presence of Ca2+-activated K+ current (I K(Ca)) in GT1–1 neurons was explored by testing the effect of removal of extracellular Ca2+([Ca2+]e) on outward K+ current. Removal of [Ca2+]e reduced both transient and sustained outward currents (Fig.3). A series of experiments were then conducted in which the Ca2+ buffering capacity in the pipette was varied while the fraction of current eliminated by removal of [Ca2+]e was measured. When 5.5 mM BAPTA was used as the Ca2+chelating agent in the pipette, removal of [Ca2+]e had no effect on the outward currents (Fig. 3 D), strongly suggesting that allI K(Ca) currents were eliminated in the presence of BAPTA. Reducing the buffering capacity of the pipette by using 10 and 0.5 mM EGTA, respectively, caused an increase in the amount of I K(Ca) currents that could be eliminated by removal of [Ca2+]e (Fig.3 D). The reduction of both peak and sustained currents by removal of [Ca2+]e was significant when the pipette contained 0.5 mM EGTA compared with 5.5 mM BAPTA. There was also a significant reduction in peak currents when the pipette contained 10 mM EGTA compared with 5.5 mM BAPTA. These data indicate that I K(Ca) currents contribute to both the transient and sustained components of current. The inability of intracellular EGTA and the ability of intracellular BAPTA to eliminate I K(Ca) currents is in agreement with data from chromaffin cells (Marty and Neher 1985). This suggests that the rapid nature of Ca2+ chelation by BAPTA is required to eliminateI K(Ca).
K+ channel antagonists blocked fractions of the transient and sustained currents (Fig.4). TEA (2 mM) blocked a sustained component of the current (Fig. 4 A); concentrations up to 10 mM did not block larger fractions of current. 4-Aminopyridine (4-AP) had dual effects: 0.75 mM blocked components of both the transient and sustained currents while 1.5 mM caused a further block of the transient fraction of the current (Fig. 4 B). Ba2+ (100 μM) blocked a small but significant fraction of both the transient and sustained components (Fig.4 C and Table 1). Higher concentrations of Ba2+ (0.5–1 mM) caused blockade of larger fractions of both the transient and sustained currents up to approximately 50% (data not shown). There was no effect of apamin or charybdotoxin on outward K+ currents despite the fact that there was a large fraction ofI K(Ca) current. The magnitude of the effects of the different K+ channel antagonists on the peak and sustained currents is summarized in Table 1.
An inwardly rectifying K+ current (I K(IR)) was also present in GT1–1 cells (Fig. 5). Addition of 100 μM Ba2+ completely blocked theI K(IR) currents, leaving only the linear leak current (Fig. 5 A). The I-V curve for the I K(IR) currents show inward-rectification at voltages more negative than −20 mV (Fig.5 B). I K(IR) was not affected by 1.5 mM 4-AP or 2 mM TEA (Fig. 5, C andD).
K+ channel antagonists affected Ca2+signaling in GT1–1 neurons
Bath application of K+ channel antagonists had distinct effects on the pattern of spontaneous Ca2+ signaling in GT1–1 cells (Figs.6−8). The effect of the addition of 100 μM Ba2+ on Ca2+oscillations is illustrated in the raster plot and line tracings of Fig. 6, A and B. Exposure to Ba2+ resulted in an increase in the frequency of spontaneous Ca2+ oscillations, as well as an increase in baseline [Ca2+]i. Exposure to Ba2+ also resulted in an increased synchronization of the activity in individual cells. This can be seen in the activity of two exemplary cells (a) and (c). Before Ba2+, they displayed the same number of Ca2+ oscillations, although they were not well synchronized. After Ba2+, they became highly synchronized. The results shown from this experiment are typical for GT1–1 cells (n = 15 culture dishes). In this experiment, an increase in the frequency of Ca2+oscillations of 3.25 ± 1.43 fold was seen in 30 of 37 randomly picked spontaneously active cells. No change in frequency was seen in three cells while four cells exhibited a decrease in frequency (0.65 ± 0.22 fold). TEA altered the Ca2+signaling in a manner distinct from Ba2+ (Fig. 6,C and D). The amplitude of individual Ca2+ oscillations was increased and changes in cell excitability occurred in three phases. The first phase, which lasted for approximately 60–120 s, included the induction of a train of Ca2+ oscillations and a stacking of individual Ca2+ oscillations. In the second phase, the cells entered a period of reduced excitability that remained for several minutes. In the third phase, the Ca2+oscillations continued with increased amplitude and approximately the same period as prior to the addition of TEA. The differential effects of TEA and Ba2+ on the pattern of Ca2+ signaling led us to investigate the possibility of an additive effect of these blockers. The effect of Ba2+ and TEA were separable; Fig. 6 Eillustrates the effect of the addition of 100 μM Ba2+ after the addition of 2 mM TEA. TEA increased Ca2+ oscillation amplitude and subsequent addition of Ba2+ in the presence of TEA caused an increase in oscillation frequency similar to that observed by the addition of Ba2+ alone. The reverse experiment, addition of 2 mM TEA in the presence of 100 μM Ba2+, obscured the effect of the TEA because the increase in oscillation frequency dominated (data not shown).
The effects of Ba2+ were concentration-dependent; the frequency of oscillations increased with increasing concentration (Fig. 7). Increasing the extracellular Ba2+ concentration [Ba2+]o incrementally from 0 to 700 μM (Fig. 7 A) caused the frequency to increase (nonlinearly) and the baseline [Ca2+]i to rise until the oscillations appeared to cease at 700 μM Ba2+. The majority of GT1–1 cell dishes exhibited spontaneous Ca2+ signaling, but some did not. Figure7 B shows the effect of Ba2+ on previously quiescent cells. The addition of Ba2+to dishes lacking spontaneous activity always resulted in the induction of Ca2+ oscillations and waves (n= 10). The resulting Ca2+ oscillations were indistinguishable from those seen in spontaneously active groups of cells in the presence of Ba2+. Note the high degree of synchrony of the Ca2+ oscillations at increasing Ba2+ concentrations in both the spontaneously active and previously quiescent cells. The K+ channel blocker 4-AP did not affect Ca2+ signaling at concentrations up to 1.0 mM (Fig. 8, A and B). Higher concentrations of 4-AP (1.5 mM) caused a transient increase in the baseline levels of [Ca2+]i (Fig.8 C).
K+ channel antagonists affect the electrical activity that underlies Ca2+ signaling
Each Ca2+ oscillation in spontaneously active GT1–1 cells is preceded by a burst of action currents, and the amplitude of each Ca2+ oscillation depends on the duration of the accompanying action current burst (Costantin and Charles 1999). The increases in Ca2+oscillation frequency and amplitude caused by Ba2+ and TEA, respectively, were further studied by simultaneous measurement of action currents and Ca2+ oscillations. The effects of 40, 140, and 700 μM Ba2+ on action currents and Ca2+ oscillations are shown (Fig.9). Ba2+ caused the typical increase in the frequency of Ca2+oscillations as well as an increase in the basal levels of Ca2+. After 40 and 140 μM Ba2+, a discrete burst of action currents continued to underlie each Ca2+ oscillation. Addition of 700 μM Ba2+ caused a large sustained increase in [Ca2+]i, while the action current bursting activity continued. The simultaneous recording configuration was then used to investigate the effects of TEA on action current bursting. The effects were consistent with each Ca2+ oscillation being accompanied by a discrete burst of action currents. Figure 10shows two experiments where TEA was added during simultaneous recording. TEA caused a train of Ca2+oscillations that was accompanied by a train of action current bursts (Fig. 10 A). A TEA-induced train of action current bursts also occurred in the cell shown in Fig. 10 B; in this experiment the Ca2+ oscillations stacked to form what appeared to be a single large Ca2+oscillation. On closer examination, the small peaks seen in the Ca2+ oscillation tracing correspond to the individual action current bursts shown in the on-cell recording. In addition to causing a train of action currents, TEA increased the duration of individual action current bursts. The action current burst duration in Fig. 10 A increased from 0.69 ± 0.12 s (n = 4) to 1.48 ± 0.43 s (n= 6), while the burst duration in Fig. 10 B increased from 0.67 ± 0.28 s (n = 10) to 2.13 ± 0.99 s (n = 14). Apamin and CTx, toxins which specifically inhibit the SK and BK Ca2+-activated K+ channels, respectively, were tested for their effect on Ca2+ signaling (data not shown). In most cells, apamin had no effect. In a small subset of cells (<5%), apamin (1 μM) increased the amplitude of the Ca2+ oscillations without the initial train of Ca2+ oscillations caused by TEA. CTx had no effect in concentrations between 10 and 500 nM. Concentrations of CTx above 1 μM caused a sustained increase in [Ca2+]i with no oscillations (data not shown). At these concentrations, CTx is not specific for Ca2+-activated K+ channels but blocks other voltage-gated K+ channels as well (Perez-Cornejo et al. 1998).
These studies demonstrate that Ba2+ and TEA selectively inhibited two different K+ currents in GT1–1 neurons and that these K+ channel blockers also caused distinct changes in the frequency or amplitude of Ca2+ oscillations. Ba2+inhibited I K(IR) currents which are active near the resting potential, while TEA inhibited depolarization-activated K+ currents opened by the firing of action potentials. Biochemical regulation targeting these two populations of K+ channels in GnRH secreting neurons could provide a mechanism underlying the episodic fluctuations in electrical activity of GnRH neurons that has been linked to the pulsatile release of GnRH.
Spontaneously active cells maintain a delicate balance between small inward and outward currents that lead to pacemaker potentials. Multiple inward currents may contribute to the pacemaker potentials in GnRH neurons, including a persistent TTX-insensitive Na+ current (Oka 1995, 1996), a capacitative Ca2+ entry current (Van Goor et al. 1999a), and a cAMP-gated channel current (Vitalis et al. 2000). These inward currents are offset by the outward current through K+ channels, including the inward rectifier K+ channel, which is open near the resting potential. GT1–1 neurons typically exhibit bursts of action potentials separated by periods of quiescence (Costantin and Charles 1999). The increase in frequency of action potential bursts caused by Ba2+ suggests that action potential threshold is reached more rapidly during the pacemaker potential due to blockade of theI K(IR) channel. The frequency increase is analogous to that seen in molluscan ganglion cells caused by the injection of depolarizing current (Connor and Stevens 1971). The frequency of oscillations in spontaneously active GT1–1 neurons increased with the extracellular Ba2+ concentration up to approximately 200 μM Ba2+. At concentrations above 200 μM Ba2+, a secondary increase in baseline Ca2+ began to occur possibly due to a lack of recovery from previous Ca2+ oscillations. This may be due to blockade of depolarization-activated K+ currents in addition to the inward rectifier. Addition of 1 mM Ba2+ caused a blockade of ∼40% of the sustained currents and ∼50% of the transient currents. At this concentration, Ba2+ is less specific and blocks outward current magnitude near the levels of 2 mM TEA and 1.5 mM 4-AP. The addition of Ba2+ to previously quiescent cells consistently initiated Ca2+ oscillations. These results suggest that increasing the extracellular Ba2+ concentration up to 200 μM caused incremental increases in the magnitude of the depolarization. The direct measurement of action currents in the presence of Ba2+ confirmed that each Ca2+ oscillation was accompanied by a burst of action currents. Action potential bursts continued even when there was a sustained increase in [Ca2+]i, indicating that bursts could continue even when Ca2+ clearance mechanisms in the cell were overwhelmed. The increase in oscillation frequency was sustained over many minutes, suggesting that the inhibition of I K(IR) was not compensated for by activation of other K+channels. Modulation of I K(IR) could therefore result in a sustained increase in oscillation frequency that could underlie a sustained pulse of hormone release occurring over several minutes in vivo.
TEA caused an increase in the amplitude of individual Ca2+ oscillations as well as transient effects on the oscillation frequency. Simultaneous recording revealed that TEA caused an increase in the duration of individual bursts of action currents. The longer burst and possibly wider individual action potentials (Tasaki and Hagiwara 1957) would allow increased Ca2+ entry and larger amplitude oscillations. Action potential bursting has been described in a variety of neurons and neuroendocrine cells including molluscan neurons (Adams 1985; Adams and Levitan 1985;Smith and Thompson 1987), pancreatic beta cells (Henquin et al. 1979; Rosario et al. 1993), and mammalian hypothalamic neurons (Andrew 1987a,b; Andrew and Dudek 1984). The mechanisms responsible for the prolonged depolarization that causes bursts are still controversial (Ashcroft and Rorsman 1995). After burst initiation, burst termination may be caused by an accumulation of [Ca2+]i, followed by activation of I K(Ca) channels and subsequent hyperpolarization (Atwater et al. 1983). Alternatively, the bursts may be caused by a plateau potential driven by a slowly inactivating (persistent) inward current (Cook et al. 1991). The increase in burst duration by TEA suggests that burst termination in GT1–1 neurons occurs at least in part by the activation of TEA-sensitive K+ channels. The blockade of these K+ channels by TEA while increasing the burst duration does not eliminate bursting behavior, suggesting that other mechanisms for the bursting are involved or that the fraction of K+ current not blocked by TEA (60–80%) is sufficiently activated to cause burst termination. TEA-sensitive K+ currents have also been shown to contribute to the repolarizing phase of pacemaker potentials (Abe and Oka 1999). The identity of the K+ channels blocked by TEA in GT1–1 neurons are most likely I K(DR) andI K(Ca) channels; theI K(IR) current was unaffected by TEA. Identifying the contribution of I K(DR)versus I K(Ca) in the TEA prolongation of bursts in these cells is difficult given the lack of sensitivity of the I K(Ca) currents to apamin or CTx.
The outward K+ currents presented here have similarities and differences with other reported K+ currents in GnRH neurons. Bosma (1993) has reported in GT1–7 neurons individual cells that have a mixture of transient components and a slow sustained component as well as individual cells with only the slow component present. The predominant current in our cells is consistently a mixture of the slower sustained and transient currents. We did not observe the slow sustained currents as the predominant current in individual cells as reported by Bosma (1993), although we have observed similar currents in cells measured within 2 days after plating. Transient 4-AP and sustained TEA-sensitive current components have been reported in GnRH cells in the fish terminal nerve (Abe and Oka 1999). The I K(Ca) current in GT1–1 neurons constitutes a significant fraction of currents even with 0.5 or 10 mM EGTA in the pipette. They were eliminated by inclusion of 5.5 mM BAPTA in the pipette. Several groups have reported a decrease in the slope conductance of the I-V curve for K+ currents in the region of the reversal potential for voltage-gated Ca2+ channels. The whole-cell currents and the isolatedI K(Ca) I-V curves never displayed the characteristic N-shaped I-V curve as reported in adrenal chromaffin cells (Marty and Neher 1985) and pancreatic β cells (Smith et al. 1990). The small convexity of the I-V relationship reported by Smith et al. (1990) may have been present in some cells but was not obvious. Perhaps this is due to different Ca2+buffering capacities of GT1–1 neurons compared with chromaffin cells or β cells. The I K(Ca) component extracted by the removal of [Ca2+]e was similar to that reported in the sister GT1–7 cell line (Spergel et al. 1996), except we did not find any sensitivity to CTx. The potency of our CTx was confirmed by blockade of BK channels expressed in Xenopus oocytes (data not shown). The presence of CTx-insensitive I K(Ca) channels in GT1–1 neurons is a finding that warrants more detailed investigation. CTx-insensitive BK channels have been reported in rat brain and neurohypophysis (Reinhart et al. 1989; Wang et al. 1992) and a recent report indicates that the presence of particular accessory β subunits can confer varying degrees of sensitivity to the BK channel (Meera et al. 2000). In addition to the increase in Ca2+ oscillation amplitude caused by TEA, there was a robust increase and then decrease in excitability followed by a return to an oscillation period near the control level before TEA addition. The initial increase in excitability is likely due to a rapid inhibition ofI K(DR) andI K(Ca) channels by TEA. The subsequent decrease in excitability may be due to multiple factors. One possibility is that there is a compensatory up-regulation of other K+ conductances that “overshoots” and thereby decreases excitability. This behavior contrasts with the sustained frequency increase in the presence of Ba2+, where there does not appear to be any compensatory response.
A working model for the spontaneous action potential bursting associated with Ca2+ signaling in GT1–1 neurons includes an oscillating membrane pacemaker potential caused by persistent inward currents opposed by outward K+currents including I K(IR). TTX-sensitive action potentials fire with spontaneous depolarization of the membrane potential above threshold. Membrane repolarization occurs by activation of TEA-sensitive I K(DR)and possibly apamin- and CTx-insensitiveI K(Ca) channels. During action potential bursts, accumulation of [Ca2+]i recruits additional I K(Ca), which ultimately terminates the burst. Thus, GT1–1 neurons could achieve a frequency modulation (FM) or amplitude modulation (AM) of Ca2+ oscillations by regulation ofI K(IR) or TEA-sensitiveI K(DR) andI K(Ca) channels, respectively. Extracellular Ca2+ that enters the cell via plasma membrane voltage-gated Ca2+ channels is responsible for the rise in [Ca2+]i with little or no contribution from intracellular stores (Charles and Hales 1995; Charles et al. 1996). GT1–1 neurons may therefore be considered a neuronal model of a relative pure form of electrically driven intracellular Ca2+ signaling (Costantin and Charles 1999). In this model, the modulation of action potential burst duration or burst frequency through the regulation of plasma membrane ion channels directly controls the amplitude or frequency of Ca2+oscillations in individual cells.
Van Goor et al. (1999a) have reported a greater role for intracellular Ca2+ stores and capacitative Ca2+ entry in the electrical activity of GT1 neurons after the application of ligand. Our studies indicate that these sources of Ca2+ are not required for spontaneous Ca2+ signaling. However, we do not exclude the possibility that these Ca2+ sources could modulate the Ca2+ signaling as proposed in our model and could act as a pathway for ligand-evoked modulation of spontaneous signaling. Van Goor et al. (1999b) also have proposed a model in which mode switching in GT1–1 neurons occurs that is dependent on the degree of hyperpolarization between action potentials. They conclude that cells with depolarized baseline membrane potentials have inactivated Na+ channels, whereas relief of inactivation of Na+ channels occurs in cells with more hyperpolarized membrane potentials. The depolarized cells fire broad, low-magnitude Ca2+ action potentials, while the hyperpolarized cells fire narrower, high-magnitude sharper action potentials due the participation of TTX-sensitive Na+ channels. We have not observed TTX-insensitive action currents or Ca2+ signaling in intact cells (Charles et al. 1996; Costantin and Charles 1999; Hales et al. 1994). We conclude that the membrane potential in our cells is sufficiently hyperpolarized to allow voltage-gated Na+channels to play a primary role in spontaneous Ca2+ signaling. However, we do not exclude the possibility that external ligands may result in a different mode of signaling where Ca2+ action potentials play a primary role.
The FM and AM of Ca2+ oscillations has been shown to control cellular functions, including cell differentiation, gene transcription, and secretion (Berridge 1997;Clapham 1995; Putney 1998). FM of Ca2+ oscillations has been shown to regulate protein kinase activity (De Koninck and Schulman 1998), glycogen metabolism in liver (Woods et al. 1986), the transcription of specific genes (Dolmetsch et al. 1998;Li et al. 1998), and the expression of ion channels or neurite extension during neuronal differentiation (Spitzer and Gu 1997). AM of Ca2+ oscillations have recently been shown to regulate differential gene activation in B-lymphocytes (Berridge 1997; Dolmetsch et al. 1997). Our laboratory has recently shown that increased Ca2+ oscillation frequency is correlated with an increase in prolactin release from GH3 cells (Charles et al. 1999). The present studies indicate that particular subsets of K+ channels are capable of underlying specific changes in the FM or AM of Ca2+ signaling in neuroendocrine cells.
AM or FM of Ca2+ oscillations in GnRH neurons may be important in the pulsatile release of GnRH. In vivo studies in rhesus monkey have shown that gonadotropin pulses are correlated with increases in the multiunit electrical activity in the mediobasal hypothalamus, due to an increase in the frequency of action potentials in individual cells (Cardenas et al. 1993). It has also been proposed that episodic release of GnRH may be due to a combination of pulsatile gene expression combined with rapid mRNA turnover (Maurer and Wray 1997). AM or FM of Ca2+ oscillations could lead to pulsatile GnRH secretion by changes in gene expression that increase the amount of hormone produced and secreted or by episodically increasing the frequency of secretory events in individual cells. Potassium channels may therefore represent regulatory targets that modulate pulsatile secretion through their specific effects on patterns of action potential bursts and Ca2+ oscillations.
This work was supported by National Institute of Neurological Disorders and Stroke Grants R29-NS-32283 and P01-NS-02808 to A. C. Charles.
A. C. Charles (E-mail:).
- Copyright © 2001 The American Physiological Society