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1.
Regulation of the inward K+ -channels in the guard cell plasma membranes plays impotant roles in regulation of stomatal movement in responses to exogenous and endogenous signals. It is well-known that elevation of cytosolic Ca2+ in guard cells inactivates these inward K + channels, and consequently inhibits stomatal opening or induces stomatal closing, yet the downstream molecular mechanism for the Ca2 + -mediated inhibition of the inward K+ channels remains unknown. The calmodulin-like domain protein kinases (CDPKs) have been identified as an unique group of protein kinases in higher plant cells. As a downstream regulator, CDPK may play roles in mediating Ca2+ regulation on the inward K+ -channels in stomatal guard cells. The authors have applied the patchclamp technique to investigate if CDPK be involved in the regulation of the inward K+ -channels in Vicia faba guard cells by cytosolic Ca2+ . The presence of the 1.5 μmol/L intracellular Ca2 + result-ed in inhibition of the inward K+ channel activity by 60%, while the addition of purified CDPK from the cytoplasmic side resulted in greater inhibition than Ca2+ alone. Histone Ⅲ-S and protamine, which is the substrate and substrate competitive inhibitor of CDPKs respectively, completely reversed the Ca2+ -induced inhibition of the inward K+ channel activities. These results are the first reported evidences for that CDPKs are involved in the Ca2+ -mediated inward K+ -channel regulation in guard cells. 相似文献
2.
3.
Distinct abscisic acid signaling pathways for modulation of guard cell versus mesophyll cell potassium channels revealed by expression studies in Xenopus laevis oocytes
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Regulation of guard cell ion transport by abscisic acid (ABA) and in particular ABA inhibition of a guard cell inward K(+) current (I(Kin)) is well documented. However, little is known concerning ABA effects on ion transport in other plant cell types. Here we applied patch clamp techniques to mesophyll cell protoplasts of fava bean (Vicia faba cv Long Pod) plants and demonstrated ABA inhibition of an outward K(+) current (I(Kout)). When mesophyll cell protoplast mRNA (mesophyll mRNA) was expressed in Xenopus laevis oocytes, I(Kout) was generated that displayed similar properties to I(Kout) observed from direct analysis of mesophyll cell protoplasts. I(Kout) expressed by mesophyll mRNA-injected oocytes was inhibited by ABA, indicating that the ABA signal transduction pathway observed in mesophyll cells was preserved in the frog oocytes. Co-injection of oocytes with guard cell protoplast mRNA and cRNA for KAT1, an inward K(+) channel expressed in guard cells, resulted in I(Kin) that was similarly inhibited by ABA. However, oocytes co-injected with mesophyll mRNA and KAT1 cRNA produced I(Kin) that was not inhibited by ABA. These results demonstrate that the mesophyll-encoded signaling mechanism could not substitute for the guard cell pathway. These findings indicate that mesophyll cells and guard cells use distinct and different receptor types and/or signal transduction pathways in ABA regulation of K(+) channels. 相似文献
4.
Expression of a Cs(+)-resistant guard cell K+ channel confers Cs(+)-resistant, light-induced stomatal opening in transgenic arabidopsis. 总被引:2,自引:0,他引:2
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A M Ichida Z M Pei V M Baizabal-Aguirre K J Turner J I Schroeder 《The Plant cell》1997,9(10):1843-1857
Inward-rectifying K+ (K+in) channels in the guard cell plasma membrane have been suggested to function as a major pathway for K+ influx into guard cells during stomatal opening. When K+in channels were blocked with external Cs+ in wild-type Arabidopsis guard cells, light-induced stomatal opening was reduced. Transgenic Arabidopsis plants were generated that expressed a mutant of the guard cell K+in channel, KAT1, which shows enhanced resistance to the Cs+ block. Stomata in these transgenic lines opened in the presence of external Cs+. Patch-clamp experiments with transgenic guard cells showed that inward K+(in) currents were blocked less by Cs+ than were K+ currents in controls. These data provide direct evidence that KAT1 functions as a plasma membrane K+ channel in vivo and that K+in channels constitute an important mechanism for light-induced stomatal opening. In addition, biophysical properties of K+in channels in guard cells indicate that components in addition to KAT1 may contribute to the formation of K+in channels in vivo. 相似文献
5.
Guard cell inward K+ channel activity in arabidopsis involves expression of the twin channel subunits KAT1 and KAT2 总被引:1,自引:0,他引:1
Pilot G Lacombe B Gaymard F Cherel I Boucherez J Thibaud JB Sentenac H 《The Journal of biological chemistry》2001,276(5):3215-3221
Stomatal opening, which controls gas exchanges between plants and the atmosphere, results from an increase in turgor of the two guard cells that surround the pore of the stoma. KAT1 was the only inward K(+) channel shown to be expressed in Arabidopsis guard cells, where it was proposed to mediate a K(+) influx that enables stomatal opening. We report that another Arabidopsis K(+) channel, KAT2, is expressed in guard cells. More than KAT1, KAT2 displays functional features resembling those of native inward K(+) channels in guard cells. Coexpression in Xenopus oocytes and two-hybrid experiments indicated that KAT1 and KAT2 can form heteromultimeric channels. The data indicate that KAT2 plays a crucial role in the stomatal opening machinery. 相似文献
6.
Regulation by external K+ in a maize inward shaker channel targets transport activity in the high concentration range 总被引:2,自引:0,他引:2
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An inward Shaker K(+) channel identified in Zea mays (maize), ZmK2.1, displays strong regulation by external K(+) when expressed in Xenopus laevis (African clawed frog) oocytes or COS cells. ZmK2.1 is specifically activated by K(+) with an apparent K(m) close to 15 mM independent of the membrane hyperpolarization level. In the absence of K(+), ZmK2.1 appears to enter a nonconducting state. Thus, whatever the membrane potential, this maize channel cannot mediate K(+) influx in the submillimolar concentration range, unlike its relatives in Arabidopsis thaliana. Its expression is restricted to the shoots, the strongest signal (RT-PCR) being associated with vascular/bundle sheath strands. Based on sequence and gene structure, the closest relatives of ZmK2.1 in Arabidopsis are K(+) Arabidopsis Transporter 1 (KAT1) (expressed in guard cells) and KAT2 (expressed in guard cells and leaf phloem). Patch-clamp analyses of guard cell protoplasts reveal a higher functional diversity of K(+) channels in maize than in Arabidopsis. Channels endowed with regulation by external K(+) similar to that of ZmK2.1 (channel activity regulated by external K(+) with a K(m) close to 15 mM, regulation independent of external Ca(2+)) constitute a major component of the maize guard cell inward K(+) channel population. The presence of such channels in maize might reflect physiological traits of C4 and/or monocotyledonous plants. 相似文献
7.
Irene Marten Frederic Gaymard Guy Lemaillet Jean-Baptiste Thibaud Hervé Sentenac Rainer Hedrich 《FEBS letters》1996,380(3):229-232
Following the biophysical analysis of plant K+ channels in their natural environment, three members from the green branch of the evolutionary tree of life KAT1, AKT1 and KST1 have recently been identified on the molecular level. Among them, we focused on the expression and characterization of the Arabidopsis thaliana K+ channel KAT1 in the insect cell line Sf9. The infection of Sf9 cells with KAT1-recombinant baculovirus resulted in functional expression of KAT1 channels, which was monitored by inward-rectifying, K+-selective (impermeable to Na+ and even NH4+) ionic conductance in whole-cell patch-clamp recordings. A voltage threshold as low as −60 to −80 mV for voltage activation compared to other plant inward rectifiers in vivo, and to in vitro expression of KAT1 in Xenopus oocytes or yeast, may be indicative for channel modulation by the expression system. A rise in cytoplasmic Ca2+ concentration (up to 1 mM), a regulator of the inward rectifier in Vicia faba guard cells, did not modify the voltage dependence of KAT1 in Sf9 cells. The access to channel function on one side and channel protein on the other make Sf9 cells a suitable heterologous system for studies on the biophysical properties, post-translational modification and assembly of a green inward rectifier. 相似文献
8.
Zimmermann S Hartje S Ehrhardt T Plesch G Mueller-Roeber B 《The Plant journal : for cell and molecular biology》2001,28(5):517-527
An appreciable number of potassium channels mediating K+ uptake have been identified in higher plants. Promoter-beta-glucuronidase reporter gene studies were used here to demonstrate that SKT1, encoding a potato K+ inwardly rectifying channel, is expressed in guard cells in addition to KST1 previously reported. However, whereas KST1 was found to be expressed in essentially all mature guard cells, SKT1 expression was almost exclusively restricted to guard cells of the abaxial leaf epidermis. This suggests that different types of K+ channel subunits contribute to channel formation in potato guard cells and therefore differential regulation of stomatal movements in the two leaf surfaces. The overlapping expression pattern of SKT1 and KST1 in abaxial guard cells indicates that K+in channels of different sub-families contribute to ionic currents in this cell type, thus explaining the different properties of channels expressed solely in heterologous systems and those endogenous to guard cells. Interaction studies had previously suggested that plant K+ inward rectifiers form clusters via their conserved C-terminal domain, KT/HA. K+ channels co-expressed in one cell type may therefore form heteromers, which increase functional variability of K+ currents, a phenomenon well described for animal voltage-gated K+ channels. Co-expression of KST1 and SKT1 in Xenopus oocytes resulted in currents with an intermediate sensitivity towards Cs+, suggesting the presence of heteromers, and a sensitivity towards external Ca2+, which reflected the property of the endogenous K+in current in guard cells. Modulation of KST1 currents in oocytes by co-expressing KST1 with a SKT1 pore-mutant, which by itself was not able to confer activating K+ currents, demonstrated the possibility that KST1 and SKT1 co-assemble to hetero-oligomers. Furthermore, various C-terminal deletions of the mutated SKT1 channel restored KST1 currents, showing that the C-terminal KT motif is essential for heteromeric channel formation. 相似文献
9.
The N-terminus of the K channel KAT1 controls its voltage-dependent gating by altering the membrane electric field. 总被引:3,自引:0,他引:3
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Functional roles of different domains (pore region, S4 segment, N-terminus) of the KAT1 potassium channel in its voltage-dependent gating were electrophysiologically studied in Xenopus oocytes. The KAT1 properties did not depend on the extracellular K+ concentration or on residue H267, equivalent to one of the residues known to be important in C-type inactivation in Shaker channels, indicating that the hyperpolarization-induced KAT1 inward currents are related to the channel activation rather than to recovery from inactivation. Neutralization of a positively charged amino acid in the S4 domain (R176S) reduced the gating charge movement, suggesting that it acts as a voltage-sensing residue in KAT1. N-terminal deletions alone (e.g., delta20-34) did not affect the gating charge movement. However, the deletions paradoxically increased the voltage sensitivity of the R176S mutant channel, but not that of the wild-type channel. We propose a simple model in which the N-terminus determines the KAT1 voltage sensitivity by contributing to the electric field sensed by the voltage sensor. 相似文献
10.
A novel chloride channel in Vicia faba guard cell vacuoles activated by the serine/threonine kinase, CDPK. 总被引:10,自引:0,他引:10
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Calcium-Dependent Protein Kinases (CDPKs) in higher plants contain a C-terminal calmodulin-like regulatory domain. Little is known regarding physiological CDPK targets. Both kinase activity and multiple Ca2+-dependent signaling pathways have been implicated in the control of stomatal guard cell movements. To determine whether CDPK or other protein kinases could have a role in guard cell signaling, purified and recombinant kinases were applied to Vicia faba guard cell vacuoles during patch-clamp experiments. CDPK activated novel vacuolar chloride (VCL) and malate conductances in guard cells. Activation was dependent on both Ca2+ and ATP. Furthermore, VCL activation occurred in the absence of Ca2+ using a Ca2+-independent, constitutively active, CDPK* mutant. Protein kinase A showed weaker activation (22% as compared with CDPK). Current reversals in whole vacuole recordings shifted with the Nernst potential for Cl-and vanished in glutamate. Single channel recordings showed a CDPK-activated 34 +/- 5 pS Cl- channel. VCL channels were activated at physiological potentials enabling Cl- uptake into vacuoles. VCL channels may provide a previously unidentified, but necessary, pathway for anion uptake into vacuoles required for stomatal opening. CDPK-activated VCL currents were also observed in red beet vacuoles suggesting that these channels may provide a more general mechanism for kinase-dependent anion uptake. 相似文献
11.
An Abscisic Acid-Activated and Calcium-Independent Protein Kinase from Guard Cells of Fava Bean 总被引:16,自引:4,他引:12
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Abscisic acid (ABA) regulation of stomatal aperture is known to involve both Ca2+-dependent and Ca2+-independent signal transduction pathways. Electrophysiological studies suggest that protein phosphorylation is involved in ABA action in guard cells. Using biochemical approaches, we identified an ABA-activated and Ca2+- independent protein kinase (AAPK) from guard cell protoplasts of fava bean. Autophosphorylation of AAPK was rapidly (~1 min) activated by ABA in a Ca2+- independent manner. ABA-activated autophosphorylation of AAPK occurred on serine but not on tyrosine residues and appeared to be guard cell specific. AAPK phosphorylated histone type III-S on serine and threonine residues, and its activity toward histone type III-S was markedly stimulated in ABA-treated guard cell protoplasts. Our results suggest that AAPK may play an important role in the Ca2+-independent ABA signaling pathways of guard cells. 相似文献
12.
Patch-clamp techniques were employed to investigate if calcium-dependent protein kinases (CDPKs) be involved in the signal transduction pathways of stomatal movement regulation by the phytohormone abscisic acid (ABA) in Vicia faba. Stomatal opening was completely inhibited by external application of 1 μmol/L ABA, and such ABA inhibition was significantly reversed by the addition of CDPK inhibitor trifluoper- azine (TFP). The inward whole-cell K+-currents were inhibited by 60% in the presence of 1 μmoL/L intracellular ABA, and this inhibition was completely abolished by the addition of CDPK competitive substrate histone Ⅲ-S. The results suggest that CDPKs may be involved in the signal transduction cascades of ABA-regulated stomatal movements. 相似文献
13.
I Dreyer S Antunes T Hoshi B Müller-R?ber K Palme O Pongs B Reintanz R Hedrich 《Biophysical journal》1997,72(5):2143-2150
In plants a large diversity of inwardly rectifying K+ channels (K(in) channels) has been observed between tissues and species. However, only three different types of voltage-dependent plant K+ uptake channel subfamilies have been cloned so far; they relate either to KAT1, AKT1, or AtKC1. To explore the mechanisms underlying the channel diversity, we investigated the assembly of plant inwardly rectifying alpha-subunits. cRNA encoding five different K+ channel alpha-subunits of the three subfamilies (KAT1, KST1, AKT1, SKT1, and AtKC1) which were isolated from different tissues, species, and plant families (Arabidopsis thaliana and Solanum tuberosum) was reciprocally co-injected into Xenopus oocytes. We identified plant K+ channels as multimers. Moreover, using K+ channel mutants expressing different sensitivities to voltage, Cs+, Ca2+, and H+, we could prove heteromers on the basis of their altered voltage and modulator susceptibility. We discovered that, in contrast to animal K+ channel alpha-subunits, functional aggregates of plant K(in) channel alpha-subunits assembled indiscriminately. Interestingly, AKT-type channels from A. thaliana and S. tuberosum, which as homomers were electrically silent in oocytes after co-expression, mediated K+ currents. Our findings suggest that K+ channel diversity in plants results from nonselective heteromerization of different alpha-subunits, and thus depends on the spatial segregation of individual alpha-subunit pools and the degree of temporal overlap and kinetics of expression. 相似文献
14.
Cloning and electrophysiological analysis of KST1, an inward rectifying K+ channel expressed in potato guard cells. 总被引:18,自引:2,他引:16
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B Müller-R?ber J Ellenberg N Provart L Willmitzer H Busch D Becker P Dietrich S Hoth R Hedrich 《The EMBO journal》1995,14(11):2409-2416
Potassium uptake by guard cells represents part of the osmotic motor which drives stomatal opening. Patch-clamp measurements have identified inward rectifying K+ channels capable of mediating K+ uptake in guard cells and various other plant cell types. Here we report the molecular cloning and characterization of a voltage-dependent K+ channel (KST1) from potato (Solanum tuberosum L.) guard cells. In situ hybridization shows expression of kst1 in guard cells. Two-electrode voltage-clamp and patch-clamp studies of the gene product after cRNA injection into Xenopus oocytes identified KST1 as a slowly activating, voltage-dependent, inward rectifying K+ channel. The single channel current voltage curve was linear in the range -160 to +20 mV, with a deduced single channel conductance of 7 pS in symmetrical 100 mM K+. This channel type, modulated by pH changes within the physiological range, required ATP for activation. In line with the properties of a K(+)-selective channel, KST1 was permeable to K+, Rb+ and NH4+ and excluded Na+ and Li+. Cs+ at submillimolar concentrations blocked the channel in a voltage-dependent manner. Related studies on potato guard cell protoplasts confirmed the biophysical characteristics of the kst1 gene product (KST1) in the heterologous expression system. Therefore, KST1 represents a major K+ uptake channel in potato guard cells. 相似文献
15.
Evidence for protein phosphatase 1 and 2A regulation of K+ channels in two types of leaf cells. 总被引:13,自引:5,他引:8
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Ion channels control ion fluxes across membranes, membrane potential, and signal transduction between and within cells. Protein kinases and phosphatases are important regulators involved in stimulus-response coupling in eukaryotic organisms. We have identified in extracts of Vicia faba leaf cells protein phosphatase activities inhibited by okadaic acid (OA) and calyculin A (CA), two inhibitors of protein phosphatases 1 and 2A. Using whole-cell patch-clamp techniques, we have demonstrated that inward K+ currents in guard cells are inhibited by nanomolar concentrations of OA or CA, whereas outward K+ currents are not affected. However, the same inhibitors enhance the magnitude of outward K+ currents in mesophyll cells. A phosphatase antagonist, adenosine-5'-O-(3-thiotriphosphate), has an effect similar to OA and CA on outward K+ currents in mesophyll cells. Our findings suggest that protein phosphatases 1 and/or 2A play different physiological roles in modulating the activity of K+ channels in mesophyll cells and guard cells. 相似文献
16.
The K+ channel KZM2 is involved in stomatal movement by modulating inward K+ currents in maize guard cells
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Yong‐Qiang Gao Wei‐Hua Wu Yi Wang 《The Plant journal : for cell and molecular biology》2017,92(4):662-675
Stomata are the major gates in plant leaf that allow water and gas exchange, which is essential for plant transpiration and photosynthesis. Stomatal movement is mainly controlled by the ion channels and transporters in guard cells. In Arabidopsis, the inward Shaker K+ channels, such as KAT1 and KAT2, are responsible for stomatal opening. However, the characterization of inward K+ channels in maize guard cells is limited. In the present study, we identified two KAT1‐like Shaker K+ channels, KZM2 and KZM3, which were highly expressed in maize guard cells. Subcellular analysis indicated that KZM2 and KZM3 can localize at the plasma membrane. Electrophysiological characterization in HEK293 cells revealed that both KZM2 and KZM3 were inward K+ (Kin) channels, but showing distinct channel kinetics. When expressed in Xenopus oocytes, only KZM3, but not KZM2, can mediate inward K+ currents. However, KZM2 can interact with KZM3 forming heteromeric Kin channel. In oocytes, KZM2 inhibited KZM3 channel conductance and negatively shifted the voltage dependence of KZM3. The activation of KZM2–KZM3 heteromeric channel became slower than the KZM3 channel. Patch‐clamping results showed that the inward K+ currents of maize guard cells were significantly increased in the KZM2 RNAi lines. In addition, the RNAi lines exhibited faster stomatal opening after light exposure. In conclusion, the presented results demonstrate that KZM2 functions as a negative regulator to modulate the Kin channels in maize guard cells. KZM2 and KZM3 may form heteromeric Kin channel and control stomatal opening in maize. 相似文献
17.
Distinct molecular bases for pH sensitivity of the guard cell K+ channels KST1 and KAT1 总被引:5,自引:0,他引:5
Acid-induced potassium uptake through K+ channels is a prerequisite for stomatal opening. Our previous studies identified a pore histidine as a major component of the acid activation mechanism of the potato guard cell K+ channel KST1 (1). Although this histidine is highly conserved among all plant K+ uptake channels cloned so far, the pH-dependent gating of the Arabidopsis thaliana guard cell K+ channel KAT1 was not affected by mutations of this histidine. In both channels, KST1 and KAT1, aspartate mutants in the K+ channel consensus sequence GYGD adjacent to the histidine (KST1-D269N and KAT1-D265N) were inhibited by a rise in the extracellular proton concentration. pH changes affected the half-maximal activation voltage V(1)/(2) of the KST1 mutant, whereas in the mutant channel KAT1-D265N an acid-induced decrease in the maximum conductance gmax indicated the presence of a proton block. In contrast to the wild type KST1, the S4-mutant channel KST1-R181Q exhibited an activation upon alcalization of the extracellular solution. From our electrophysiological studies on channel mutants with respect to the pore histidine as well as the aspartate, we conclude that the common proton-supported shift in the voltage dependence of KST1 and KAT1 is based on distinct molecular elements. 相似文献
18.
Latorre R Olcese R Basso C Gonzalez C Munoz F Cosmelli D Alvarez O 《The Journal of general physiology》2003,122(4):459-469
Animal and plant voltage-gated ion channels share a common architecture. They are made up of four subunits and the positive charges on helical S4 segments of the protein in animal K+ channels are the main voltage-sensing elements. The KAT1 channel cloned from Arabidopsis thaliana, despite its structural similarity to animal outward rectifier K+ channels is, however, an inward rectifier. Here we detected KAT1-gating currents due to the existence of an intrinsic voltage sensor in this channel. The measured gating currents evoked in response to hyperpolarizing voltage steps consist of a very fast (tau = 318 +/- 34 micros at -180 mV) and a slower component (4.5 +/- 0.5 ms at -180 mV) representing charge moved when most channels are closed. The observed gating currents precede in time the ionic currents and they are measurable at voltages (less than or equal to -60) at which the channel open probability is negligible ( approximately 10-4). These two observations, together with the fact that there is a delay in the onset of the ionic currents, indicate that gating charge transits between several closed states before the KAT1 channel opens. To gain insight into the molecular mechanisms that give rise to the gating currents and lead to channel opening, we probed external accessibility of S4 domain residues to methanethiosulfonate-ethyltrimethylammonium (MTSET) in both closed and open cysteine-substituted KAT1 channels. The results demonstrate that the putative voltage-sensing charges of S4 move inward when the KAT1 channels open. 相似文献
19.
Aluminum (Al) inhibits inward K(+) channels (K(in)) in both root hair and guard cells, which accounts for at least part of the Al toxicity in plants. To understand the mechanism of Al-induced K(in) inhibition, we performed patch clamp analyses on K(in) in guard cells and on KAT1 channels expressed in Xenopus oocytes. Our results show that Al inhibits plant K(in) by blocking the channels at the cytoplasmic side of the plasma membrane. In guard cells, single-channel recording revealed that Al inhibition of K(in) occurred only upon internal exposure. Using both "giant patch" recording and single-channel analyses, we found that Al reduced KAT1 open probability and changed its activation kinetics through an internal membrane-delimited mechanism. We also provide evidence that a Ca(2)+ channel-like pathway that is sensitive to antagonists verapamil and La(3)+ mediates Al entry across the plasma membrane. We conclude that Al enters plant cells through a Ca(2)+ channel-like pathway and inhibits K(+) uptake by internally blocking K(in). 相似文献
20.
The G protein-coupled inwardly rectifying K+ channel, GIRK1/GIRK4, can be activated by receptors coupled to the Galpha(i) subunit. An opposing role for Galpha(q) receptor signaling in GIRK regulation has only recently begun to be established. We have studied the effects of m1 muscarinic acetylcholine receptor (mAChR) stimulation, which is known to mobilize calcium and activate protein kinase C (PKC) by a Galpha(q)-dependent mechanism, on whole cell GIRK1/4 currents in Xenopus oocytes. We found that stimulation of the m1 mAChR suppresses both basal and dopamine 2 receptor-activated GIRK 1/4 currents. Overexpression of Gbetagamma subunits attenuates this effect, suggesting that increased binding of Gbetagamma to the GIRK channel can effectively compete with the G(q)-mediated inhibitory signal. This G(q) signal requires the use of second messenger molecules; pharmacology implicates a role for PKC and Ca2+ responses as m1 mAChR-mediated inhibition of GIRK channels is mimicked by PMA and Ca2+ ionophore. We have analyzed a series of mutant and chimeric channels suggesting that the GIRK4 subunit is capable of responding to G(q) signals and that the resulting current inhibition does not occur via phosphorylation of a canonical PKC site on the channel itself. 相似文献