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(Circulation Research. 1996;79:302-309.)
© 1996 American Heart Association, Inc.

Articles

Angiotensin II Type 2 Receptor–Mediated Regulation of Rat Neuronal K⁺ Channels

Jeffrey R. Martens, Desuo Wang, Colin Sumners, Philip Posner, Craig H. Gelband

the Department of Physiology, University of Florida, College of Medicine, Gainesville.

Correspondence to Craig H. Gelband, PhD, Department of Physiology, University of Florida, College of Medicine, PO Box 100274, Gainesville, FL 32610. E-mail gelband@phys.med.ufl.edu.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have previously shown that angiotensin II (Ang II), via AT₂ receptors, increases whole-cell K⁺ current in cultured rat hypothalamus and brain stem neurons. We have now investigated the AT₂ receptor–mediated effects of Ang II on the activity of single delayed rectifier K⁺ channels in cell-attached membrane patches. In control recordings (bath, 5.4 mmol/L K⁺; pipette, 140 mmol/L K⁺), two voltage-dependent channels were recorded with conductances of 34±4 and 56±6 pS, respectively (n=6). When patches were excised, the channels reversed near a membrane potential expected for a K⁺ channel. In cell-attached patches (-40 mV), Ang II (100 nmol/L) increased open probability of the 56-pS K⁺ channel from 0.03±0.01 to 0.21±0.05 (n=3). The selective AT₂ receptor antagonist PD 123319 (1 µmol/L) but not the AT₁ receptor antagonist losartan (1 µmol/L) blocked the actions of Ang II (n=3). The selective AT₂ receptor agonist CGP 42112 (100 nmol/L) produced similar effects to Ang II. Kinetic analysis of the Ang II effect showed that open-time histograms were best fit by two exponential functions. Ang II increased both open-time constants relative to control (control, ₁=0.9±0.1 milliseconds, ₂=2.3±0.3 milliseconds; Ang II, ₁=3.1±0.4 milliseconds, ₂=12.1±2.4 milliseconds), and PD 123319 blocked this effect (n=3). The closed-time histogram was not affected by Ang II, PD 123319, or losartan. These results suggest that activation of AT₂ receptors modulates rat hypothalamus and brain stem neuronal whole-cell K⁺ current by increasing the open probability of a 56-pS K⁺ channel.

Key Words: angiotensin II • K⁺ channel • neuron • open probability

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
It is generally accepted that there are two major subtypes of Ang II receptors. Ang II type 1 (AT₁) receptors mediate the regulatory effects of Ang II on many biological events, including blood pressure, drinking, and aldosterone release.^{1 2} In contrast, the physiological functions and signal transduction pathways associated with Ang II type 2 (AT₂) receptors have not been fully delineated.³ In the brain, activation of AT₂ receptors may play a role in the regulation of neuronal cell development^{4 5} and the control of neuronal excitability.⁶ Recent novel evidence suggests that activation of AT₂ receptors plays a role in a number of hemodynamic mechanisms, including vasodilation,^{7 8} cerebrovascular resistance,⁹ and water clearance.¹⁰ Until recently, the molecular identity of the AT₂ receptor was completely unknown. Cloning studies using fetal rat and pheochromocytoma (PC12W) cell expression libraries have revealed that the AT₂ receptor belongs to the guanine nucleotide (GTP) binding protein–coupled class of receptor, which was sensitive to pertussis toxin.^{11 12} We have previously used neurons cultured from the hypothalamus and brain stem of neonatal rats to investigate the pharmacological, molecular, and functional properties of AT₂ receptors.^{13 14} These cultured neurons contain high densities of AT₂ receptors that are similar, from both pharmacological and molecular standpoints, to the cloned AT₂ receptors and also to AT₂ receptors present in other tissues and cells.^{11 12 13 14}

In previous studies, we investigated the receptor-mediated effects of Ang II on membrane ionic currents in cultured neurons. We determined that Ang II, via AT₂ receptors, stimulates I_K in cultured neurons.⁶ Furthermore, we have shown that this Ang II effect involves activation of a pertussis toxin–sensitive GTP binding protein (G_i) and a serine/threonine protein phosphatase type 2A (PP2A).¹⁵ The effects of Ang II can be mimicked by intracellular application of a peptide that corresponds to the third transmembrane loop of the cloned AT₂ receptor.¹⁶ In spite of pharmacological, molecular, and electrophysiological similarities, we had no direct evidence of the type of single K⁺ channel that is coupled to activation of the AT₂ receptor. Experiments were performed in cell-attached patches to examine the effect of Ang II on K⁺ channel activity. Both Ang II and the selective AT₂ receptor agonist CGP 42112 increased NP_o of a 56-pS K⁺channel. These effects were blocked by PD 123319 (an AT₂ receptor antagonist) but not by losartan (an AT₁ receptor antagonist). These results suggest that Ang II, through activation of AT₂ receptors, can modulate single K⁺ channel activity and that this channel may underlie the increase in I_K whole-cell current observed with Ang II.⁶

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
One-day-old Sprague-Dawley rats were obtained from our breeding colony, which originated from Charles River Farms (Wilmington, Mass). Losartan potassium was generously provided by Dr Ronald D. Smith of Du Pont-Merck (Wilmington, Del). PD 123319 was purchased from Research Biochemicals Inc. CGP 42112 was obtained from Bachem. DMEM was obtained from GIBCO. Crystallized trypsin (xl) was from Cooper Biomedical. PDHS, ARC, DNase I, poly-L-lysine (molecular weight, 150 000), Ang II, ATP, and HEPES were purchased from Sigma Chemical Co. All other chemicals were purchased from Fisher Scientific.

Preparation of Neuronal Cultures
Neuronal cocultures were prepared from the brain stem and a hypothalamic block of 1-day-old Sprague-Dawley rats exactly as described previously.¹⁴ Trypsin and DNase I–dissociated cells were resuspended in DMEM containing 10% PDHS and were plated in poly-L-lysine–precoated 35-mm Costar plastic tissue culture dishes. Cells were grown for 3 days at 37°C in a humidified incubator with 90% air/10% CO₂. They were then exposed to 1 µmol/L ARC for 2 days in fresh DMEM/10% PDHS. After this time, the ARC was removed, and the cells were incubated with fresh DMEM/10% PDHS for a further 9 to 12 days before use. At the time of use, cultures consisted of 90% neurons and 10% astrocyte glia, as determined by immunofluorescent staining with antibodies against neurofilament proteins and glial fibrillary acidic proteins.¹⁴

Electrophysiological Recordings
Single-channel recordings were performed at room temperature using the cell-attached and inside-out patch configurations of the patch-clamp recording technique in cultured neurons.¹⁷ Cells were bathed in Krebs' solution containing (mmol/L) NaCl 140, KCl 3.9, CaCl₂ 1.8, KH₂PO₄ 1.5, glucose 5.5, MgCl₂ 1.2, and HEPES 10. Neuronal cultures were superfused within the culture dish (2.5-mL volume) at a rate of 2.0 mL/min. Unitary currents were detected using an Axopatch-1D patch-clamp amplifier (Axon Instruments) and recorded onto VHS tape (Vetter 420, A.R. Vetter Co Inc) for subsequent off-line analysis. Recording electrodes had resistances of 3 to 5 M. The pipette solution contained (mmol/L) KCl 140, CaCl₂ 1.5, MgCl₂ 1.0, glucose 5.5, and HEPES 1.0. Seals of <5-G resistance were discarded. Single-channel currents were filtered at 2 kHz and digitized at 10 kHz. By convention, outward and inward currents are depicted by upward and downward deflections of the current trace, respectively. Values for NP_o and mean open times were obtained from 3-minute steady state recordings of data. Data analysis was performed with pCLAMP 6.0.2 software (Axon Instruments). Single-channel openings were identified by an algorithm that uses both amplitude and slope information, measured with an interactive threshold detection program in the pCLAMP software. The threshold for detecting events was set at 50% of the expected single-channel amplitude. Mean open times, NP_o, and amplitude histograms were calculated from values obtained from this program. Specifically, NP_o was determined by using the following equation:

where t_s is the total time spent at each current level corresponding to s=1, 2, . . . n, and T is the total time of the recording. pCLAMP 6.0.2 allows the investigator to select up to four single-channel current amplitude levels based on 50% of the expected single-channel amplitude. Using this algorithm, the conductance of the single channel affected by Ang II could be identified. All histograms illustrated were based on this analysis criterion.

Statistics
Results are expressed as mean±SEM. Statistical significance was evaluated by using Student's t test for unpaired observations. Differences were considered significant at P<.05; n corresponds to the number of cells examined.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell-attached patches were used to assess the ability of Ang II to regulate K⁺ channels in neuronal cultured cells. Fig 1 shows a representative example of the types of K⁺ channels recorded at different pipette voltages. When the membrane potential in the patch pipette was depolarized or hyperpolarized, two levels of single-channel current were apparent. The two single-channel current amplitudes are marked by the dashed lines. Single-channel currents appear to reverse at a transmembrane potential of 0 mV, which under our recording conditions is the reversal potential for K⁺. Mean current-voltage relationships for the two conductances when 140 mmol/L K⁺ was in the patch pipette are illustrated in Fig 2A. The conductance values for the two channel types under these conditions were 34±4 and 56±6 pS, respectively (n=12). When the patch was excised, the channels had conductance values of 12±2 and 25±3 pS (Fig 2B). Under these conditions, both single-channel conductances reversed at membrane potentials expected for a K⁺ channel (more positive than +70 mV under these conditions, n=5). Both single channels showed voltage-dependent activation upon depolarization. Fig 2C illustrates the mean NP_o-voltage relationship for the 56-pS channel. The data were well fit by a Boltzmann function. Based on the above experimental data, the two types of conductances recorded in membranes of neuronal cultures most likely represent voltage-dependent K⁺ channels.

View larger version (51K): [in this window] [in a new window]   Figure 1. Unitary K+ channels in cultured neonatal neurons. Representative traces from cell-attached patch experiments with 5 mmol/L K+ in the bath and with 140 mmol/L K+ in the patch pipette. Two different channels appear in the traces at depolarized and hyperpolarized membrane potentials. The two open channel levels (O1 and O2) are marked by dashed lines, and the closed level (C) is marked by a solid line.

View larger version (17K): [in this window] [in a new window]   Figure 2. Current-voltage and NPo-voltage relations for the single-channel currents recorded. A, Graph showing mean current-voltage relations for the small-conductance () and large-conductance () channels (n=6 patches). Mean slope conductances () were 34 and 56 pS, respectively. Data points were fit by linear regression. B, Graph of experiments using inside-out patches, showing mean current-voltage relations for small-conductance () and large-conductance () channels (n=3); SEM for some points is not shown, since it was smaller than the symbol. Mean slope conductances () were 12 and 25 pS, respectively. Data points were fit by linear regression. C, Graph of mean NPo-voltage relation of the 56-pS K+ channel in panel A (n=3).

We next investigated the actions of bath application of Ang II (100 nmol/L) on the activity of the two voltage-dependent K⁺ channels. Fig 3 shows the effect of Ang II on both single-channel recordings and single-channel current amplitude histograms. In this experiment (transmembrane potential, -40 mV), the 56-pS K⁺ channel had a NP_o of 0.02. Application of Ang II increased the NP_o of the 56-pS K⁺ channel to 0.13. The mean effect of Ang II was to increase NP_o of the 56-pS K⁺ channel from 0.03±0.01 to 0.21±0.05 (P<.05, n=6). Ang II had no significant effect on the NP_o of the 34-pS K⁺ channel (control, 0.11±0.03; Ang II, 0.08±0.02). No effect of Ang II on the single-channel current amplitude was observed for either K⁺ channel.

View larger version (25K): [in this window] [in a new window]   Figure 3. Effect of Ang II on single K+ channels. Left, Cell-attached patches containing K+ channels in control. Right, Unitary K+ channels during bath application of 100 nmol/L Ang II. At the top are recordings in the presence of 1 µmol/L losartan at a transmembrane potential of -40 mV. At the bottom are amplitude histograms. NPo, shown for the 56-pS K+ channel, shows a significant increase upon application of Ang II.

We next examined the pharmacological sensitivity of the Ang II response by using a selective AT₂ receptor antagonist, PD 123319, and a selective AT₁ receptor antagonist, losartan. Losartan (1 µmol/L) had no effect on control K⁺ channel activity; therefore, it was used in the bath during all future recordings to minimize the contribution of Ang II on activation of AT₁ receptors that might be present. Fig 4 shows single-channel records and single-channel current amplitude histograms in control and in the presence of Ang II and Ang II+PD 123319 (1 µmol/L). In control recordings (transmembrane potential, -40 mV), both large- and small-conductance channels were recorded. The NP_o of the 56-pS channel was 0.03 in this experiment. Ang II increased NP_o to 0.18, and an application of PD 123319 reversed the actions of Ang II close to control values (0.02). In a number of experiments, Ang II significantly increased NP_o of the 56-pS K⁺ channel from 0.03±0.01 to 0.19±0.03 (Fig 5A, P<.05, n=3). PD 123319 in the presence of Ang II significantly decreased NP_o to 0.07±0.02 (Fig 5A, P<.05, n=3). No significant effect of Ang II or PD 123319 was associated with the 34-pS K⁺ channel (control, 0.11±0.03; Ang II, 0.08±0.02; and PD 123319, 0.12±0.03). Similarly, no effect of PD 123319 was observed in control recordings.

View larger version (38K): [in this window] [in a new window]   Figure 4. Effect of PD 123319 on Ang II–stimulated K+ channel activity in cell-attached membrane patches. Top, Control recordings at a transmembrane potential of -40 mV. Middle, Recordings during bath application of 100 nmol/L Ang II. Bottom, Recordings during application of Ang II and 1 µmol/L PD 123319. On the left are representative traces. On the right are amplitude histograms showing the NPo of the 56-pS K+ channel.

View larger version (29K): [in this window] [in a new window]   Figure 5. Mean increase in NPo of the 56-pS K+ channel in the presence of Ang II or CGP 42112. A, Ang II (100 nmol/L) significantly increased NPo, and PD 123319 reversed the Ang II effect (*P<.05, n=3). B, CGP 42112 (100 nmol/L) significantly increased NPo, and PD 123319 reversed the CGP 42112 effect (*P<.05, n=3).

View larger version (25K): [in this window] [in a new window]   Figure 6. Effect of PD 123319 on CGP 42112–stimulated K+ channel activity in cell-attached membrane patches. Top, Control recordings at a transmembrane potential of -20 mV. Middle, Recordings during bath application of 100 nmol/L CGP 42112. Bottom, Recordings during application of CGP 42112 and 1 µmol/L PD 123319. All single-channel openings are downward deflections of current.

In order to discount a nonspecific effect of Ang II and PD 123319 on K⁺ channel activity, experiments using the selective AT₂ receptor agonist CGP 42112 were performed. Fig 6 illustrates single-channel records in control and in the presence of CGP 42112 (100 nmol/L) and CGP 42112+PD 123319 (1 µmol/L). In control recordings (transmembrane potential, -20 mV), both large- and small-conductance channels were recorded. The NP_o of the 56-pS K⁺ channel was 0.21 in this experiment. CGP 42112 increased NP_o to 0.39, and an application of PD 123319 reversed the actions of Ang II close to control values (0.19). Overall, CGP 42112 significantly increased NP_o of the 56-pS K⁺ channel from 0.22±0.19 to 0.39±0.23 (Fig 5B, P<.05, n=4). PD 123319 in the presence of CGP 42112 significantly decreased NP_o to 0.17±0.12 (Fig 5B, P<.05, n=4). No significant effect of CGP 42112 was associated with the 34-pS K⁺ channel (control, 0.22±0.03; Ang II, 0.19±0.04; PD 123319, 0.24±0.03).

Kinetic analysis of the Ang II effect on the 56-pS K⁺ channel showed that open-time histograms were best fit by the sum of two exponential functions (Fig 7). In this experiment, Ang II significantly increased both open-time constants when compared with control (control, ₁=0.9 milliseconds, ₂=2.7 milliseconds; Ang II, ₁=3.1 milliseconds, ₂=14 milliseconds), and PD 123319 (₁=1.5 milliseconds, ₂=6.6 milliseconds) reversed this effect. The closed-time constants of the time histogram were not affected by Ang II or PD 123319. The mean data are presented in the Table. Ang II significantly increased the open-time constants when compared with control (P<.05, n=3). No effect was observed on the closed-time constant. However, the number of events occupying each closed-time constant was affected by Ang II and PD 123319+Ang II. In control conditions, the distribution of closed events was 42±3% and 58±4% for the fast and slow time constants, respectively. In the presence of Ang II, the distribution of closed events was shifted to 61±4% and 39±5%. This was reversed to near control levels by PD 123319. The increase in NP_o may be due to an increase in mean open time and an increase in the number of fast closed events. The increase in the number of fast closed events would therefore increase the number of openings. This suggests that more rapid closures existed in the presence of Ang II. These results suggest that activation of AT₂ receptors modulates rat hypothalamus/brain stem neuronal whole-cell K⁺ current by increasing the NP_o of a 56-pS K⁺ channel.

View larger version (25K): [in this window] [in a new window]   Figure 7. Open-time and closed-time histograms. Shown is a graph of open-time (left) and closed-time (right) histograms for single K+ channel recordings. In all cases, the data were best fit by the sum of two exponentials. Top, Control recordings at -40 mV. Middle, Recordings in the presence of 100 nmol/L Ang II. Bottom, Recordings in the presence of Ang II and 1 µmol/L PD 123319. There was no significant change in the closed-time distributions. However, there was a significant increase in NPo with the application of Ang II, which is reversed with PD 123319.

View this table: [in this window] [in a new window]   Table 1. Effect of Ang II and PD 123319 on Open- and Closed-Time Constants of the 56-pS K+ Channel

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In the present study, we characterized two voltage-dependent K⁺ channels in neonatal neuronal cultures. In cell-attached patch-clamp experiments (bath, 3.9 mmol/L K⁺; pipette, 140 mmol/L K⁺), two different K⁺ channels, with single-channel conductances of 24±4 and 56±6 pS, respectively, were recorded. When the patches were excised, these channels showed K⁺ selectivity (Figs 1 and 2). We also demonstrated that Ang II and CGP 42112, via activation of AT₂ receptors, increase the NP_o of a 56-pS K⁺ channel (Figs 3, 4, and 6). PD 123319, an AT₂ receptor antagonist, inhibited the Ang II– and CGP 42112–stimulated increase in NP_o of the 56-pS K⁺ channel, illustrating that the Ang II stimulation is via the AT₂ receptor (Figs 4 through 6). Ang II had no significant effect on the 34-pS channel. Finally, the increase in NP_o was due to an increase in the open-time constant of the 56-pS K⁺ channel (Fig 7).

These results expand upon our previous work^{6 16} by characterizing a single K⁺ channel that may underlie the AT₂ receptor–modulated Ang II increase in whole-cell K⁺ current observed in our cultured neurons. Previously, a 30% increase in whole-cell K⁺ current (holding potential, -80 mV; test potential, +10 mV) was demonstrated with Ang II.^{6 16} It can be assumed that whole-cell current (I) is proportional to the product of the single-channel current amplitude (i), the number of functional channels in the patch (N), and the Ang II–stimulated NP_o. Using the following equation and assuming that i and NP_o in control conditions have values of 4.0 pA and 0.41 at +10 mV, respectively (Fig 2B and 2C), then I is at least 2.1 pA in control.

This value would reflect the whole-cell current in a patch of membrane. Our data show that in the presence of Ang II, NP_o of the 56-pS K⁺ channel approximately triples at membrane potentials of -40 and -20 mV (Figs 3, 4, and 6). Therefore, we can hypothesize that Ang II increases NP_o to 0.4 to 0.6 at +10 mV. Assuming that the patch of membrane was 1 µm² and that the surface area of our cells was 30 µm², a whole-cell current of 270 pA would be generated. Therefore, the results obtained from these experiments suggest that modulation of the 56-pS K⁺ channel may account for the 300- to 400-pA increase in whole-cell K⁺ current previously demonstrated.⁶ This value can vary based on the number of channels per cell. However, this value may be underestimated or overestimated, since the NP_o values observed in our experiments were performed with K⁺ gradients opposite from those of Kang et al.⁶ It is known that NP_o values of some K⁺ channels (ie, inward rectifier and A current) can be altered in different K⁺ gradients.¹⁸ The change in NP_o may be due to rectification properties of the channel, a change in mean open time, or cation block of the channel. Since there is very little evidence that voltage-gated delayed rectifier K⁺ channels have an altered NP_o when the K⁺ gradient is changed, we believe that the Ang II–stimulated increase in NP_o of the 56-pS K⁺ underlies our previous whole-cell data.⁶

The effect of Ang II on the NP_o of a voltage-dependent K⁺ channel could have relevant effects on neuronal activity. In the brain, an increase in K⁺ channel activity could potentially reduce the refractory period of the neuronal action potential and hence increase neuronal activity. This is consistent with the observation of Ambuhl et al,¹⁹ who showed AT₂ receptor–mediated stimulation of neuronal activity in inferior olivary neurons. Palovcik and Phillips,²⁰ using hippocampal slices, demonstrated that opening K⁺ channels may also lead to a membrane hyperpolarization, therefore causing an inhibition of neuronal activity. This hypothesis is consistent with novel reports demonstrating an AT₂-mediated depressor effect in the periphery.^{6 7} In mice lacking the gene encoding the AT₂ receptor, the blood pressure response to low doses of Ang II was enhanced. This suggests that AT₂ receptor activation may antagonize the well-established AT₁-mediated pressor action of Ang II (eg, increased blood pressure, increased water intake, and altered baroreceptor function).¹⁴ This antihypertensive action may be consistent with a hyperpolarization of neurons resulting from the AT₂ receptor–mediated increase in K⁺ channel NP_o.

The finding that the increase in NP_o with Ang II was due to an increase in the open-time distribution with no significant change in the closed-time distribution indicates that Ang II influences the open state of this 56-pS channel. This is consistent with a role for second messengers or other signal transduction processes in the regulation of the 56-pS K⁺ channel. We observed a similar phenomenon in our previous studies. A latency period before AT₂ receptor-mediated changes in I_K was observed,^{6 16} and the involvement of an inhibitory GTP-binding protein in the AT₂ receptor stimulation of neuronal K⁺ currents was demonstrated.²⁰

In conclusion, we have shown that Ang II, via the AT₂ receptor, increases single K⁺ channel activity. Our single-channel data are consistent with previous whole-cell current data showing an Ang II increase in I_K.⁶ The novel results suggesting that AT₂ receptor activation is important in blood pressure regulation^{7 8} indicate that more investigation is required to understand the intracellular pathways involved in the Ang II regulation of K⁺ channel activity.

	Selected Abbreviations and Acronyms

 

= time constant Ang II = angiotensin II ARC = cytosine arabinoside IK = delayed rectifier K+ current NPo = channel Po, where N is the number of functional channels in a patch PDHS = plasma-derived horse serum

	Acknowledgments

 
This study was supported by National Institutes of Health grant HL-49310 (Drs Sumners and Posner), an Initial Investigatorship from the American Heart Association, Florida Affiliate, Inc (Drs Gelband and Martens), and a postdoctoral fellowship from the American Heart Association, Florida Affiliate, Inc (Dr Wang). We would like to thank Jennifer Moore for technical assistance.

Received March 20, 1996; accepted May 9, 1996.

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