Receptor-mediated regulation of IsK, a very slowly activating, voltage-dependent K+ channel in Xenopus oocytes

Vol. 184, No. 3, 1992

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May 15, 1992

R e c e p t o r - m e d i a t e d r e g u l a t i o n of IsK, a very slowly activating, voltage-dependent K ÷ c h a n n e l in Xenopus o o c y t e s E r i c HONORE, B e r n a r d A T r A L T ; F l o r i a n LESAGE, J a c q u e s BARHANIN and M i c h e l L A Z n U N S K I Institut de Pharmacologie Mol6culaire et Cellulaire, CNRS, 660 route des Lucioles, Sophia Antipolis, 06560 Valbonne, France Received March 25, 1992

SUMMARY. Expression of IsK in Xenopus oocytes has been obtained in 2 ways : (i) by injection of cardiac polyA+ RNA from neonatal mouse heart; (ii) by injection of a cRNA synthesized in vitro. It was observed that polyA + RNA not only directs the expression of the IsK channel b u t also contains purinergic P2 and endothelin receptors. Stimulation of these receptors, that produce intracellular Ca 2+ increase together with diacylglycerol production activating protein kinase C, increases IsK activity. The same type of results and the same conclusions were obtained by co-injecting cRNA's corresponding to the 5-HT2 receptor and the IsK channel into oocytes. This stimulatory effect was shown to be due to Ca 2+ via a calmodulin-dependent kinase process. Conversely, activation of protein kinase C p a t h w a y alone by phorbol esters leads to inhibition of IsK activity. ¢ 1992Academic P ..... Inc.

INTRODUCTION. The slowly activating voltage-dependent K + (IsK) channel was originally cloned in rat kidney and is mainly found in epithelial cells (1). A cDNA encoding this channel was also cloned from a neonatal mouse h e a r t cDNA library and its properties were studied after expression of the c o m p l e m e n t a r y RNA in Xenopus oocytes (2). IsK is also p r e s e n t in rat uterus and neonatal heart (3, 4). Very recently, IsK was found to be present in h u m a n T lymphocytes (5). The IsK protein consists of 129-130 amino acids depending on the animal species, with a single putative t r a n s m e m b r a n e domain and its structure differs completely from that of the other known ion channel proteins (for a review see 6, 7). The IsK protein, because of its small size, is an attractive channel to study structure-function relationships and to

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investigate molecular mechanisms involved in the regulation of this very slow voltage-dependent K + channel activity (8, 9). The present report examines how stimulation of receptors coupled to phospholipase C and coexpressed with IsK in Xenopus oocytes regulates the slow K ÷ channel activity. This regulation is probably physiologically important in diverse cells t h a t express this channel such as cardiac cells and h u m a n T lymphocytes.

1VLATERIALSAND METHODS The methods of polyA ÷ RNA preparation, cloning IsK protein, in vitro RNA synthesis, oocyte isolation, injection and electrophysiology have been previously reported (2, 5). Briefly, total RNA was extracted from newborn mouse heart (10) and then polyA + isolated. For Xenopus oocyte injection, a s i n g l e - s t r a n d e d cRNA sense was s y n t h e s i z e d from cDNA i n s e r t e d in Bluescript using the T7 polymerase (RNA transcription kit from Stratagene). Xenopus laevis were purchased from C.R.B.M. (Montpellier, France). Pieces of the ovary were surgically removed and individual oocytes were dissected away in a modified Barth's solution : 88 mM NaC1, 1 mM KC1, 2.4 mM NaHCO3, 0.33 mM Ca(NO3)2, 0.41 mM CaC12, 0.82 mM MgSO4; 10 mM HEPES; pH 7.4 with NaOH. Stage V and VI oocytes were treated for 2 hours with collagenase (1 mg/ml, Sigma) in Barth's medium to discard follicular cells. 50 nl RNA solution were injected per oocyte using a pressure microinjector. Oocytes were then kept for 2 to 6 days in Barth's medium supplemented with 100 UI/ml penicillin and 100 pg/ml streptomycin. In a 0.3 ml perfusion chamber, a single oocyte was impaled with two standard glass microelectrodes (0.5-2.0 M ~ resistance) filled with 3M KCI and maintained under voltage clamp using Dagan TEV-200 amplifier. Stimulation of the preparation, data acquisition and analyses were performed using pClamp software (Axon Instruments; USA). Drugs were applied either externally by addition to the superfusate (Gilson peristaltic pump; flow rate : 3 ml/min) or internally using a pressure injection. A saline solution (ND 96) of the following composition was used in all procedures unless otherwise stated : 96 mM NaC1, 2 mM KC1, 1.8 mM CaC12, 2 mM MgC12, 5 mM H E P E S (pH 7.4 with NaOH).

RESULTS AND DISCUSSION As previously described (2), microinjection of newborn mouse cardiac polyA + RNA into Xenopus oocytes directed the expression of a large longlasting outward K + current (IsK) which did not undergo inactivation (Fig. 1A). The current-voltage relationship of this K + current displayed a marked outward going-rectification when the potential was made more positive than -50 mV (Fig. 1B). Non-injected or tRNA-injected oocytes did not express this 1136

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Fig. 1, Regulation of the IsK channel by purinergic and endothelin receptors in cardiac mRNA-injected Xenopus oocytes. Xenopus oocytes were microinjected with 20 ng polyA+ RNA extracted from newborn mouse heart. Membrane currents were recorded after 3 days. The holding potential was -60 mV and the oocytes were depolarized to +30 mV every minute. A, 100 ~M extracellular ATP were added to the superfusing medium for 2 minutes. The ATP-induced stimulation of IsK was reversed with 30 pM W7. B, I-V relationship in control conditions ([B) and in the presence of 100 ~M external ATP (w). C, 100 nM endothelin I applied for 3 minutes stimulated IsK. This effect was reversed with 30 pM W7:

outward current. When the injected oocytes were challenged for 2 minutes with external ATP, IsK was largely potentiated (Fig. 1A). Non-injected oocytes were not sensitive to ATP (not shown). Clearly, the injection of mouse c a r d i a c RNA into oocytes directed the expression of both the IsK channel and of purinergic receptors. The expressed purinergic receptor was sensitive to ATP a n d ADP but not to AMP and adenosine, thus corresponding to a P2 purinoceptor (11). The potentiation of IsK by brief exposure to ATP was maintained up to 1 hour. The I-V relationship illustrated in Fig. 1B shows t h a t the voltage threshold for activation was not modified by ATP and t h a t stimulation was observed at all potentials. Addition of the calmodulin antagonist W7 (or of trifluoperazine, not shown) inhibited the stimulation of IsK by ATP (Fig. 1A). Fig. 1C demonstrates t h a t IsK activity was also increased by external application of endothelin I. The endothelin receptor was not p r e s e n t in control oocytes (not shown). The expressed endothelin receptor Was stimulated by endothelin I and endothelin II, but not by endothelin III, t h u s corresponding to a type Ia endothelin receptor (12). Similarly to purines, endothelin effects were reversed by the calmodulin antagonist W7 (Fig. 1C). Both purinergic and endothelin stimulations involve phospholipase C activation and consequently inositol trisphosphate (InsP3) and diacylglycerol (DG) productions (11-14). Therefore, a more detailed study of the modulation of IsK c h a n n e l activity by second messengers, was carried out after 1137

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F i g . 2. Ca2+-calmodulin-dependent stimulation of IsK. Oocytes were microinjected with 5 ng of synthetic RNA encoding IsK channel. A, Intracellular injection of InsP3 (final concentration lpM) potentiated IsK. This effect was blocked by 30 gM W7. B, 1 ~M ionomycin stimulated IsK and 30 ~M W7 blocked this effect. C. The stimulation of IsK by ionomycin is reversed by intracellular injection of 0.5 unit of alkaline phosphatase (Sigma, type XXX-L). In these experiments the holding potential was -60 mV and the oocytes depolarized every 60 s to +30 mV.

expression of the cloned IsK protein and analysis of the individual effects of InsP3 and of protein kinase C activation respectively. Fig. 2A shows t h a t i n t r a c e l l u l a r microinjection of InsP3 into oocytes s t i m u l a t e d IsK. This s t i m u l a t i o n was again reversed by W7. Fig. 2B shows t h a t the Ca 2+ ionophore ionomycin mimicked the effect of InsP3 injection. In t h a t case, the stimulation of IsK channel activity was also blocked by W7 (Fig. 2B). Fig. 3B shows t h a t microinjection of alkaline phosphatase not only reversed the ionomycin-induced increase of IsK activity but t h a t it also depressed the intrinsic activity of IsK by about 60%. These results suggest t h a t u n d e r normal conditions of expression of IsK in Xenopus oocytes, a substantial proportion of the channel is already activated by phosphorylation. If this i n t e r p r e t a t i o n is correct, t h e n t r e a t m e n t s t h a t produce dephosphorylation are expected to suppress this activation. All these results suggest t h a t Ca 2+ release from InsP3-sensitive stores stimulates IsK by a Ca2+-calmodulin d e p e n d e n t phosphorylation. The IsK protein sequence contains a Ca 2+c a l m o d u l i n - d e p e n d e n t kinase II consensus site j u s t u p s t r e a m of the hydrophobic domain. H u m a n and mouse IsK genes are divergent in the region of this putative consensus sequence (in single-letter code, the mouse sequence a t r e s i d u e s 35-41 is SQLRDDSK; the h u m a n sequence is SPRSSDGK). Therefore, the h u m a n IsK would not be expected to be p h o s p h o r y l a t e d by Ca2+-calmodulin kinase II. However, it has been previously observed t h a t the h u m a n IsK channel was also activated by intracellular increases of internal Ca 2+ (5). Therefore, it appears likely t h a t the regulation of the IsK channel by kinase II m a y be indirect. 1138

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Fig. 3. Protein kinase C activation does not participate in the serotoninmediated regulation of IsK. Oocytes were injected with 5 ng of synthetic RNAs encoding both the IsK channel and the 5-HT2 receptor. A, The oocyte was exposed for 2 minutes to t ~M serotonin and the amplitude of IsK was measured every minute. B, The effect of serotonin was reversed with 30 pM W7. C, The oocyte was pretreated for 15 minutes with 30 ~M W7 and then challenged for 1 hour with 1 ~M serotonin. In the presence of 30 pM W7, application of 30 nM PMA for 20 minutes depressed IsK. D, The oocyte was injected with EGTA (final concentration 5 mM) and challenged with 1 ~M serotonin for 1 hour.

5-HT receptors are also coupled to phospholipase C through G proteins (15). The cloned 5-HT2 receptor (16) was coexpressed with the cloned IsK channel in oocytes (Fig. 3A). Fig. 3A shows t h a t a brief exposure of oocytes to serotonin induced a large and rapid increase in IsK amplitude. This increase was sustained for up to 60 minutes. Serotonin did not affect controls or oocytes expressing only IsK (in the absence of the 5-HT2 receptor, not shown). Activation of the serotonin receptor also stimulated IsK channel activity by a Ca2+-calmodulin-dependent process since oocytes p r e t r e a t e d with W7 or with the Ca 2+ chelator EGTA were not sensitive to serotonin (Fig. 3C and D). It has been previously shown t h a t kinase C inhibits the activity of mouse Isk (2). Fig. 3C shows t h a t p r e t r e a t m e n t of oocytes with W7 did not abolish the inhibitory effect of the phorbol ester PMA (although W7 had possibly some depressive action on protein kinase C which probably prevents observation of an inhibitory effect of serotonin in the presence of W7). Activation of the 5-HT2 receptor by serotonin leads to both intracellular increase of Ca 2+ via InsP3 production and to kinase C activation via diacylglycerol 1139

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formation. Results in Fig. 3 show that when both Ca 2+ increases and kinase C is activated, the dominant effect is produced by Ca 2+ (which activates) rather than by kinase C (which inhibits) since the overall effect is an activation. This conclusion is consistent with results presented in Fig. 1 for stimulations of the purinergic P2 receptor and the endothelin receptors. In conclusion, results found in this work indicate t h a t all hormone, n e u r o t r a n s m i t t e r or growth factor receptors that lead to an activation of phospholipase C will stimulate IsK mainly because they induce an increase of intracellular Ca 2+ which causes an activation of Ca2+-calmodulin kinase II required for the (probably indirect) activatory effect. Situations can arise, for other types of receptors, for which production of diacylglycerol and activation of kinase C will occur without substantial increase in intracellular Ca 2+ concentrations (for example receptors t h a t activate p h o s p h o l i p a s e A2 i n s t e a d of phospholipase C). U n d e r t h e s e conditions, the dominating effect is expected to be an inhibition of IsK since kinase C itself, or diacylglycerol or phorbol esters (this work and 2) all inhibit IsK activity. It is expected that the regulatory properties described in this work will be important in cardiac cells that express this channel. Activation or inhibition of the IsK channel would lead to drastic changes of the duration of the action potential and of the heart beating rate. The IsK channel has also been shown to be p r e s e n t in h u m a n T-lymphocytes which are well known to have numerous receptors coupled to phospholipase C or phospholipase A2. Since it was suggested that the IsK channel may be important for T-lymphocyte activation (5), it is also expected t h a t the modulation of IsK activity described in this work will also have consequences in T lymphocyte physiology. We are grateful to Dr. E. Van Obberghen-Schilling for providing the Chinese h a m s t e r 5-HT2 receptor clone. This work was s u p p o r t e d by the Centre National de la Recherche Scientifique, the Association pour la Recherche sur le Cancer, and the Association pour la Recherche sur la Scl~rose en Plaques. B.A. is a recipient of an ARC fellowship. We t h a n k F. Aguila and C. Roulinat for expert technical assistance. Acknowledgments.

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