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Cerebellar metabotropic glutamate receptor 1 (mGluR1) expressed by Purkinje cells may play an important role in learning-related cerebellar plasticity. Eyeblink conditioning (EBC) is a well-studied form of Pavlovian learning that engages discrete areas of cerebellar cortex and deep cerebellar nuclei. EBC is impaired in mGluR1 knockout mice. Here, we show that infusion of the mGluR1/5 agonist DHPG into the lobulus simplex region of cerebellar cortex facilitates EBC in rats. Infusion was made prior to Sessions 1 and 2 of EBC but the facilitatory effects persisted throughout subsequent, noninfusion sessions. The facilitatory effects were confined to frequency of eyeblink conditioned responses (CRs); there were no effects on amplitude or latency of CRs. There were also no effects on reflexive responding to the tone conditioned stimulus or eyelid stimulation unconditioned stimulus. The current results provide further evidence that cerebellar mGluR1 plays a role in cerebellar-dependent associative learning and complement previous studies using mGluR1 knockout mice. (PsycInfo Database Record (c) 2021 APA, all rights reserved).

Numerous experiments using ex vivo electrophysiology suggest that mammalian learning and memory involves regulation of voltage-gated ion channels in terms of changes in function. Yet, little is known about learning-related regulation of voltage-gated ion channels in terms of changes in expression. In two experiments, we examined changes in cell surface expression of the voltage-gated potassium channel alpha-subunit Kv1.2 in a discrete region of cerebellar cortex after eyeblink conditioning (EBC), a well-studied form of cerebellar-dependent learning. Kv1.2 in cerebellar cortex is expressed almost entirely in basket cells, primarily in the axon terminal pinceaux (PCX) region, and Purkinje cells, primarily in dendrites. Cell surface expression of Kv1.2 was measured using both multiphoton microscopy, which allowed measurement confined to the PCX region, and biotinylation/western blot, which measured total cell surface expression. In the first experiment, rats underwent three sessions of EBC, explicitly unpaired stimulus exposure, or context-only exposure and the results revealed a decrease in Kv1.2 cell surface expression in the unpaired group as measured with microscopy but no change as measured with western blot. In the second experiment, the same three training groups underwent only one half of a session of training, and the results revealed an increase in Kv1.2 cell surface expression in the unpaired group as measured with western blot but no change as measured with microscopy. In addition, rats in the EBC group that did not express conditioned responses (CRs) exhibited the same increase in Kv1.2 cell surface expression as the unpaired group. The overall pattern of results suggests that cell surface expression of Kv1.2 is changed with exposure to EBC stimuli in the absence, or prior to the emergence, of CRs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Protein kinase M zeta (PKM-ζ), a constitutively active N-terminal truncated form of PKC-ζ, has long been implicated in a cellular correlate of learning, long-term potentiation (LTP). Inhibition of PKM-ζ with zeta-inhibitory peptide (ZIP) has been shown in many brain structures to disrupt maintenance of AMPA receptors, irreversibly disrupting numerous forms of learning and memory that have been maintained for weeks. Delay eyeblink conditioning (EBC) is an established model for the assessment of cerebellar learning; here, we show that PKC-ζ and PKM-ζ are highly expressed in the cerebellar cortex, with highest expression found in Purkinje cell (PC) nuclei. Despite being highly expressed in the cerebellar cortex, no studies have examined how regulation of cerebellar PKM-ζ may affect cerebellar-dependent learning and memory. Given its disruption of learning in other brain structures, we hypothesized that ZIP would also disrupt delay EBC. We have shown that infusion of ZIP into the lobulus simplex of the rat cerebellar cortex can indeed significantly disrupt delay EBC. (PsycINFO Database Record, ((c) 2016 APA, all rights reserved).)

Insulin signaling plays a central role in the regulation of facilitative glucose transporters (GLUTs) in humans. To establish Caenorhabditis elegans (C. elegans) as a model to study the mechanism underlying insulin regulation of GLUT, we identified that FGT-1 is most likely the only functional GLUT homolog in C. elegans and is ubiquitously expressed. The FGT-1-mediated glucose uptake was almost completely defective in insulin/IGF-like signaling (IIS) mutants daf-2 and age-1, and this defect mainly resulted from the down-regulated FGT-1 protein expression. However, glycosylation may also be involved because OGA-1, an O-GlcNAcase, was essential for the function of FGT-1. Thus, our study showed that C. elegans can be a new powerful model system to study insulin regulation of GLUT.

We have previously shown that intracerebellar infusion of the neuropeptide secretin enhances the acquisition phase of eyeblink conditioning (EBC). Here, we sought to test whether endogenous secretin also regulates EBC and to test whether the effect of exogenous and endogenous secretin is specific to acquisition. In Experiment 1, rats received intracerebellar infusions of the secretin receptor antagonist 5-27 secretin or vehicle into the lobulus simplex of cerebellar cortex immediately prior to sessions 1-3 of acquisition. Antagonist-infused rats showed a reduction in the percentage of eyeblink CRs compared with vehicle-infused rats. In Experiment 2, rats received intracerebellar infusions of secretin or vehicle immediately prior to sessions 1-2 of extinction. Secretin did not significantly affect extinction performance. In Experiment 3, rats received intracerebellar infusions of 5-27 secretin or vehicle immediately prior to sessions 1-2 of extinction. The secretin antagonist did not significantly affect extinction performance. Together, our current and previous results indicate that both exogenous and endogenous cerebellar secretin modulate acquisition, but not extinction, of EBC. We have previously shown that (1) secretin reduces surface expression of the voltage-gated potassium channel α-subunit Kv1.2 in cerebellar cortex and (2) intracerebellar infusions of a Kv1.2 blocker enhance EBC acquisition, much like secretin. Kv1.2 is almost exclusively expressed in cerebellar cortex at basket cell-Purkinje cell pinceaus and Purkinje cell dendrites; we propose that EBC-induced secretin release from PCs modulates EBC acquisition by reducing surface expression of Kv1.2 at one or both of these sites., (© 2014 Fuchs et al.; Published by Cold Spring Harbor Laboratory Press.)

Caenorhabditis elegans (C. elegans) is an attractive animal model for biological and biomedical research because it permits relatively easy genetic dissection of cellular pathways, including insulin/IGF-like signaling (IIS), that are conserved in mammalian cells. To explore C. elegans as a model system to study the regulation of the facilitative glucose transporter (GLUT), we have characterized the GLUT gene homologues in C. elegans: fgt-1, R09B5.11, C35A11.4, F53H8.3, F48E3.2, F13B12.2, Y61A9LA.1, K08F9.1 and Y37A1A.3. The exogenous expression of these gene products in Xenopus oocytes showed transport activity to unmetabolized glucose analogue 2-deoxy-D-glucose only in FGT-1. The FGT-1-mediated transport activity was inhibited by the specific GLUT inhibitor phloretin and exhibited a Michaelis constant (Km) of 2.8 mM. Mannose, galactose, and fructose were able to inhibit FGT-1-mediated 2-deoxy-D-glucose uptake (P < 0.01), indicating that FGT-1 is also able to transport these hexose sugars. A GFP fusion protein of FGT-1 was observed only on the basolateral membrane of digestive tract epithelia in C. elegans, but not in other tissues. FGT-1::eGFP expression was observed from early embryonic stages. The knockdown or mutation of fgt-1 resulted in increased fat staining in both wild-type and daf-2 (mammalian insulin receptor homologue) mutant animals. Other common phenotypes of IIS mutant animals, including dauer formation and brood size reduction, were not affected by fgt-1 knockdown in wild-type or daf-2 mutants. Our results indicated that in C. elegans, FGT-1 is mainly a mammalian GLUT2-like intestinal glucose transporter and is involved in lipid metabolism.

The potassium channel Kv1.2 α-subunit is expressed in cerebellar Purkinje cell (PC) dendrites where its pharmacological inhibition increases excitability (Khavandgar et al., 2005). Kv1.2 is also expressed in cerebellar basket cell (BC) axon terminals (Sheng et al., 1994), where its blockade increases BC inhibition of PCs (Southan and Robertson, 1998a). Secretin receptors are also expressed both in PC dendrites and BC axon terminals (for review, see (Yuan et al., 2011). The effect of secretin on PC excitability is not yet known, but, like Kv1.2 inhibitors, secretin potently increases inhibitory input to PCs (Yung et al., 2001). This suggests secretin may act in part by suppressing Kv1.2. Receptor-mediated endocytosis is a mechanism of Kv1.2 suppression (Nesti et al., 2004). This process can be regulated by protein kinase A (PKA) (Connors et al., 2008). Since secretin receptors activate PKA (Wessels-Reiker et al., 1993), we tested the hypothesis that secretin regulates Kv1.2 trafficking in the cerebellum. Using cell-surface protein biotinylation of rat cerebellar slices, we found secretin decreased cell-surface Kv1.2 levels by modulating Kv1.2 endocytic trafficking. This effect was mimicked by activating adenylate cyclase (AC) with forskolin, and was blocked by pharmacological inhibitors of AC or PKA. Imaging studies identified the BC axon terminal and PC dendrites as loci of AC-dependent Kv1.2 trafficking. The physiological significance of secretin-regulated Kv1.2 endocytosis is supported by our finding that infusion into the cerebellar cortex of either the Kv1.2 inhibitor tityustoxin-Kα, or of the Kv1.2 regulator secretin, significantly enhances acquisition of eyeblink conditioning in rats.

Glucose is an essential substrate for lactose synthesis and an important energy source in milk production. Glucose uptake in the mammary gland, therefore, plays a critical role in milk synthesis. Facilitative glucose transporters (GLUT) mediate glucose uptake in the mammary gland. Glucose transporter 1 (GLUT1) is the major facilitative glucose transporter expressed in the bovine mammary gland and has been shown to localize to the basolateral membrane of mammary epithelial cells. Glucose transporter 1 is, therefore, thought to play a major role in glucose uptake during lactation. The objective of this study was to determine the transport kinetic properties and substrate specificity of bovine GLUT1 using the Xenopus oocyte model. Bovine GLUT1 (bGLUT1) was expressed in Xenopus oocytes by microinjection of in vitro transcribed cRNA and was found to be localized to the plasma membrane, which resulted in increased glucose uptake. This bGLUT1-mediated glucose uptake was dramatically inhibited by specific facilitative glucose transport inhibitors, cytochalasin B, and phloretin. Kinetic analysis of bovine and human GLUT1 was conducted under zero-trans conditions using radio-labeled 2-deoxy-D-glucose and the principles of Michaelis-Menten kinetics. Bovine GLUT1 exhibited a Michaelis constant (K(m)) of 9.8 ± 3.0mM for 2-deoxy-d-glucose, similar to 11.7 ± 3.7 mM for human GLUT1. Transport by bGLUT1 was inhibited by mannose and galactose, but not fructose, indicating that bGLUT1 may also be able to transport mannose and galactose. Our data provides functional insight into the transport properties of bGLUT1 in taking up glucose across mammary epithelial cells for milk synthesis., (Copyright © 2012 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The voltage-gated potassium channel, Kv1.3, plays an important role in regulating membrane excitability in diverse cell types ranging from T-lymphocytes to neurons. In the present study, we test the hypothesis that the C-terminal PDZ binding domain modulates the function and localization of Kv1.3. We created a mutant form of Kv1.3 that lacked the last three amino acids of the C-terminal PDZ-binding domain (Kv1.3ΔTDV). This form of Kv1.3 did not bind the PDZ domain containing protein, PSD95. We transfected wild type and mutant Kv1.3 into HEK293 cells and determined if the mutation affected current, Golgi localization, and surface expression of the channel. We found that cells transfected with Kv1.3ΔTDV had greater current and lower Golgi localization than those transfected with Kv1.3. Truncation of the C-terminal PDZ domain did not affect surface expression of Kv1.3. These findings suggest that PDZ-dependent interactions affect both Kv1.3 localization and function. The finding that current and Golgi localization changed without a corresponding change in surface expression suggests that PDZ interactions affect localization and function via independent mechanisms., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Kv1.2 is a member of the Shaker family of voltage-sensitive potassium channels and contributes to regulation of membrane excitability. The electrophysiological activity of Kv1.2 undergoes tyrosine kinase-dependent suppression in a process involving RhoA. We report that RhoA elicits suppression of Kv1.2 ionic current by modulating channel endocytosis. This occurs through two distinct pathways, one clathrin-dependent and the other cholesterol-dependent. Activation of Rho kinase (ROCK) via the lysophosphatidic acid (LPA) receptor elicits clathrin-dependent Kv1.2 endocytosis and consequent attenuation of its ionic current. LPA-induced channel endocytosis is blocked by the ROCK inhibitor Y27632 or by clathrin RNA interference. In contrast, steady-state endocytosis of Kv1.2 in unstimulated cells is cholesterol dependent. Inhibition of basal ROCK signaling with Y27632 increased surface Kv1.2, an effect that persists in the presence of clathrin small interfering RNA and that is not additive to the increase in surface channel levels elicited by the cholesterol sequestering drug filipin. Temperature block experiments show that ROCK affects cholesterol-dependent trafficking by modulating the recycling of endocytosed channel back to the plasma membrane. Both receptor-stimulated and steady-state Kv1.2 trafficking modulated by RhoA/ROCK required the activation of dynamin as well as the ROCK effector Lim-kinase, indicating a key role for actin remodeling in RhoA-dependent Kv1.2 regulation.

Kv1.3 channels are known to modulate many aspects of neuronal function. We tested the hypothesis that Kv1.3 modulates the function of postganglionic sympathetic neurons. RT-PCR, immunoblot, and immunohistochemical analyses indicated that Kv1.3 channels were expressed in these neurons. Immunohistochemical analyses indicated that Kv1.3 protein was localized to neuronal cell bodies, processes, and nerve fibers at sympathetic neurovascular junctions. Margatoxin (MgTX), a specific inhibitor of Kv1.3, was used to assess the function of the channel. Electrophysiological analyses indicated that MgTX significantly reduced outward currents [P < 0.05; n = 18 (control) and 15 (MgTX)], depolarized resting membrane potential, and decreased the latency to action potential firing [P < 0.05; n = 11 (control) and 13 (MgTX)]. The primary physiological input to postganglionic sympathetic neurons is ACh, which activates nicotinic and muscarinic ACh receptors. MgTX modulated nicotinic ACh receptor agonist-induced norepinephrine release (P < 0.05; n >or= 6), and MgTX-sensitive current was suppressed upon activation of muscarinic ACh receptors with bethanechol (P < 0.05; n = 12). These data indicate that Kv1.3 affects the function of postganglionic sympathetic neurons, which suggests that Kv1.3 influences sympathetic control of cardiovascular function. Our data also indicate that modulation of Kv1.3 is likely to affect sympathetic control of cardiovascular function.

The Shaker family potassium channel, Kv1.2, is a key determinant of membrane excitability in neurons and cardiovascular tissue. Kv1.2 is subject to multiple forms of regulation and therefore integrates cellular signals involved in the homeostasis of excitability. The cyclic AMP/protein kinase A (PKA) pathway enhances Kv1.2 ionic current; however, the mechanisms for this are not fully known. Here we show that cAMP maintains Kv1.2 homeostasis through opposing effects on channel trafficking. We found that Kv1.2 is regulated by two distinct cAMP pathways, one PKA-dependent and the other PKA-independent. PKA inhibitors elevate Kv1.2 surface levels, suggesting that basal levels of cAMP control steady-state turnover of the channel. Elevation of cAMP above basal levels also increases the amount of Kv1.2 at the cell surface. This effect is not blocked by PKA inhibitors, but is blocked by inhibition of Kv1.2 endocytosis. We conclude that Kv1.2 levels at the cell surface are kept in dynamic balance by opposing effects of cAMP.

Voltage-dependent potassium (Kv) and calcium (VDCC) channels play an important role in the regulation of membrane potential and intracellular calcium concentration in cerebral artery myocytes. Recent evidence suggests VDCC activity is increased and Kv channel activity is decreased in cerebral arteries following subarachnoid hemorrhage (SAH), promoting enhanced constriction. We have examined the impact of the blood component oxyhemoglobin on Kv and VDCC function in small (100-200 microm) diameter cerebral arteries. Acute (10 min) exposure of oxyhemoglobin caused cerebral artery constriction and Kv current suppression that was abolished by tyrosine kinase inhibitors and a Kv channel blocker. Although short-term oxyhemoglobin application did not directly alter VDCC activity, five-day exposure to oxyhemoglobin was associated with enhanced expression of voltage-dependent calcium channels. This work suggests that acute and chronic effects of oxyhemoglobin act synergistically to promote membrane depolarization and increased VDCC activity in cerebral arteries. These actions of oxyhemoglobin may contribute to the development of cerebral vasospasm following aneurysmal subarachnoid hemorrhage.

Ion channels are key determinants of membrane excitability. The actin cytoskeleton has a central role in morphology, migration, intracellular transport, and signaling. In this article, we show that the actin-binding protein cortactin regulates the potassium channel Kv1.2 and thereby provides a direct link between actin dynamics and membrane excitability. In previous reports, we showed that the tyrosine phosphorylation-mediated suppression of Kv1.2 ionic current occurs by endocytosis of the channel protein. Pull-down assays using recombinant-purified cortactin and Kv1.2 demonstrated that their interaction is direct and reduced by tyrosine phosphorylation of Kv1.2. This finding suggests a link between cortactin and Kv1.2 endocytosis. Here, we confirm that relationship and identify the molecular mechanisms involved. We use FRET to demonstrate that Kv1.2 and cortactin interact in vivo. By manipulating the cortactin-binding site within Kv1.2, we confirm that cortactin proximity influences channel function. We used flow cytometry in conjunction with cortactin gene replacement to identify C-terminal tyrosines, the fourth repeat actin-binding domain, and the N-terminal Arp2/3-binding region, as critical to Kv1.2 regulation. Surprisingly, cortactin's dynamin-binding Src homology 3 domain is not required for Kv1.2 endocytosis, despite that process being dynamin-dependent. These findings predict that cortactin-mediated actin remodeling in excitable cells is not only important for cell structure, but may directly impact membrane excitability.

Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences. Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca(2+)-selective and K(+)-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K(+) channel (K(v)) currents by approximately 30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca(2+) channels or large conductance Ca(2+)-activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of K(v) channel currents and oxyhb-induced constriction of isolated cerebral arteries. The K(v) channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in K(v) channel activity could represent either channel block, or a decrease in K(v) channel density on the plasma membrane. To explore whether oxyhb altered trafficking of K(v) channels to the plasma membrane, we used an antibody generated against an extracellular epitope of K(v)1.5 channels. In the presence of oxyhb, staining of K(v)1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with K(v)1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of K(v) currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.

The voltage-gated potassium channel Kv1.2 undergoes tyrosine phosphorylation-dependent suppression of its ionic current. However, little is known about the physical mechanism behind that process. We have found that the Kv1.2 alpha-subunit protein undergoes endocytosis in response to the same stimuli that evoke suppression of Kv1.2 ionic current. The process is tyrosine phosphorylation-dependent because the same tyrosine to phenylalanine mutation in the N-terminus of Kv1.2 that confers resistance to channel suppression (Y132F) also confers resistance to channel endocytosis. Overexpression of a dominant negative form of dynamin blocked stimulus-induced Kv1.2 endocytosis and also blocked suppression of Kv1.2 ionic current. These data indicate that endocytosis of Kv1.2 from the cell surface is a key mechanism for channel suppression by tyrosine kinases.

Tyrosine phosphorylation evokes functional changes in a variety of ion channels. Modulation of the actin cytoskeleton also affects the function of some channels. Little is known about how these avenues of ion channel regulation may interact. We report that the potassium channel Kv1.2 associates with the actin-binding protein cortactin and that the binding is modulated by tyrosine phosphorylation. Immunocytochemical and biochemical analyses show that Kv1.2 and cortactin co-localize to the cortical actin cytoskeleton at the leading edges of the cell. Binding assays using purified recombinant proteins reveal a 19-amino acid span within the carboxyl terminus of Kv1.2 that is necessary for direct cortactin binding. Phosphorylation of specific tyrosines within the C terminus of Kv1.2 attenuates that binding. In HEK293 cells, activation of the M1 muscarinic acetylcholine receptor evokes tyrosine phosphorylation-dependent suppression of Kv1.2 ionic current. We show that M1 receptor activation also reduces the interaction of cortactin with Kv1.2 and that mutant Kv1.2 channels deficient for cortactin binding exhibit strongly attenuated ionic current. These results demonstrate a dynamic, phosphorylation-dependent interaction between Kv1.2 and the actin cytoskeleton-binding protein cortactin and suggest a role for that interaction in the regulation of Kv1.2 ionic current.

Elevation of intravascular pressure causes depolarization and constriction (myogenic tone) of small arteries and arterioles, and this response is a key element in blood flow regulation. However, the nature of pressure-induced depolarization has remained elusive. In the present study, we provide evidence that a transient receptor potential channel (TRPC6) homologue has a major role in this depolarizing response to pressure. Antisense oligodeoxynucleotides to TRPC6 decreased TRPC6 protein expression and greatly attenuated arterial smooth muscle depolarization and constriction caused by elevated pressure in intact cerebral arteries. Suppressing the expression of this channel protein also reduced the current density of a major cation current in resistance artery smooth muscle cells. We propose that TRPC6 channels play an essential role in regulation of myogenic tone.

The human tumor necrosis factor alpha (TNF-alpha) gene is rapidly activated in response to multiple signals of stress and inflammation. We have identified transcription factors present in the TNF-alpha enhancer complex in vivo following ionophore stimulation (ATF-2/Jun and NFAT) and virus infection (ATF-2/Jun, NFAT, and Sp1), demonstrating a novel role for NFAT and Sp1 in virus induction of gene expression. We show that virus infection results in calcium flux and calcineurin-dependent NFAT dephosphorylation; however, relatively lower levels of NFAT are present in the nucleus following virus infection as compared to ionophore stimulation. Strikingly, Sp1 functionally synergizes with NFAT and ATF-2/c-jun in the activation of TNF-alpha gene transcription and selectively associates with the TNF-alpha promoter upon virus infection but not upon ionophore stimulation in vivo. We conclude that the specificity of TNF-alpha transcriptional activation is achieved through the assembly of stimulus-specific enhancer complexes and through synergistic interactions among the distinct activators within these enhancer complexes.

The phosphorylation state of a given tyrosine residue is determined by both protein tyrosine kinase (PTK) and protein tyrosine phosphatase (PTP) activities. However, little is known about the functional interaction of these opposing activities at the level of an identified effector molecule. G protein-coupled receptors (GPCRs), including the m1 muscarinic acetylcholine receptor (mAChR), regulate a tyrosine kinase activity that phosphorylates and suppresses current generated by the Kv1.2 potassium channel. We examined the possibility that PTPs also participate in this signaling pathway since the tyrosine phosphatase inhibitor vanadate increases the extent of both Kv1.2 phosphorylation and suppression. We show that an endogenous transmembrane tyrosine phosphatase, receptor tyrosine phosphatase alpha (RPTPalpha), becomes tyrosine phosphorylated and co-immunoprecipitates with Kv1.2 in a manner dependent on m1 receptor activation. The N- and C-termini of Kv1.2 are shown to bind RPTPalpha in vitro. Overexpression of RPTPalpha in Xenopus oocytes increases resting Kv1.2 current. Biochemical and electrophysiological analysis reveals that recruiting RPTPalpha to Kv1.2 functionally reverses the tyrosine kinase-induced phosphorylation and suppression of Kv1.2 current in mammalian cells. Taken together, these results identify RPTPalpha as a new target of m1 mAChR signaling and reveal a novel regulatory mechanism whereby GPCR-mediated suppression of a potassium channel depends on the coordinate and parallel regulation of PTK and PTP activities.

Tyrosine kinases activated by G protein-coupled receptors can phosphorylate and thereby suppress the activity of the delayed rectifier potassium channel Kv1.2. Using a yeast two-hybrid screen, we identified the small GTP-binding protein RhoA as a necessary component in this process. Coimmunoprecipitation experiments confirmed that RhoA associates with Kv1.2. Electrophysiological analyses revealed that overexpression of RhoA markedly reduced the basal current generated by Kv1.2 expressed in Xenopus oocytes. Furthermore, in 293 cells expressing Kv1.2 and ml muscarinic acetylcholine receptors, inactivating RhoA using C3 exoenzyme blocked the ability of ml receptors to suppress Kv1.2 current. Therefore, these results demonstrate that RhoA regulates Kv1.2 activity and is a central component in the mechanism of receptor-mediated tyrosine kinase-dependent suppression of Kv1.2.

Intracellular tyrosine kinases link the G protein-coupled m1 muscarinic acetylcholine receptor (mAChR) to multiple cellular responses. However, the mechanisms by which m1 mAChRs stimulate tyrosine kinase activity and the identity of the kinases within particular signaling pathways remain largely unknown. We show that the epidermal growth factor receptor (EGFR), a single transmembrane receptor tyrosine kinase, becomes catalytically active and dimerized through an m1 mAChR-regulated pathway that requires protein kinase C, but is independent of EGF. Finally, we demonstrate that transactivation of the EGFR plays a major role in a pathway linking m1 mAChRs to modulation of the Kv1.2 potassium channel. These results demonstrate a ligand-independent mechanism of EGFR transactivation by m1 mAChRs and reveal a novel role for these growth factor receptors in the regulation of ion channels by G protein-coupled receptors.

Background: One of the principal mechanisms by which G-protein-coupled receptors evoke cellular responses is through the activation of phospholipase C (PLC) and the subsequent release of Ca2+ from intracellular stores. Receptors that couple to pertussis toxin (PTX)-insensitive G proteins typically evoke large increases in PLC activity and intracellular Ca2+ release. In contrast, receptors that use only PTX-sensitive G proteins usually generate weak PLC-dependent responses, but efficiently regulate a second effector enzyme, adenylyl cyclase. For example, in many cell types, agonist binding by the m4 muscarinic acetylcholine receptor (m4 receptor) results in a strong inhibition of adenylyl cyclase and very little stimulation of PLC activity or release of intracellular Ca2+. We have investigated whether the weak, PTX-sensitive stimulation of PLC activity by the m4 receptor can play a significant role in the generation of cellular responses., Results: We report here that PTX-sensitive Ca2+ release mediated by the m4 receptor in transfected Chinese hamster ovary cells is greatly enhanced when endogenous purinergic receptors simultaneously activate a PTX-insensitive signaling pathway. Furthermore, m4-receptor-induced transcription of the c-fos gene (a Ca(2+)-sensitive response) is similarly potentiated when purinergic receptors are coactivated. These enhanced m4-receptor-dependent Ca2+ responses do not require an influx of external Ca2+, and occur in the absence of detectable purinergic-receptor-stimulated Ca2+ release; they apparently require the activation of both PTX-sensitive and PTX-insensitive G-protein pathways. Measurements of phosphoinositide hydrolysis indicate that the enhancement of m4-receptor-mediated Ca2+ signaling by purinergic receptors is due to a synergistic increase in agonist-stimulated PLC activity., Conclusions: These studies demonstrate that the potency of m4-receptor-mediated PLC signaling is highly dependent upon the presence or absence of other PLC-activating agonists. The ability of the m4 receptor to evoke a strong, but conditional, activation of PLC may allow this type of receptor to participate in a coincidence-detection system that amplifies simultaneous PLC-activating signals through a mechanism involving crosstalk between PTX-sensitive and PTX-insensitive G-protein pathways.

The insulin-stimulated cation channel previously identified in patch-clamped muscle preparations is here shown to be responsible for bulk Na+ entry into the cell. The mainly Na+ current of the channel was shown to be accompanied by an inhibitory Ca2+ component responsible for oscillations. Here, using quantitative fluorescence imaging of Fura-2- and SBFI-loaded soleus muscle, we measure changes in [Na+]i and [Ca2+]i related to channel function. Insulin increased [Na+]i and [Ca+]i in a transient spike of < 1-min duration. There was a momentary dip in [Na+]i related to inhibition of the channel by the Ca2+ spike, and changes in external Ca2+ were shown to alter [Na+]i via the cation channel, all effects being blocked by the specific channel inhibitor mu-conotoxin, but not by tetrodotoxin. The [Ca2+]i spike could also be induced by 8-bromo cyclic-guanosine 5'-monophosphate, an analogue of the channel-activator cyclic-guanosine 5'-monophosphate (cGMP). In addition it was noted that insulin reduced the [Ca2+]i rise upon subsequent muscle depolarization by a factor of 3.5. Insulin could be substituted with phorbol ester for the same effect and HA1004, a protein kinase inhibitor, blocked the reduction.

Cardiac beta-adrenergic receptors accelerate heart rate by modulating ionic currents through a pathway involving cyclic AMP-dependent protein kinase A (PKA). Previous studies have focused on the regulation of Ca2+ channels by PKA; however, due to the heterogeneity of K+ channels expressed within the heart, little is known about the mechanism by which PKA modulates individual K+ channels. Here we report that PKA strongly enhanced the activity of a cloned delayed rectifier K+ channel that is normally expressed in cardiac atria. This effect required a single PKA consensus phosphorylation site located near the amino terminus of the channel protein. Furthermore, patch clamp analysis revealed that PKA phosphorylation increased the open time that single channels spend in higher conductance states. These studies provide evidence that hormonal modulation of a cardiac K+ channel involves direct phosphorylation by PKA.

Neurotransmitter receptors alter membrane excitability and synaptic efficacy by generating intracellular signals that ultimately change the properties of ion channels. Through expression studies in Xenopus oocytes and mammalian cells, we found that the G protein-coupled m1 muscarinic acetylcholine receptor potently suppresses a cloned delayed rectifier K+ channel through a pathway involving phospholipase C activation and direct tyrosine phosphorylation of the K+ channel. Furthermore, analysis of neuroblastoma cells revealed that a similar tyrosine kinase-dependent pathway links endogenous G protein-coupled receptors to suppression of the native RAK channel. These results suggest a novel mechanism by which neurotransmitters and hormones may regulate a specific type of K+ channel that is widely expressed in the mammalian brain and heart.

Food avoidance learning in the mollusc Pleurobranchaea entails reduction in the responsiveness of key brain interneurons in the feeding neural circuitry, the paracerebral feeding command interneurons (PCNs), to the neurotransmitter acetylcholine (AcCho). Food stimuli applied to the oral veil of an untrained animal depolarize the PCNs and induce the feeding motor program (FMP). Atropine (a muscarinic cholinergic antagonist) reversibly blocks the food-induced depolarization of the PCNs, implicating AcCho as the neurotransmitter mediating food detection. AcCho applied directly to PCN somata depolarizes them, indicating that the PCN soma membrane contains AcCho receptors and induces the FMP in the isolated central nervous system preparation. The AcCho response of the PCNs is mediated by muscarinic-like receptors, since comparable depolarization is induced by muscarinic agonists (acetyl-beta-methylcholine, oxotremorine, pilocarpine), but not nicotine, and blocked by muscarinic antagonists (atropine, trifluoperazine). The nicotinic antagonist hexamethonium, however, blocked the AcCho response in four of six cases. When specimens are trained to suppress feeding behavior using a conventional food-avoidance learning paradigm (conditionally paired food and shock), AcCho applied to PCNs in the same concentration as in untrained animals causes little or no depolarization and does not initiate the FMP. Increasing the concentration of AcCho 10-100 times, however, induces weak PCN depolarization in trained specimens, indicating that learning diminishes but does not fully abolish AcCho responsiveness of the PCNs. This study proposes a cellular mechanism of long-term associative learning--namely, postsynaptic modulation of neurotransmitter responsiveness in central neurons that could apply also to mammalian species.