The erg inwardly rectifying K+ current and its modulation by thyrotrophin-releasing hormone in giant clonal rat anterior pituitary cells

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The erg inwardly rectifying K⁺ current and its modulation by thyrotrophin-releasing hormone in giant clonal rat anterior pituitary cells

Christiane K. Bauer

Physiologisches Institut, Universitätskrankenhaus Eppendorf, Martinistraße 52, D-20246 Hamburg, Germany

Received 7 November 1997; accepted after revision 20 March 1998.

ABSTRACT

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Abstract
Introduction
Methods
Results
Discussion
References

The voltage-dependent inwardly rectifying K⁺ current (I_K,IR) of clonal rat anterior pituitary cells (GH₃/B₆) was investigated in solutions with physiological K⁺ gradient using giant polynuclear cells.

I_K,IR was isolated by the use of the selective erg (ether-à-go-go-related gene) channel blocker E-4031. In external 5 mM K⁺ solution, I_K,IR carried steady-state outward current in the potential range between -60 and 0 mV, with a maximum current amplitude at -40 mV. Negative to the K⁺ equilibrium potential, E_K, large transient inward currents occurred.

A selective pharmacological block of I_K,IR induced a sustained depolarization of the membrane potential when Ca²⁺ action potentials were blocked, confirming the contribution of I_K,IR to the resting membrane potential of GH₃/B₆ cells.

Thyrotrophin-releasing hormone (TRH) reduced effectively the sustained outward and the transient inward I_K,IR. The magnitude of a TRH-induced depolarization of the membrane potential was consistent with an almost complete reduction of I_K,IR.

The results demonstrate that the TRH-induced reduction of I_K,IR is able to mediate the resting potential depolarization, suggesting that the increase in the frequency of action potentials occurring during the second phase of the TRH response in GH cells should be sustained by I_K,IR inhibition. Moreover, this is the first evidence of a ligand-induced physiological modulation of an erg-mediated current.

INTRODUCTION

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Abstract
Introduction
Methods
Results
Discussion
References

Lactotroph cells of the anterior pituitary respond to the hypothalamic neuropeptide thyrotrophin-releasing hormone (TRH) with an increase in prolactin secretion. The underlying intracellular signal cascade activated by TRH has been studied mainly in the somatomammotrophic GH cell lines. Since TRH induces secretion predominantly via an increase in the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i), the TRH-induced biphasic secretory response is mirrored in a biphasic increase in [Ca²⁺]_i, which is accompanied by a biphasic electrical response. The hyperpolarization due to opening of Ca²⁺-dependent K⁺ channels during the first phase results from the release of stored intracellular Ca²⁺ producing a transient rise in [Ca²⁺]_i, whereas the second phase of the electrical response causes or at least contributes to the subsequent plateau-like increase in [Ca²⁺]_i (for reviews, see Ozawa & Sand, 1986; Corrette, Bauer & Schwarz, 1995). The second-phase electrical response is characterized by a depolarization or an onset of action potentials in silent cells and an increase in the frequency of action potentials in spontaneously active cells. These effects have been explained by a decrease in K⁺ conductance (Ozawa & Sand, 1986).

The voltage-dependent inwardly rectifying K⁺ current I_K,IR is suggested to contribute to the resting membrane potential of GH cells (Bauer, Meyerhof & Schwarz, 1990). A similar voltage-dependent inwardly rectifying K⁺ current is present in native rat lactotrophs (Corrette, Bauer & Schwarz, 1996). It has already been shown that I_K,IR can be modulated by TRH (Bauer et al. 1990; Barros, Delgado, del Camino & de la Peña, 1992; Corrette et al. 1996; Barros, del Camino, Pardo, Palomero, Giráldez & de la Peña, 1997), but these studies have been carried out in high external potassium to increase the current amplitude. Under these conditions, the physiologically relevant I_K,IR outward currents could not be investigated.

Recently it has been shown that I_K,IR exhibits a high sensitivity to several class III antiarrhythmic agents, which made it possible to isolate I_K,IR as a drug-sensitive current (Weinsberg, Bauer & Schwarz, 1997; Barros et al. 1997). These antiarrhythmics, including methanesulphonanilides like E-4031 and WAY-123,398, are suggested to be specific blockers of the rapidly activating component of the cardiac delayed rectifier current (I_Kr) and HERG(human ether-à-go-go-related gene) K⁺ channels which are believed to be the basis of I_Kr (Spector, Curran, Keating & Sanguinetti, 1996). I_Kr and the heterologously expressed HERG current exhibit time-dependent inward rectification. This distinctive property is suggested to result from rapid inactivation being faster than activation at depolarized potentials and to recovery from inactivation being faster than deactivation (Shibasaki, 1987; Wang, Liu, Morales, Strauss & Rasmusson, 1997). We have recently cloned the rat homologue of HERG (rerg) and shown that the I_K,IR channel in GH₃/B₆ cells is most probably encoded by rerg (Bauer, Engeland, Wulfsen, Ludwig, Pongs & Schwarz, 1998a). As a result of this finding, the voltage-dependent gating of I_K,IR which has been described up to now using the classical inward rectifier model (e.g. activation for the current increase upon hyperpolarization) will now be described using the HERG current model (e.g. recovery from inactivation for the current increase upon hyperpolarization; Smith, Baukrowitz & Yellen, 1996).

In the present study, specific erg channel blockers were used to investigate I_K,IR in solutions with physiological K⁺ gradients in polynuclear giant GH₃/B₆ cells which exhibit a high I_K,IR amplitude. It was demonstrated that the TRH-induced reduction of I_K,IR occurred within a potential range that is important for the control of the electrical activity of the cells.

Part of this work has been published in abstract form (Bauer, 1997).

METHODS

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Abstract
Introduction
Methods
Results
Discussion
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Cell culture

Giant clonal rat anterior pituitary (GH₃/B₆) cells were routinely cultured in Ham's F10 medium supplemented with 15 % horse serum and 2�5 % fetal calf serum. The cells were incubated at 37�C in an atmosphere of 95 % air and 5 % CO₂. GH₃ cells, which contain fewer giant polynuclear cells, were grown under identical conditions.

Solutions and chemicals

The external '150 mM K⁺' solution contained (mM): 140 KCl, 4 MgCl₂, 1 CaCl₂, 2�5 EGTA, 10 Hepes, 10 glucose, 10 KOH to adjust pH to 7�3. The standard external '5 mM K⁺' solution contained (mM): 5 KCl, 135 NaCl, 4 MgCl₂, 1 CaCl₂, 2�5 EGTA, 10 Hepes, 10 glucose, 10 NaOH to adjust pH to 7�3. TTX (500 nM) was added to both external solutions to block Na⁺ channels. The concentration of free Ca²⁺ was low (estimated free Ca²⁺ was 75 nM; EQCAL, Biosoft, Cambridge, UK) to reduce Ca²⁺ currents and Ca²⁺-dependent K⁺ currents. NiCl₂ was added to the 5 mM K⁺ solution at 200 µM, a concentration which does not affect I_K,IR (Bauer et al. 1990), to block Ca²⁺ channels. Patch pipettes were filled with (mM): 140 KCl, 2 MgCl₂, 1 CaCl₂, 2�5 EGTA, 10 Hepes, 10 glucose, 10 KOH to adjust pH to 7�3. The estimated free Ca²⁺ concentration of the pipette solution was 66 nM (EQCAL). Where indicated, MgCl₂ was omitted; the contamination of this intracellular solution with Mg²⁺ was estimated to be less than 1 µM. The estimated free Mg²⁺ in the normal intracellular solution was 1�8 mM (EQCAL). For most TRH experiments, 2 mM MgATP and 0�2 mM Na₂GTP were added to the intracellular solution. There was no difference in the observed TRH response with and without these nucleotides in the pipette solution.

The antiarrhythmic drugs E-4031 and WAY-123,398 were generous gifts from Eisai, Tokyo, Japan, and from Wyeth-Ayerst, Princeton, NJ, USA, respectively. Both drugs were dissolved in water to yield a stock solution of 10 mM.

Electrophysiology

Cells were used 1-5 days after passaging. Electrophysiological recordings were performed in the whole-cell mode to render the TRH-induced reduction of I_K,IR irreversible (Bauer et al. 1990; Barros, Mieskes, del Camino & de la Peña, 1993). This was necessary to obtain the complete I-V relationship of the TRH-sensitive current component. In addition, voltage-dependent, spontaneous or TRH-induced changes in [Ca²⁺]_i were reduced in the whole-cell recording mode. The pipette resistance ranged from 2�5 to 4 M $Omega$ . Prior to recording, fast and slow capacitances were compensated; the value of the slow capacitance was taken to represent the cell capacitance. Series resistance errors were compensated to at least 70 %. Stimulation, data acquisition and analysis were carried out with an EPC9 patch-clamp amplifier and the 'Pulse' software (HEKA Elektronik, Lambrecht, Germany). All experiments were performed at room temperature (20-26�C).

RESULTS

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Abstract
Introduction
Methods
Results
Discussion
References

Occurrence of giant polynuclear GH₃/B₆ cells and recording of I_K,IR in isotonic KCl

In cell cultures of GH₃/B₆ cells, large round cells of up to 40 µm diameter occur infrequently, but regularly. These cells contain several nuclei (Fig. 1). Recordings of hyperpolarization-elicited K⁺ currents in external 150 mM K⁺ solution demonstrated that these giant cells exhibited high amplitudes of the voltage-dependent inwardly rectifying K⁺ current (I_K,IR) previously described in 'normal' mononuclear GH₃/B₆ cells (Bauer et al. 1990). In the standard pulse protocol, a 2 s depolarizing pulse to +20 mV preceded every test pulse in order to achieve maximum I_K,IR amplitudes due to a maximum steady-state activation of the erg channels (Wang et al. 1997).

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Figure 1. Hyperpolarization-elicited K⁺ currents recorded from a mononuclear and a giant polynuclear GH₃/B₆ cell in isotonic KCl
Left: micrograph of GH₃/B₆ cells with a giant polynuclear cell which contained at least 12 nuclei. Right: membrane currents recorded from a big mononuclear cell (20 pF) and from a giant polynuclear cell (60 pF) in symmetrical (150 mM) K⁺ solution. The K⁺ inward currents were elicited with the standard pulse protocol shown at the top; in this and subsequent figures voltages are given in mV. The complete pulse sequence consisted of variable 200 ms test pulses to potentials between 0 and -120 mV preceded by a 2 s prepulse to +20 mV and a 500 ms pulse to -20 mV. Test pulses were followed by a 100 ms pulse to -20 mV before returning to the holding potential of -40 mV.

A strong dependence of the deactivation process on external divalent cations has been found in two other native erg-mediated currents (Faravelli, Arcangeli, Olivotto & Wanke, 1996; Ho, Earm, Lee, Brown & Noble, 1996), but not in heterologously expressed HERG currents (Wang et al. 1997). Therefore, additional experiments were performed in external 150 mM K⁺ solution containing 2 mM Ca²⁺ and 4 mM Mg²⁺ (but without EGTA) and in a solution without free divalent cations (CaCl₂ and MgCl₂ omitted, 2�5 mM EGTA added). The increase in external free Ca²⁺ made the recordings of I_K,IR more difficult due to an increase in overlapping currents, but did not induce a clear acceleration of the I_K,IR deactivation process ( $tau$ _deact_,-120: 30 � 3�2 ms (n = 4) vs . 35�7 � 4�0 ms in external solution with 75 nM free Ca²⁺(n = 6); time constants obtained by fitting single exponential curves to the current decay during the hyperpolarization to -120 mV; series resistance compensation 90 %). In the absence of external divalent cations, I_K,IR deactivation at potentials more negative than -60 mV was still evident ( $tau$ _deact,-120: 63 � 18 ms, n = 3; series resistance compensation 50-70 %), although the recordings were impeded by huge steady-state and tail currents, probably due to K⁺ permeation through Ca²⁺ channels. These results suggested that I_K,IR deactivation is not very sensitive to external divalent cations.

Isolation of I_K,IR as an E-4031-sensitive current in solutions with a physiological K⁺ gradient

Giant polynuclear GH₃/B₆ cells were used for recording in external 5 mM K⁺ solution because of they exhibit high-amplitude I_K,IR. Figure 2A demonstrates the presence of I_K,IR in the control currents: upon hyperpolarization, a time-dependent current increase occurred, which reversed direction at about -80 mV. Application of 10 µM E-4031, which has been shown to block I_K,IR fast and completely (Weinsberg et al. 1997), abolished this current increase, confirming that it was due to I_K,IR.

Since the rerg channels were most probably 'fully activated' (Wang et al. 1997; Bauer et al. 1998a) by the depolarizing pulse prior to the test pulses, I_K,IR isolated as the E-4031-sensitive current exhibited, during the 200 ms test pulses, sustained outward currents in the potential range between -60 and 0 mV with a maximum amplitude at about -40 mV (Fig. 2C and D). At more negative potentials I_K,IR deactivated and demonstrated a strong voltage dependence of the deactivation kinetics. The I-V relationship of the peak currents showed a clear inward rectification. Similar results were obtained when I_K,IR was isolated as a WAY-123,398-sensitive current.

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Figure 2. Isolation of I_K,IR as an E-4031-sensitive current recorded in physiological K⁺ gradients
A, inward and outward membrane currents recorded from a giant GH₃/B₆ cell (54 pF) in external 5 mM K⁺ solution using the standard pulse protocol described in Fig. 1. The holding potential was -40 mV. B, membrane currents recorded from the same cell as in A after addition of 10 µM E-4031. C, isolation of I_K,IR as the E-4031-sensitive current obtained by subtraction of the currents recorded in the presence of E-4031 (B) from the control currents shown in A. D, I-V plot of the peak and late (measured at the end of the 200 ms test pulses) outward and inward E-4031-sensitive currents shown in C. E, time- and voltage-dependent increase and decrease of E-4031-sensitive currents recorded from two cells using intracellular solutions prepared with differing amounts of MgCl₂. The '0' mM Mg²⁺ solution was estimated to contain less than 1 µM free Mg²⁺. F, I-V plots of the peak and late outward and inward E-4031-sensitive currents recorded with differing amounts of Mg²⁺ in the intracellular solutions; shown are mean current values � S.D. obtained from each 5 giant GH₃/B₆ cells with a comparable range of cell sizes (43�2 � 6�9 pF and 43�6 � 5�5 pF for 1�8 mM and '0' mM Mg²⁺, respectively). In the voltage range from -50 to 0 mV, peak values were omitted because they did not differ from late current values.

In contrast to classical inward rectifiers, the gating of HERG channels has been shown to be independent of intracellular Mg²⁺ (Smith et al. 1996). To investigate the Mg²⁺ dependence of the rerg-mediated I_K,IR, experiments were performed with and without MgCl₂ in the pipette solution. The kinetics of inactivation (time constant, $tau$ _inact) and recovery from inactivation (time constant, $tau$ _recov) were similar in the two groups (Fig. 2E; $tau$ _recov at -40 mV: 6�9 � 1�1 ms vs. 8�0 � 1�3 ms; $tau$ _recov at -50 mV: 7�2 � 1�0 ms vs. 8�6 � 1�4 ms; $tau$ _inact at -20 mV: 11�0 � 2�5 ms vs. 12�8 � 3�1 ms; data obtained for 1�8 mM (n = 5) and '0' mM (n = 5) free Mg²⁺, respectively). Also the voltage dependence of peak and late current amplitudes of I_K,IR (Fig. 2F) did not significantly differ.

A few experiments were performed on GH₃ cells. In this cell line, giant polynuclear cells occurred less frequently. The characteristics of I_K,IR recorded as the E-4031-sensitive or WAY-123,398-sensitive current in 5 mM K⁺ from giant GH₃ cells were identical to those observed in GH₃/B₆ cells.

Correlation of I_K,IR with the membrane potential of the cells

In order to relate a specific block of I_K,IR to changes in membrane potential, experiments were performed in which alternating recordings were made of membrane currents and membrane potential in the voltage-clamp and current-clamp mode, respectively. The standard external 5 mM K⁺ solution was used in which Ca²⁺ action potentials were blocked. Figure 3 shows one of five experiments, where 10 µM WAY-123,398 induced a depolarization which could be clearly attributed to a block of I_K,IR. The average depolarization amounted to 4�4 � 0�96 mV, from -45�6 to -41�2 mV (n = 5). Most experiments of this kind could not be evaluated due to either an increase or a decrease in the amplitude of a hyperpolarization-activated current (I_h) during the critical period of membrane potential recording.

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Figure 3. Block of I_K,IR depolarizes the membrane potential
Recordings of membrane currents and membrane potential from a 70 pF giant GH₃/B₆ cell in external 5 mM K⁺ solution. The voltage-clamp recordings in the absence (A) and presence of WAY-123,398 (C) were separated by a period in which the effect of 10 µM WAY-123,398 on the membrane potential was monitored in the current-clamp mode (B).

Effects of TRH on I_K,IR

Membrane currents of giant GH₃/B₆ cells were recorded with the standard pulse protocol before and about 3 min after the addition of TRH. In between, the TRH response was traced with a double-pulse sequence applied every 10 s. The standard protocol lasted less than 1 min and TRH was applied within 3 min of patch rupture to enable measurement of the effects of TRH on I_K,IR in the whole-cell recording mode (Barros et al. 1993). The time course of the TRH-induced reduction of I_K,IR was similar to that previously described for experiments in isotonic KCl (Bauer, Davison, Kubasov, Schwarz & Mason, 1994), with a maximal reduction of I_K,IR achieved about 150 s after the application of TRH. Figure 4A-D shows that the TRH-sensitive current closely resembled I_K,IR, isolated as the E-4031-sensitive current. This implies that TRH selectively reduced I_K,IR under these experimental conditions, and that this effect occurred over the whole voltage range.

The amount of TRH-induced I_K,IR reduction is most probably underestimated in the above experiments, since they were carried out with a depolarizing prepulse and we have previously shown that the magnitude of a TRH-induced reduction of I_K,IR can be decreased by a preceding depolarization (Bauer et al. 1990). Double pulses with and without a prepulse to +20 mV (see Fig. 4, top right) were used to investigate whether this phenomenon also occurred in cells in 5 mM K⁺ solution exhibiting outward I_K,IR. Before application of TRH, the membrane currents recorded with and without a depolarizing prepulse were almost identical (Fig. 4E) in the majority of the cells. TRH reduced inward and outward membrane currents and a difference between current traces elicited with and without prepulse became evident (Fig. 4F). Both the TRH-sensitive and the small prepulse-sensitive current components (Fig. 4G and H) exhibited characteristic inactivation of I_K,IR upon depolarization and recovery upon hyperpolarization. In most experiments of this kind, after application of TRH, the current response to hyperpolarization to -100 mV without preceding depolarization resembled tail currents only (Fig. 4F), with no indication of the characteristic hook representing recovery from inactivation which is observed with erg channels. This suggested that, in the absence of depolarizing pulses to potentials more positive than those occurring physiologically in GH₃/B₆ cells, TRH produces an almost complete reduction of I_K,IR.

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Figure 4. TRH selectively reduces I_K,IR
A, membrane currents were recorded in a giant GH₃/B₆ cell (39 pF) using the standard pulse protocol described in Fig. 1. The holding potential was -40 mV. B, membrane currents recorded from the same cell as in A about 3 min after the application of 500 nM TRH. C, TRH-sensitive current obtained by subtraction of the currents recorded after the addition of TRH (B) from the control currents shown in A. D, I-V plot of the peak and late outward and inward TRH-sensitive currents shown in C. E-H, time- and voltage-dependent increase and decrease of membrane currents recorded from a 43 pF giant cell before (E) and after (F) addition of 500 nM TRH. The TRH-sensitive current (G) and the prepulse-sensitive current after TRH (H) were obtained by subtraction (G = E - F, H = F* - F). The asterisks mark current traces elicited by pulse sequences with depolarizing prepulses. The sequence of voltage pulses is shown on top. A sequence with a 1�5 s depolarizing prepulse to +20 mV delivered 2 s before the pulse to -20 mV was followed by two pulse sequences without a prepulse. Pulse sequences were repeated every 10 s.

Another set of experiments was therefore performed to investigate the effects of TRH on the resting membrane potential induced by a specific reduction of I_K,IR. Before and after a period of monitoring the effects of TRH application in the current clamp mode, membrane currents were recorded in the standard 5 mM K⁺ solution using a holding potential of -20 mV, a potential at which rerg currents are almost 'fully activated' and no deactivation occurs (Bauer et al. 1998a). The pulse protocol consisted of 200 ms test pulses to different potentials followed by a short pulse to -100 mV. Using this protocol, an estimation could be made of the deactivation of I_K,IR during the test pulses by measuring the amplitude of the current which could be recovered during hyperpolarization. In experiments in which the presence of I_K,IR was clearly visible in the control currents (Fig. 5A), TRH (500 nM or 1 µM) induced an irreversible depolarization of the membrane potential (Fig. 5B). The depolarization induced by TRH amounted to 4�4 � 0�4 mV (n = 3; values obtained from the experiment shown in Fig. 5 and two comparable experiments), a value which is similar to the depolarizing effect of WAY-123,398 and which was due to a complete block of I_K,IR. In one further experiment, where I_K,IR clearly dominated the control currents, the TRH-induced depolarization amounted to 7�3 mV. A distinct TRH-induced first-phase hyperpolarization was observed in only one of these experiments, suggesting that the recording conditions (EGTA-buffered low Ca²⁺ concentrations in the extracellular and the pipette solutions) inhibited a rise in [Ca²⁺]_i produced by the TRH-induced release of Ca²⁺ from intracellular stores. Membrane currents recorded after the addition of TRH showed no indications of the presence of I_K,IR (Fig. 5C) in three of four experiments. The TRH-sensitive currents always exhibited the characteristic properties of I_K,IR (Fig. 5E), demonstrating that the TRH-induced depolarizations were produced by a selective inhibition of I_K,IR. As already shown in the above experiments, a long depolarizing prepulse was able to partially restore I_K,IR (Fig. 5D and F). This effect might be explained by a strong TRH-induced shift of the voltage dependence of activation of I_K,IR to more positive potentials. This was indicated by a shift to more positive potentials of the inflection points of Boltzmann functions fitted to the values of the peak inward currents during hyperpolarization to -100 mV after the addition of TRH (Fig. 5F, from -59�6 to -48�2 mV). Due to contamination by tail currents resulting from the deactivation of other K⁺ currents (visible in Fig. 5C and F : cir ), these curves do not accurately represent the voltage dependence of I_K,IR activation. For the TRH-sensitive currents, the mean value of the inflection point of the fitted Boltzmann functions was -78�2 � 2�9 mV (n = 3).

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Figure 5. The TRH-induced reduction of I_K,IR depolarizes the membrane potential
Recordings of membrane currents and membrane potential from a 38 pF giant GH₃/B₆ cell in external 5 mM K⁺ solution. The voltage-clamp recordings shown in A and C were separated by a period in which the effect of 1 µM TRH on the membrane potential was monitored in the current-clamp mode (B). Membrane currents in A and C were elicited by the pulse protocol shown below A without a depolarizing prepulse, whereas the membrane currents in D were obtained with a 2 s depolarizing prepulse to +20 mV. The gap between prepulse and test pulses was 500 ms. The holding potential was -20 mV. E, TRH-sensitive current obtained by subtraction of the currents recorded after the addition of TRH, shown in C, from the control currents, shown in A; note the expanded current scale in E. F, peak amplitudes of the currents obtained with the constant 20 ms pulse to -100 mV are plotted against the preceding test pulse potential. Continuous and dotted lines represent Boltzmann functions fitted to the data points; values for the inflection point and steepness are, respectively: -59�6 mV and -17�5 mV (Control; continuous line), -48�2 mV and -11�5 mV (TRH*), -75�1 mV and -9�15 mV (TRH-sensitive). Please note that the current level at -100 mV differed slightly in A and D due to a moderate activation of I_h in D.

DISCUSSION

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Abstract
Introduction
Methods
Results
Discussion
References

The investigation of I_K,IR in solutions with a physiological K⁺ gradient was enabled by the combined use of erg-specific pharmacological blockers and giant polynuclear GH₃/B₆ cells, which exhibit a high I_K,IR amplitude. It could be demonstrated that I_K,IR reaches its maximum outward value in the range of the resting potential of these cells, and that TRH drastically reduces the physiologically relevant sustained outward I_K,IR. Nevertheless, in future investigations of I_K,IR in normal-sized cells, external solutions with elevated K⁺ concentrations will need to be used because of the low I_K,IR density.

Another endogenous erg current has been described in neuroblastoma cells using a high-K⁺ external solution (Arcangeli et al. 1995), and recently this erg current has been shown to be crucial for spike-frequency adaptation in F-11 dorsal root ganglion neurone × neuroblastoma hybrid cells (Chiesa, Rosati, Arcangeli, Olivotto & Wanke, 1997). Like neuroblastoma cells, lactotroph cells lack classical inward rectifier currents. Therefore I_K,IR is able to contribute to the resting potential of these cells which ranges from about -60 to -40 mV.

In giant GH₃/B₆ cells, the average I_K,IR density at -40 mV amounted to only 1�5 pA pF^-1 (64 pA/43 pF). Therefore, the importance of I_K,IR depends strongly on the input resistance of the cells, which is probably linked to their physiological state. In cells with high basal [Ca²⁺]_i, for example, Ca²⁺-dependent K⁺ channels contribute essentially to the resting conductance of the cells. This might explain why Weinsberg et al. (1997) observed an effect of E-4031 on the membrane potential of normal GH₃/B₆ cells in only two out of three experiments. In comparable experiments performed in giant GH₃/B₆ cells, only one out of nine cells did not respond to E-4031, and TRH was unable to produce a second phase in this cell (A. Winkelmann, C. K. Bauer & J. R. Schwarz, unpublished observations). The lack of a second phase in the TRH response has been described in about one out of six GH cells (reviewed in Corrette et al. 1995).

Pharmacological block of I_K,IR induced only a moderate depolarization of the membrane potential of giant GH₃/B₆ cells, from about -45�5 to -41 mV. Nevertheless, the fact that the threshold for the initiation of action potentials is close to -40 mV in these cells, just the potential where I_K,IR has its maximum outward current, suggests that this level of depolarization could be highly effective. In the physiological voltage range, TRH almost completely reduced outward and inward I_K,IR and induced a depolarization of the membrane potential comparable to that induced by complete pharmacological block of I_K,IR. Since the TRH-sensitive current is almost identical to I_K,IR, TRH exerts its maximum I_K,IR-mediated effects on the resting conductance in a voltage range critical for the electrical activity of the cell. These data, together with the sensitivity of the TRH-induced reduction of I_K,IR to a depolarizing prepulse, suggest that the I_K,IR-mediated effects of TRH are most apparent in silent cells.

Normal rat lactotrophs have been shown to possess an inwardly rectifying K⁺ current quite similar to I_K,IR (Corrette et al. 1996). Using E-4031 as a specific blocker of this current, we have recently found that this inwardly rectifying K⁺ current is involved in the maintenance of the resting potential of normal lactotrophs and in the control of prolactin secretion (Bauer et al. 1998b).

In cardiac cells, erg channels are involved in action potential repolarization, and a selective block of I_Kr results in a prolongation of the action potential (Curran, Splawski, Timothy, Vincent, Green & Keating, 1995). This proven role for HERG channels suggests an additional new role for the rerg-mediated I_K,IR: the reduction of I_K,IR by TRH might help to mediate a prolongation of Ca²⁺ action potentials, an effect which occurs often during the second phase of the TRH response. Up to now, this effect has been suggested to be mediated solely by an inhibition of the Ca²⁺- and voltage-dependent K⁺ channel BK (reviewed in Corrette et al. 1995).

The mechanism by which TRH exerts its effects on I_K,IR is not known, but it has been suggested that it is mediated by a phosphorylation, which is produced neither by protein kinase C nor by protein kinase A (Barros et al. 1993; Bauer et al. 1994). The irreversibility of the TRH-induced reduction of I_K,IR in the whole-cell recording mode has been proposed to be due to a wash-out of phosphatases, because the reduction of I_K,IR by TRH is completely reversible in perforated-patch experiments, and is partially reversible in whole-cell experiments where protein phosphatase 2A has been added to the pipette solution (Barros et al. 1993). The assumption that TRH-induced phosphorylation of erg channels mediates the reduction of I_K,IR agrees well with the present results, which indicate that TRH shifts the voltage dependence of I_K,IR activation to more positive potentials, since phosphorylation of voltage-dependent ion channels has been suggested to result in a shift of the activation curve towards more positive potentials simply by electrostatic interactions (Perozo & Bezanilla, 1990). The partial reversal of the TRH-induced reduction of I_K,IR by a depolarizing prepulse is reminiscent of the depolarization-induced reversal of G-protein-mediated inhibition of Ca²⁺ currents (reviewed by Dolphin, 1996). This phenomenon has been interpreted as depolarization-induced dissociation of a complex formed by direct interaction of inhibitory G-proteins with the Ca²⁺ channels. However, it is suggested that the (much slower) TRH-induced reduction of I_K,IR is not a membrane-delimited event and involves a diffusible second messenger, since the magnitude of the TRH response decreases with prolonged intracellular dialysis during whole-cell recordings (Barros et al. 1993).

Recently, it has been suggested that the cardiac I_Kr is mediated by a minK-HERG complex (McDonald et al. 1997). MinK mRNA is not present in GH₃/B₆ cells (I. Wulfsen, J. R. Schwarz & C. K. Bauer, unpublished observations). This suggests a direct modulatory action of a TRH-activated second messenger on the rerg protein. Up to now, I_K,IR is the only example of an erg-mediated current which is modulated by a ligand. This implies that the importance of the TRH-induced reduction of I_K,IR is not confined to the study of prolactin secretion, and GH₃/B₆ cells might serve as a model system for the study of neuropeptide-induced modulation of erg channels.

				M. Mewe, I. Wulfsen, A. M. E. Schuster, R. Middendorff, G. Glassmeier, J. R. Schwarz, and C. K. Bauer Erg K+ channels modulate contractile activity in the bovine epididymal duct Am J Physiol Regulatory Integrative Comp Physiol, March 1, 2008; 294(3): R895 - R904. [Abstract] [Full Text] [PDF]

				N. M. Kirchberger, I. Wulfsen, J. R. Schwarz, and C. K. Bauer Effects of TRH on heteromeric rat erg1a/1b K+ channels are dominated by the rerg1b subunit J. Physiol., February 15, 2006; 571(1): 27 - 42. [Abstract] [Full Text] [PDF]

				A. Secondo, A. Pannaccione, M. Cataldi, R. Sirabella, L. Formisano, G. Di Renzo, and L. Annunziato Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents Am J Physiol Cell Physiol, January 1, 2006; 290(1): C233 - C243. [Abstract] [Full Text] [PDF]

				P. Miranda, T. Giraldez, P. de la Pena, D. G Manso, C. Alonso-Ron, D. Gomez-Varela, P. Dominguez, and F. Barros Specificity of TRH receptor coupling to G-proteins for regulation of ERG K+ channels in GH3 rat anterior pituitary cells J. Physiol., August 1, 2005; 566(3): 717 - 736. [Abstract] [Full Text] [PDF]

				P. Sturm, S. Wimmers, J. R Schwarz, and C. K Bauer Extracellular potassium effects are conserved within the rat erg K+ channel family J. Physiol., April 15, 2005; 564(2): 329 - 345. [Abstract] [Full Text] [PDF]

				M. Lecchi, E. Redaelli, B. Rosati, G. Gurrola, T. Florio, O. Crociani, G. Curia, R. R. Cassulini, A. Masi, A. Arcangeli, et al. Isolation of a Long-Lasting eag-Related Gene-Type K+ Current in MMQ Lactotrophs and Its Accommodating Role during Slow Firing and Prolactin Release J. Neurosci., May 1, 2002; 22(9): 3414 - 3425. [Abstract] [Full Text] [PDF]

				S.-N. Wu, Y.-K. Lo, B. I.-T. Kuo, and H.-T. Chiang Ceramide Inhibits the Inwardly Rectifying Potassium Current in GH3 Lactotrophs Endocrinology, November 1, 2001; 142(11): 4785 - 4794. [Abstract] [Full Text] [PDF]

				F. Van Goor, D. Zivadinovic, and S. S. Stojilkovic Differential Expression of Ionic Channels in Rat Anterior Pituitary Cells Mol. Endocrinol., July 1, 2001; 15(7): 1222 - 1236. [Abstract] [Full Text] [PDF]

				A. Secondo, M. Taglialatela, M. Cataldi, G. Giorgio, M. Valore, G. Di Renzo, and L. Annunziato Pharmacological Blockade of ERG K+ Channels and Ca2+ Influx through Store-Operated Channels Exerts Opposite Effects on Intracellular Ca2+ Oscillations in Pituitary GH3 Cells Mol. Pharmacol., November 1, 2000; 58(5): 1115 - 1128. [Abstract] [Full Text]

				M. E. Freeman, B. Kanyicska, A. Lerant, and G. Nagy Prolactin: Structure, Function, and Regulation of Secretion Physiol Rev, October 1, 2000; 80(4): 1523 - 1631. [Abstract] [Full Text] [PDF]

				H. I. Akbarali, H. Thatte, X. D. He, W. R. Giles, and R. K. Goyal Role of HERG-like K+ currents in opossum esophageal circular smooth muscle Am J Physiol Cell Physiol, December 1, 1999; 277(6): C1284 - C1290. [Abstract] [Full Text] [PDF]

				J. R. Schwarz and C. K. Bauer The ether-a-go-go-Related Gene K+ Current: Functions of a Strange Inward Rectifier Physiology, August 1, 1999; 14(4): 135 - 142. [Abstract] [Full Text] [PDF]