Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating IsK channels expressed in Xenopus oocytes

Kidney International, VoL 49 (1996), pp. 112—116

Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating 'sK channels expressed in Xenopus oocytes SIEGFRIED WALDEGGER, GERTRAUD RABER, HARTMUT SUBBRICH, J. PETER RUPPERSBERG, B. FAKLER,

HEINI MURER, FLORIis LAr'4G, and ANDREAS E. BUSCH Institute of Physiology, and Department of Sensoty Biophysics, Eberhard-Karls-Universitdt Tubingen, Tubingen, Germany, and Institute of Physiology, University of Zurich, Zurich, Switzerland

Coexpression and stimulation of parathyroid hormone receptor positively regulates slowly activating 'K channels expressed in Xenopus oocytes. Expression of the 'K protein in Xenopus oocytes induced the characteristically slow, voltage-dependent outward currents. Superfusion with the parathyroid hormone (PTH) peptide 1—34 had no effect on 'K when expressed alone, but increased 'sK when 'K was coexpressed with the

PTH-receptor. PTH receptor stimulation caused a shift of 'K conductance-voltage relationship to more negative potentials, and a decrease of both the rate of 'sK activation and deactivation. 'K regulation by PTH was independent of extracellular Ca2, and was also present in 'SK protein mutants lacking the protein kinase C consensus site. However, regulation of 'K by PTH was mimicked by activators of protein kinase A (PKA) and greatly reduced in the presence of the kinase inhibitors staurosporine and

kinase A have also been shown to regulate 'sK [10]. As PTH receptors and the 'sK protein are both expressed in cells of proximal tubules, we were interested in a putative regulation of the 'K protein by PTH receptor stimulation. To this end, we have coexpressed the PTH receptor and the 'sK protein in Xenopus oocytes, and studied regulation of the 'K protein by PTH receptor stimulation and the possible mechanism underlying such regulation.

Methods Handling and injection of Xenopus oocytes has been described H89. These results suggest that PTH regulates 'sK by a mechanism previously in detail [11]. The two-microelectrode voltage clamp involving phosphorylation independent of protein kinase C (PKC). Such regulation may play a role in proximal tubule cells of the kidney, where configuration was used to record currents from Xenopus laevis both PTH receptor and the LK protein are expressed. oocytes previously injected with cRNA transcribed in vitro from linearized plasmids encoding the 'SK protein from rat (r-ISK, [4]), human (h-ISK, [12]) and the rat 'sK protein mutant r-I del E 102(deletion of the intracellular domain from amino acid 102 to the Reabsorption of bicarbonate and inorganic phosphate (P3 end, [13]) using the appropriate RNA-polymerases (T3 for h-ISK substrates via membranes of the proximal tubule is reduced by and T7 for PTH receptor, r-ISK and its mutants). If not otherwise parathyroid hormone [1]; these effects are related to activation of cRNA encoding the PTH receptor [31 was coinjected. All PTH receptors and subsequent inhibition of apically located stated, cRNAs were capped and injected at I ng/oocyte. In two sets of Na/H exchange and NaIP1 cotransport. In rabbit proximal experiments the delayed rectifier K channel Kvl.1 [14] and the tubule cells PTH was shown to regulate a C1 conductance via an inward rectifying K channel IRK 1 [15, 16] were coexpressed activation of both PKA and PKC [2]. Stimulation of a cloned PTH with the PTH receptor. 'sK was usually activated by 15 second receptor has been shown to couple to both adenylate cyclase and voltage steps from —80 mV holding potential to —10 mV every 30 phospholipase C [3]. The gene encoding the 'sK protein has been seconds. Recordings were performed at 22°C using a Geneclamp originally cloned from rat kidney [4], where it is expressed in the brush border membrane of proximal tubule cells [5]. The 'SK amplifier (Axon Instruments, Foster City, USA) and MacLab D/A protein induces a slowly activating, voltage-dependent potassium conductance upon expression in Xenopus oocytes, but its molecular structure is unrelated to other K channel proteins. The 'SK

converter and software for data acquisition and analysis (ADInstruments, Castle Hill, Australia). The control solution contained (mM): NaCl 96, KC1 2, CaC12 1.8, MgC12 1, HEPES 5 (pH

7.5). The microelectrodes were filled with 3 M KCI solution and

protein has been shown to be the target for I regulation by had resistances between 0.5 to 1.1 M(1. Chemicals were added peroxides [6] and protein kinase C-mediated phosphotylation [7]. 'sK activity is also heavily influenced by changes in the cytoskeletal

organization and [Ca2] [8, 9]. Furthermore, activators of protein

Received for publication April 14, 1995 and in revised form August 7, 1995 Accepted for publication August 7, 1995

© 1996 by the International Society of Nephrology

from stock solutions into the superfusion solution as indicated. Chemicals used were diltiazem, parathyroid hormone fragment 1 to 34, dibutyiyl-cAMP (DB-cAMP), phorbol-didecanoate (PDD), staurosporine (Sigma) and H89 (Calbiochem). The recorded currents were leak-corrected and the amplitudes were measured at the end of the depolarizing voltage steps. To estimate activation kinetics (Tart) of 'sK' a single exponential function was fitted to the current traces obtained at 20 mV. A single exponential function was also fitted to the tail currents at —80 mV after depolarizations

112

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Waldegger et al: PTH regulation of 'vK

PTH

A

Control

Control

PTH

0.6 jiA

8s B

C

1800 -

PTH

1700 -

5O-

Fig. 1. (A) Original tracings showing the effect of PTH -receptor stimulation on I,-ampIitude in h-4,<-expressing control oocytes (left) and in oocytes coexpressing h-I,<-channels and PTHreceptors (right). 'SK was activated by a 15 second voltage step to —10 mV from a holding potential of —80 mV. PTH-receptor stimulation was achieved by application of 10 ns PTHfragment 1—34. (B) Graphical illustration of the effect of 10 nvi PTH-fragment 1—34 on oocytes

1600 -

• 1500 1400 -

I 20

1300 -

I—

0

wash

0 0 0

1200 -

0

-

(S

—10

expressing human h-ISK and rat r-L either alone (, —PTH-r) or cocxpressed with PTH-

1100 h-IsK

r-IsK

0 5 10 15 20 25 30 35 Time, minutes

receptors ( +PTH-r). (C) Time course of PTH-fragment 1—34 (10 nM) effects in a representative oocyte coexpressing h-ISK and

PTH-receptor.

to —20 mV to give a deactivation time constant (Tdeact). The (V0) was shifted from 6.5 1.6 mV under control conditions conductance of oocytes was calculated by assuming an EK of —100 (N = 5) to —0.5 1.8 mV after PTH (Fig. 2A; N = 5). mV. Data are presented as means with standard errors (sEM), Furthermore, both time constants for h-ISK activation (at 20 mV) where N represents the number of experiments performed. A and deactivation (analyzed at —80 mV after prior activation to paired Student's t-test was used to test for statistical significance, —20 mV) were significantly decreased (P < 0.05), indicating a faster activation and deactivation of 'sK channels after PTH which was obtained for P < 0.05. regulation (Fig. 2 B, C). Results Previously, r-ISK was shown to be negatively regulated by PKC Slowly activating, voltage-dependent human 'sK (h-ISK) or rat mediated phosphorylation of Ser 103 [7] and positively regulated 'SK (r-ISK) channels were expressed in Xenopus oocytes and by PKA [10]. The cloned PTH receptor has been shown to activate induced the characteristically slowly activating outward currents both protein kinase C and A [3]. Stimulation of PKA with upon depolarization. Superfusion of oocytes expressing h-LK or forskolin and DB-cAMP alone increased r-ISK by 83.3 16.5% rISK with PTH 1 to 34 (10 nM) did not alter these currents (N = 4), while simultaneous stimulation of PKA and PKC by a significantly (Fig. 1 A, B). In contrast, in oocytes in which the PTH receptor was coexpressed, the same concentration of PTH (10 nM) increased both h- and r-ISK by 48.5 it 12.0% (N = 6) and 51.1

6.7% (N = 21), respectively (Fig. 1 A, B). Upon washout for 15 minutes the amplitude returned to about 125% of control (N = 6;

cocktail of DB-cAMP (1 mM), forskolin (1 fLM) and PDD (50 nM)

resulted in a decrease of h-ISK by —76.8

4.4% (N =

5).

Similarly, PDD (50 nM) mediated activation of PKC caused an 'sK decrease after 30 minutes of about —60% (N = 5). Further, PTH

receptor stimulation caused also a current increase in oocytes

Fig. 1C). At 1 nM PTH did not produce a significant effect on r-ISK (N = 5), but 3 nM PTH increased r-ISK (26.9 9.1%; N = 4). The

expressing r-ISK del E102—, a mutant lacking the protein kinase C

effects occurred slowly and reached a steady state after about 10

4).

6.8%; N = These results exclude a role of PKC in the PTH induced

regulation site previously described for r-l5< (43.5

minutes.

modulation. In addition, the effects of the kinase inhibitor stau-

The effects of PTH on 'sK conductance-voltage relationship, activation and deactivation kinetics were analyzed in detail. Performing 20 second voltage steps from —40 to 40 mV (increment 20 mV) the voltage needed to evoke half-maximal r-ISK

rosporine (1 jIM) which inhibits both PKA and PKC, were studied. Superfusion of r-ISK with staurosporine (1 jiM) decreased r-ISK by

about 50% (Fig. 3B). In the presence of staurosporine, PTH regulation was greatly abolished (Fig. 3 A, D). H89 (5 jiM), a more

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et al: PTH regulation of 1K

A

1.2 1—

0.8 x

0.6 -

0)

0)

0.4 -

0.2 0 —50

—30

10

—10

30

50

Voltage, mV

C

B

6-

0.7

5-

0.6

> E

C C"

Fig. 2. (A) Statistical analysis of the effect of 10 nM PTH-fragment 1—34 on r-lK conductancevoltage relationship. Twenty second depolarizing voltage steps from —40 to +40 mV (increment 20 mV) were performed from a holding potential of —80 mV at an interval of 60

seconds. V indicates the calculated voltage needed to evoke half-maximal r-I currents (N

= 5). (B) Effect of 10 nM PTH-fragment 1—34 on h-INK activation kinetics in h-ISK expressing control oocytes (LI control) and in h-ISK and

0.5

(.)

PTH-receptor coexpressing oocytes (U PTH). The, time constant for h-IK activation (tau.act) was analyzed at a depolarizing step from —80 to +20 mV for 20 seconds (N = 5). A single exponential function was fitted to the current at that voltage. (C) Effect of 10 nM PTH-fragment

0.4 U)

0.3

0 (T

l

1—34 on h-ISK deactivation kinetics in h-IRK expressing LI control oocytes and in h-ISK and U PTH-receptor coexpressing oocytes. The time constant for h-IK deactivation (tau.deact) was analyzed at —80 mV (using a single exponential function) after prior activation to —20 mV for 20 seconds (N = 5).

0.2 1

0

0.1

0

specific protein kinase A inhibitor, decreased r-IK by 58.0 3.3% (Fig. 3C; N = 5). Similar to staurosporine, H89 greatly prevented PTH regulation (Fig. 3D; N = 5). These results support the

distinct K channels. We therefore coexpressed the PTH receptor either with the delayed rectifier K channel Kvl.1 [141 and the inwardly rectifying K channel IRK1 [15]. K currents through

PKA.

depolarizing pulses from —80 to —10 mV every 20 seconds, while

hypothesis that PTH mediates 'K regulation by activation of the delayed rectifier K channel were evoked with 500 ms Previously it was shown that an increase in [Ca211 causes inward K currents in oocytes expressing IRK1 were recorded at acceleration of 'sK activation [91. An endogeneous L-type-like hyperpolarizing pulses (500 ms pulses from —50 to —100 mV). Ca2-channel exists in the membrane of Xenopus oocytes [171. We However, stimulation of PTH receptors did not alter the current consequently investigated whether a PTH-mediated Ca2 influx amplitudes for Kvl.1 and IRK1 channels (Fig. 4; N = 4 and 4, through such channels may play a role in 'sK regulation. Experiments were therefore performed in the absence of Ca2 (0 Ca2 at 3 mtvt Mg2) or in the presence of the Ca2-channel antagonist

respectively).

Discussion

diltiazem (0.1 mM) in the superfusion solution. Deletion of PTH plays a major role in the regulation of mineral metabolism extracellular Ca2 did not alter 'sK amplitude (N = 4), while and transport in osteoblasts and renal cells [18]. Effects of PTH on diltiazem (0.1 mM) decreased 'sK by about 50% (Fig. 3B; N = 4). However, PTH increased 'sK also in the absence of Ca2 (by 72.4 10.8%; N = 4) or in the presence of diltiazem (Fig. 3 B, D). Finally, we tested for the specificity of PTH mediated effects on

the membrane potential of osteoblasts have been well characterized [19], but reports on electrophysiological effects of PTH in renal cells are rare [2]. Interestingly, expression of a single cloned

PTH-receptor in COS cells can act via two distinct signaling

115

Waldegger et a!: PTH regulation of 1K

A

B

stauro/PTH stauro

diItIPTH

dilt

Fig. 3. (A) Original tracings showing the effect of staurosporine on the PTH-fragment 1—34 mediated regulation of 'sK amplitude. The

I0.3 8s

0 C,,

10 nM PTH-fragment 1—34. IRK-current was

activated by a 15 second voltage step to —10 mY from a holding potential of —80 mV. (C) Inhibition of 'sK in IK-/PTH-receptorcoexpressing oocytes by diltiazem (0.1 mivi, U), staurosporine (1 j.rM, 1.) and H89 (5 ILM; LI) in proportion to ISK-amplitude under control conditions. Note the absence of the control traces in A and B. (D) Increase of 'SK amplitude mediated by 10 nM PTH-fragment 1—34 (PTH) in the presence of diltiazem (U 0.1 mM), staurosporine (LI 1 ELM) and H89 (LI 5 !.LM) in L-/PTH-receptor-coexpressing oocytes

I

0

Effects of PTH on Isk, %

0

01

Isk inhibition, %

C,

01

0

I•ii,I

8s

experiment was performed on an IK-/PTHreceptor-coexpressing oocyte superfused with staurosporine (1 !.LM) in the absence (stauro) and in the presence (stauro/PTH) of 10 nri PTH-fragment 1—34. IK-current was activated by a 15 seconds voltage step to —10 mV from a holding potential of —80 mV. (B) Original tracings showing the effect of diltiazem on the PTH-fragment 1—34 mediated regulation of 'K amplitude. The experiment was performed on an IK-/PTH-receptor-coexpressing oocyte superfused with diltiazem (0.1 mM) in the absence (dilt) and in the presence (diltIPTH) of

pathways [31: (a) activation of phospholipase C (PLC) with a

subsequent increase in [Ca2] and activation of PKC, and (b) activation of adenylate-cyclase with a subsequent activation of

(ISK-activation as described in A, N 4).

presence of the kinase inhibitor staurosporine. Because the specific protein kinase A inhibitor H89 [211 prevented 'sK regu-

lation, this kinase seems to be activated after PTH receptor

PKA. All of these PTH signals have been shown to differentially stimulation. Interestingly, both kinase inhibitors staurosporine regulate cloned 'sK channels [7—10], which are also expressed in and H89 decreased 'sK' indicating a possible regulation of LK brush border membranes of renal proximal tubule cells [5]. The under control conditions. Previously, positive regulation of 'sK was present study was undertaken (a) to investigate whether PTH described after PKA stimulation [10] and the PTH receptor has receptor stimulation can regulate 'sK channels expressed in Xeno- also been shown to activate PKA [3]. Taking our findings into pus oocytes, and (b) find out about the signaling pathway of PTH account, it seems that PTH receptor stimulation increases 'sK via activation of PKA. Since simultaneous activation of PKA and receptors in Xenopus oocytes. This study demonstrates a positive regulation of 'sK channels by PKC resulted in this study in a decrease of 'sK' PTH seems to stimulation of coexpressed PTH receptors. Positive regulation is exert its effects exclusively via PKA stimulation in Xenopus mainly reflected by an increase in the maximal conductance, a oocytes. Consequently, coupling of PTH receptors in Xenopus shift of the conductance-voltage relationship to more negative oocytes seems to be different than in COS cells, where they couple potentials and by a decrease in the rate of 'sK deactivation, For a to both PKA and PKC [31. This conclusion is also supported by number of reasons these effects are not likely to be caused by our recent finding that neither PTH or selective activation of PKA stimulation of PLC and subsequent increase in [Ca2I1 and regulate the rat Na/P1 cotransporter NaPi-2, while activation of activation of PKC. (1) PTFI also increased an 'sK mutant lacking PKC with phorbol esters potently inhibits NaPi-2 [22]. Therefore, the PKC phosphorylation site. (2) Stimulation of PTH did not PTH receptors do not seem to couple to PKC in Xenopus oocytes. To our knowledge there is no information yet on effects of PTH activate endogeneous Ca2 activated C1 channels in Xenopus oocytes which are usually observed due to an increase in [Ca2] on K conductances in proximal tubule cells and nobody has after activation of receptors coupling to PLC [11]. (3) activation of studied slowly activating K conductances in these cells in detail. receptors coupling to PLC negatively regulate Kvl.1 [20], while However, in frog proximal tubule cells cAMP, a second messenger activation of PTH receptors had no effect on Kvl.1. In this study, involved in PTH action, induces membrane hyperpolarization activators of PKC decreased h-ISK, while PTH increased h-ISK. [23]. In the same cells Na coupled transport slowly activates a However, PTH-mediated 'sK regulation seems to involve stimu- K conductance due to cell depolarization, which indicates the lation of a kinase, since 'sK increase by PTH was inhibited in the presence of an ISK like conductance [24]. Thus, PTH could

116

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Waldegger et al: PTH regulation of 1sK

4. TAKUMI T, OHKUBO H, NAKANISHI S: Cloning of a membrane protein

Kvl.1 +/—PTH

that induces a slow voltage-gated potassium current. Science 242: 1042—1045, 1988 5. SUGIMOTO T, TANABE Y, SHIGEMOTO R, IwAl M, TAKUMI T, OHKUBO

H, NAKANISHI S: Immunohistochemical study of a rat membrane protein which induces a selective potassium permeation: Its localization in the apical membrane portion of epithelial cells. J Membr Biol 113:39—47, 1990

2p.A

6. BUSCH AE, WALDEGGER S, HERZER T, RABER G, GULBINS E, TAKUMI T, MORIYOSHI K, NAKANISHI S, LANG F: Molecular basis of I,< protein

regulation by oxidation and chelation. J Biol Chem 270:3638—3641, 1995 7. BUSCH AE, VARNUM MD, NORTH RA, ADELMAN JP: A single amino

lOOms

acid mutation in a potassium channel protein prevents inhibition by protein kinase. Cell Sci 255:1705—1707, 1992a 8. BUSCH AE, VARNUM MD, ADELMAN JP, NORTH RA: Hypotonic

B IRK1 +1— PTH

solution increases the slowly activating potassium channel 'sK expressed in Xenopus oocytes. Biochem Biophys Res Comm 184:804— 810, 1992

9. BUSCH AE, LANG F: Effects of [Ca2] and temperature on minK

1 jtA

lOOms Fig. 4. (A) Original tracings showing the currents through the delayed rectifier K channel Kvl. 1, both in the absence and presence of 10 flM PTH-fragment 1—34 in Kvl. 1-/PTH-receptor-coexpressing oocytes. K cur-

rents were activated with 500 ms depolarizing pulses from —80 to —10 mV

evely 20 seconds (N = 4). (B) Original tracing showing the currents through the inwardly recti'ing K channel IRK1, both in the absence and

presence of 10 flM PTH-fragment 1—34 in IRK1-/PTH-receptor-coexpressing oocytes. K currents were recorded at hyperpolarizing pulses (500 ms pulses from —50 to —120 mV, N = 4). The dashed line indicates 0 current for both A and B. For both Kvl.1 and IRK1 superfusion with P1'H has no

effect on the current amplitudes.

influence the resting potentials of proximal tubule cells by regulating substrate transport as well as 'sK

channels expressed in Xenopus oocytes. FEBS Lett 334:221—224, 1993 10. BLUMENTHAL EM, KACZMAREK LK: Modulation by cAMP of a slowly

activating potassium channel expressed in Xenopus oocytes. J Neurosci 12:290—296, 1992 11. BUSCH AE, KAVANAUGH MP, VARNUM MD, ADELMAN JP, NORTH

RA: Regulation of the slowly activating, voltage-dependent potassium expressed in Xenopus oocytes. J Physiol 450:491—502, 1992b 12. MURAl T, KA.luzuI A, TAKUMI T, OHKUBO H, NAKANISHI S:

Molecular cloning and sequence analysis of human genomic DNA encoding a novel membrane protein which exhibits a slowly activating potassium channel activity. Biochem Biophys Res Commun 161:176— 181, 1989 13. TAKUMI T, MORLYOSHI K, ARAMORI I, ISI-Ill T, OIKI S, OKADA Y, OHKUBO H, NAKANISHI S: Alteration of channel activities and gating

by mutations of slow 'SK potassium channel. J Biol Chem 266:22192— 22198, 1991 14. CHRISTIE MJ, ADELMAN JP, DOUGLASS J, NORTH RA: Expression of cloned rat potassium channels in Xenopus oocytes. Science 244:221— 224, 1989

15. Kuao Y, BALDWIN Ti, J YN, JAIc LY: Primary structure and functional expression of a mouse inward rectifier potassium channel. Nature 362:127—133, 1993

Acknowledgments

16. FAKLER B, BRANDLE U, GLOWATZKI E, ZENNER HP, RUPPERSBERG

A.E. Busch is a Heisenberg-Fellow. The work was supported by grants from the Deutsche Forschungsgemeinschaft (Bu 704/3 to 1 to AEB). We thank Dr. Segre for providing the PTH receptor clone. The authors are indebted to Dr. R. Swanson and Dr. S. Nakanishi for providing several 'K

by protein kinases and ATP-hydrolysis. Neuron 13:1413—1420, 1994 17. DASCAL N: The use of Xenopus oocytes for the study of ion channels. CRC Crit Rev Biochem 22:317—387, 1987 18. AGUS ZS, WASSERSTEIN A, GOLDFARB S: PTH, calcitonin, cyclic nucleotides and the kidney. Annu Rev Physiol 43:583—595, 1981 19. EDELMAN A, FRITSCH J, BALSAN S: Short term effects of PTH on cultured rat osteoblasts: Changes in membrane potential. Am J Physiol

clones, and to Drs. A. Muller, G.L. Busch and P. Hausen for their discussion of the manuscript. We thank B. Noll and R. Vesenmeier for their support in the preparation and handling of oocytes.

JP: Kir 2.1 inward rectifier K channels are independently regulated

251:C483—C490, 1986

Reprint requests to Dr. Andreas E. Busch, Physiologisches Institut I,

20. KAVANAUGI-I MP, CHRISTIE M, OSBORNE P, BUSCH AE, SHEN K, WU

Eberhard-Karls-Universitat Thingen, Gmelinstr. 5, D-72076 Thbingen, Germany. e-mail: andreas.busch @uni-tuebingen.de

voltage-dependent K channels expressed in Xenopus oocytes. Bio-

Y, SEEBURG PH, ADELMAN JP, NORTH RA: Transmitter regulation of chem J 277:899—902, 1991

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membrane potential and intracellular ion activities in promal tubule of