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Results for “coupled patch model”

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PubMed2006

Lectin anchored stabilized biodegradable nanoparticles for oral immunization 1. Development and in vitro evaluation.

The investigation comprises development of a stable and targeted formulation of HBsAg for the oral immunization against Hepatitis B. PLGA nanoparticles bearing HBsAg was prepared by double emulsion method. The antigen was protected from organic/aqueous interface by using protein stabilizer, trehalose. The acidic environment generated within PLGA nanoparticles was neutralized by co-encapsulation of a basic additive, Mg(OH)(2) which provides an additional stabilization to the antigen especially against acid induced antigen inactivation. Furthermore, lectin from Arachis hypogaea (PNA) was anchored on to the surface of the HBsAg loaded nanoparticles in order to enhance their affinity towards the antigen presenting cells of the Peyer's patches. The developed system was characterized for shape, size and loading efficiency. The antigen integrity was assessed by using SDS-PAGE followed by isoelectric focusing analysis. Bovine submaxillary mucin (BSM) was used as a biological model for in vitro ligand affinity determination and activity studies. The lectin anchored nanoparticles exhibited 52.18+/-4.73% loading while ligand density was estimated to be of 17.90+/-1.14 microg/mg. The results suggest that HBsAg can be successfully stabilized by co-encapsulation of an appropriate protein stabilizer, i.e. trehalose and a basic additive, Mg(OH)(2). The ligand-coupled nanoparticles demonstrated approximately four folds increase in degree interaction with the BSM as compared to plain nanoparticles. Additionally, the nanoparticles maintained their intrinsic sugar specificity as associated due to lectin (PNA).

Administration, Oral↗

PubMed2002

Ischemia-induced brain damage depends on specific gap-junctional coupling.

Ischemic brain injury results in neuronal loss and associated neurologic deficits. Although there is some evidence that intercellular communication via gap junctions can spread oxidative cell injury, the possible role of gap-junctional communication in ischemia-induced cell death is the object of debate. Because gap junctions directly connect the cytoplasms of coupled cells, they offer a way to propagate stress signals from cell to cell. The authors investigated the contribution of gap-junctional communication to cell death using an in vitro ischemia model, which was reproduced by submersion of organotypic hippocampal slices into glucose-free deoxygenated medium. The gap-junctional blocker carbenoxolone significantly decreased the spread of cell death, as measured by propidium iodide staining, over a 48-hour period after the ischemic episode. Carbenoxolone ameliorated the hypoxia-induced impairment of the intrinsic neuronal electrophysiologic characteristics, as measured by whole-cell patch clamp recordings. To determine whether specific connexins were involved in the spread of postischemic cell death, the authors partially reduced the synthesis of specific connexins using antisense oligodeoxynucleotides. Simultaneous knockdown of two connexins localized mostly in neurons, connexins 32 and 26, resulted in significant neuroprotection 48 hours after the hypoxic-hypoglycemic episode. Similarly, partial reduction of the predominant glial connexin 43 significantly decreased cell death. These results indicate that gap-junctional communication contributes to the propagation of hypoxic injury and that specific gap junctions could be a novel target to reduce brain damage.

Animals↗

PubMed1995

Sodium-dependent norepinephrine-induced currents in norepinephrine-transporter-transfected HEK-293 cells blocked by cocaine and antidepressants.

Transport of norepinephrine (NE+) by cocaine- and antidepressant-sensitive transporters in presynaptic terminals is predicted to involve the cotransport of Na+ and Cl-, resulting in a net movement of charge per transport cycle. To explore the relationship between catecholamine transport and ion permeation through the NE transporter, we established a human norepinephrine transporter (hNET) cell line suitable for biochemical analysis and patch-clamp recording. Stable transfection of hNET cDNA into HEK-293 (human embryonic kidney) cells results in lines exhibiting (1) a high number of transporter copies per cell (10(6)), as detected by radioligand binding and hNET-specific antibodies, (2) high-affinity, Na(+)-dependent transport of NE, and (3) inhibitor sensitivities similar to those of native membranes. Whole-cell voltage-clamp of hNET-293 cells reveals NE-induced, Na(+)-dependent currents blocked by antidepressants and cocaine that are absent in parental cells. In addition to NE-dependent currents, transfected cells posses an NE-independent mode of charge movement mediated by hNET. hNET antagonists without effect in non-transfected cells abolish both NE-dependent and NE-independent modes of charge movement in transfected cells. The magnitude of NE-dependent currents in these cells exceeds the expectations of simple carrier models using previous estimates of transport rates. To explain our observations, we propose that hNETs function as ion-gated ligand channels with an indefinite stoichiometry relating ion flux to NE transport. In this view, external Na+ and NE bind to the transporter with finite affinities in a cooperative fashion. However, coupled transport may not predict the magnitude or the kinetics of the total current through the transporter. We propose instead that Na+ gates NE transport and also the parallel inward flux of an indeterminate number of ions through a channel-like pore.

Adrenergic alpha-Agonists↗

PubMed2003

Amplification of receptor signalling by Ca2+ entry-mediated translocation and activation of PLCgamma2 in B lymphocytes.

In non-excitable cells, receptor-activated Ca2+ signalling comprises initial transient responses followed by a Ca2+ entry-dependent sustained and/or oscillatory phase. Here, we describe the molecular mechanism underlying the second phase linked to signal amplification. An in vivo inositol 1,4,5-trisphosphate (IP3) sensor revealed that in B lymphocytes, receptor-activated and store-operated Ca2+ entry greatly enhanced IP3 production, which terminated in phospholipase Cgamma2 (PLCgamma2)-deficient cells. Association between receptor-activated TRPC3 Ca2+ channels and PLCgamma2, which cooperate in potentiating Ca2+ responses, was demonstrated by co-immunoprecipitation. PLCgamma2-deficient cells displayed diminished Ca2+ entry-induced Ca2+ responses. However, this defect was canceled by suppressing IP3-induced Ca2+ release, implying that IP3 and IP3 receptors mediate the second Ca2+ phase. Furthermore, confocal visualization of PLCgamma2 mutants demonstrated that Ca2+ entry evoked a C2 domain-mediated PLCgamma2 translocation towards the plasma membrane in a lipase-independent manner to activate PLCgamma2. Strikingly, Ca2+ entry-activated PLCgamma2 maintained Ca2+ oscillation and extracellular signal-regulated kinase activation downstream of protein kinase C. We suggest that coupling of Ca2+ entry with PLCgamma2 translocation and activation controls the amplification and co-ordination of receptor signalling.

Animals↗

PubMed1994

Coordination of plasma membrane and vacuolar membrane ion channels during stomatal movement.

Transduction of extracellular signals in guard cells requires coupling of ion fluxes across the vacuolar and plasma membrane. Changes in cytoplasmic proton and Ca2+ concentrations are important messages during stomatal movement. Here we summarize the different biophysical properties of voltage- and ligand-dependent ion channels in both the vacuolar and plasma membranes of guard cells, focusing on how cytoplasmic Ca2+ and proton concentrations direct ion fluxes towards stomatal opening or closure. Particular attention is given to the patch clamp analysis of plasma membrane cation channels (GCKCin, GCKCout and Ca2+ channels), the anion channel (GCAC1) and the vacuolar SV-type channels. A working model is presented of how the ion channels of both membrane systems could communicate to synchronize potassium salt release during stomatal closure.

Animals↗

PubMed1991

A massively parallel computer model of propagation through a two-dimensional cardiac syncytium.

A computer model of electrical propagation through a two-dimensional (2D) sheet of cardiac tissue has been developed to run on the massively parallel processor Connection Machine (CM-2) computer. The transmembrane ionic currents in each of 16,384 (128 x 128) 100 x 100 microns 2 patches of cardiac tissue are described by modified Beeler-Reuter membrane equations. These equations, along with the parabolic differential equation derived from 2D cable theory, are solved in parallel to study normal and abnormal 2D propagation. The sheet is paced with planar waves at a basic cycle length of 500 msec (control). When a premature ectopic stimulus of sufficient strength and appropriate timing is then applied to a local region of the syncytium, one of two types of reentry is observed: (a) stable figure-of-eight reentry, or (b) unstable but self-sustaining "fibrillation-like" reentry. During this fibrillatory activity, action potential durations are 79.8 +/- 36.8 msec (control = 244.9 +/- 0.9 msec) and coupling intervals average 96.7 +/- 31.3 msec (control = 500 +/- 0 msec). We also observed that passive electrotonically-induced depolarization of already refractory tissue extended the refractory period of that tissue, and that the duration of this extension depended on the magnitude of the electrotonic effect.

Action Potentials↗

PubMed2002

Voltage-sensing mechanism is conserved among ion channels gated by opposite voltages.

Hyperpolarization-activated cyclic-nucleotide-gated (HCN) ion channels are found in rhythmically firing cells in the brain and in the heart, where the cation current through HCN channels (called I(h) or I(f)) causes these cells to fire repeatedly. These channels are also found in non-pacing cells, where they control resting membrane properties, modulate synaptic transmission, mediate long-term potentiation, and limit extreme hyperpolarizations. HCN channels share sequence motifs with depolarization-activated potassium (Kv) channels, such as the fourth transmembrane segment S4. S4 is the main voltage sensor of Kv channels, in which transmembrane movement of S4 charges triggers the opening of the activation gate. Here, using cysteine accessibility methods, we investigate whether S4 moves in an HCN channel. We show that S4 movement is conserved between Kv and HCN channels, which indicates that S4 is also the voltage sensor in HCN channels. Our results suggest that a conserved voltage-sensing mechanism operates in the oppositely voltage-gated Kv and HCN channels, but that there are different coupling mechanisms between the voltage sensor and activation gate in the two different channels.

Amino Acid Motifs↗

PubMed1997

Cost-effectiveness of treating nicotine dependence: the Mayo Clinic experience.

OBJECTIVE: To estimate the cost-effectiveness of treating nicotine dependence, expressed as cost per net year of life gained by smoking cessation. DESIGN: A cost-effectiveness analysis was conducted of a cohort of consecutive adult patients treated for nicotine dependence from April 1988 through December 1992 at the Mayo Clinic Nicotine Dependence Center (NDC). MATERIAL AND METHODS: The study cohort consisted of 5,544 patients (50.8% female; mean age, 47.8 years) with a mean baseline smoking rate of 25.4 cigarettes per day. After an initial consultation, a nonphysician counselor developed an individual nicotine dependence treatment plan, which could include follow-up counseling, nicotine replacement therapy (patches or gum), group therapy, or an inpatient program. A relapse-prevention program included telephone calls and a series of letters to the patient. We computed the years of life gained for each person specific to age, gender, smoking rate at entry, and 6-month smoking status by using published mortality rates for current and former cigarette smokers. The 6-month smoking status was assumed to be applicable at 1 year. For subsequent determinations, we modeled by computer simulation the year-by-year (to age 100) smoking status by using published relapse and late cessation rates. Coupled with treatment costs, this information allowed the expression of cost per net year of life gained by stopping smoking. Net years of life gained, discounted 0, 3, and 5%, were computed with use of cessation and relapse rates expected for patients not seen in the NDC. Treatment costs were based on 1993 rates for the intervention services but did not include any tobacco product cost savings associated with smoking cessation. RESULTS: The 1-year smoking-cessation rate was 22.2%. With all NDC patients included, the estimated net years of life gained, with use of a 5% rate of discount for benefits, was 0.058, and the corresponding cost was $6,828 per net year of life gained. CONCLUSION: In comparison with the cost-effectiveness of other medical services, the cost of $6,828 per net year of life gained by treatment of nicotine dependence is relatively inexpensive. Such cost-outcome data are important as economic considerations are applied for optimal allocation of limited health-care resources. Nonphysician health-care professionals can assume a key role in the provision of cost-effective nicotine dependence intervention.

Adult↗

PubMed2006

Photo processes on self-associated cationic porphyrins and plastocyanin complexes 1. Ligation of plastocyanin tyrosine 83 onto metalloporphyrins and electron-transfer fluorescence quenching.

The spectroscopic properties of the self-associated complexes formed between the anionic surface docking site of spinach plastocyanin and the cationic metalloporphyrins, in which the tyrosine 83 (Y83) moiety is placed just below the docking site, tetrakis(N-methyl-4-pyridyl)porphyrin (Pd(II)TMPyP(4+) and Zn(II)TMPyP(4+)), have been studied and reported herein. The fluorescence quenching phenomenon of the self-assembled complex of Zn(II)TMPyP(4+)/plastocyanin has also been discovered. The observed red-shifting of the Soret and Q-bands of the UV-visible spectra, ca. 9 nm for Pd(II)TMPyP(4+)/plastocyanin and ca. 6 nm for the Zn(II)TMPyP(4+)/plastocyanin complexes, was explained in terms of exciton theory coupled with the Gouterman model. Thus, the hydroxyphenyl terminus of the Y83 residue of the self-associated plastocyanin/cationic porphyrin complexes was implicated in the charge-transfer ligation with the central metal atoms of these metalloporphyrins. Moreover, ground-state spectrometric-binding studies between Pd(II)TMPyP(4+) and the Y83 mutant plastocyanin (Y83F-PC) system proved that Y83 moiety of plastocyanin played a critical role in the formation of such ion-pair complexes. Difference absorption spectra and the Job's plots showed that the electrostatic attractions between the cationic porphyrins and the anionic patch of plastocyanin, bearing the nearby Y83 residue, led to the predominant formation of a self-associated 1:1 complex in the ground-state with significantly high binding constants (K = (8.0 +/- 1.1) x 10(5) M(-1) and (2.7 +/- 0.8) x 10(6) M(-1) for Pd(II)TMPyP(4+) and zinc variant, respectively) in low ionic strength buffer, 1 mM KCl and 1 mM phosphate buffer (pH 7.4). Molecular modeling calculations supported the formation of a 1:1 self-associated complex between the porphyrin and plastocyanin with an average distance of ca. 9 A between the centers of mass of the porphyrin and Y83 positioned just behind the anionic surface docking site on the protein surface. The photoexcited singlet state of Zn(II)TMPyP(4+) was quenched by the Y83 residue of the self-associated plastocyanin in a static mechanism as evidenced by steady-state and time-resolved fluorescence experiments. Even when all the porphyrin was complexed (more than 97%), significant residual fluorescence from the complex was observed such that the amplitude of quenching of the singlet state of uncomplexed species was enormously obscured.

Algorithms↗

PubMed1997

Regulation of cation-selective channels in liver cells.

In liver cells, cation-selective channels are permeable to Ca2+ and have been postulated to represent a pathway for receptor-mediated Ca2+ influx. This study examines the mechanisms involved in the regulation of these channels in a model liver cell line. Using patch-clamp recording techniques, it is shown that channel open probability is a saturable function of cytosolic [Ca2+], with half-maximal opening at 660 nm. By contrast, channel opening is not affected by membrane voltage or cytosolic pH. In intact cells, reduction of cytosolic [Cl-], a physiological response to Ca2+-mobilizing hormones and cell swelling, is also associated with an increase in channel opening. Finally, channel opening is inhibited by intracellular ATP through a mechanism that does not involve ATP hydrolysis. These findings suggest that opening of cation-selective channels is coupled to the metabolic state of the cell and provides a positive feedback mechanism for regulation of receptor-mediated Na+ and Ca2+ influx.

Animals↗

PubMed2002

Comparison of coupled and uncoupled currents during glutamate uptake by GLT-1 transporters.

The transport of glutamate across the plasma membrane is coupled to the movement of cations (Na+, K+, and H+) that are necessary for glutamate uptake and transporter cycling as well as anions that are uncoupled from the flux of glutamate. Although the relationship between these coupled (stoichiometric) and uncoupled (anion) transporter currents is poorly understood, transporter-associated anion currents often are used to monitor transporter activity. To define the kinetic relationship between these two components, we have recorded transporter currents associated with stoichiometric and anion charge movements occurring in response to the rapid application of l-glutamate to outside-out patches from human embryonic kidney cells expressing GLT-1 transporters. Transporter-associated anion currents were approximately twice as slow to rise and decay as stoichiometric transport currents, but the presence of permeant anions did not slow transporter cycling. A kinetic model for GLT-1 was developed to simulate the behavior of both components of the transporter current and to estimate the capture efficiency of GLT-1. In this model the K+ counter-transport step was defined as rate-limiting, consistent with the slowing of transporter cycling after the substitution of internal K+ with Cs+ or Na+. The model predicts that in physiological conditions approximately 35% of GLT-1 transporters function as buffers, releasing glutamate back into the extracellular space after binding.

Biological Transport↗

PubMed2005

Computational model of electrically coupled, intrinsically distinct pacemaker neurons.

Electrical coupling between neurons with similar properties is often studied. Nonetheless, the role of electrical coupling between neurons with widely different intrinsic properties also occurs, but is less well understood. Inspired by the pacemaker group of the crustacean pyloric network, we developed a multicompartment, conductance-based model of a small network of intrinsically distinct, electrically coupled neurons. In the pyloric network, a small intrinsically bursting neuron, through gap junctions, drives 2 larger, tonically spiking neurons to reliably burst in-phase with it. Each model neuron has 2 compartments, one responsible for spike generation and the other for producing a slow, large-amplitude oscillation. We illustrate how these compartments interact and determine the dynamics of the model neurons. Our model captures the dynamic oscillation range measured from the isolated and coupled biological neurons. At the network level, we explore the range of coupling strengths for which synchronous bursting oscillations are possible. The spatial segregation of ionic currents significantly enhances the ability of the 2 neurons to burst synchronously, and the oscillation range of the model pacemaker network depends not only on the strength of the electrical synapse but also on the identity of the neuron receiving inputs. We also compare the activity of the electrically coupled, distinct neurons with that of a network of coupled identical bursting neurons. For small to moderate coupling strengths, the network of identical elements, when receiving asymmetrical inputs, can have a smaller dynamic range of oscillation than that of its constituent neurons in isolation.

Animals↗

PubMed1994

A comparison between muscarinic receptor occupancy, inositol 1,4,5-trisphosphate accumulation and Ca2+ mobilization in permeabilized SH-SY5Y neuroblastoma cells.

Electrically permeabilized SH-SY5Y neuroblastoma cells have been used to examine the relationship between receptor occupation by muscarinic agonists, inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) accumulation and Ca2+ mobilization from intracellular stores. The kinetics, concentration-dependence and guanine nucleotide-sensitivity of these responses have been characterized for the agonists, carbachol, arecoline and oxotremorine. Carbachol stimulated Ins(1,4,5)P3 accumulation and Ca2+ mobilization with an EC50 value approximately 50 microM, only slightly lower than the apparent affinity of this agonist for the "free" receptor (100 microM). Arecoline and oxotremorine were partial agonists, mobilizing 45 and 21% of the Ca2+ mobilized by carbachol, and yielded EC50 values for both Ins(1,4,5)P3 and Ca2+ responses, similar to their binding affinity. Guanosine 5'-O-3 thio-triphosphate (GTP gamma S) markedly enhanced the responses elicited by all three agonists. Carbachol became significantly more potent for both Ins(1,4,5)P3 accumulation (EC50 = 4.1 microM) and Ca2+ mobilization (EC50 = 0.25 microM), revealing a separation of the dose-response relationships. GTP gamma S caused a smaller separation of the responses elicited by arecoline (Ca2+ mobilization EC50 = 0.9 microM; Ins(1,4,5)P3 accumulation EC50 = 3.6 microM), and only enhanced maximal responses for oxotremorine. These data reveal that the functional coupling of muscarinic receptors to activation of phosphoinositidase C and subsequent Ca2+ mobilization from intracellular stores is maintained after electrical permeabilization. Furthermore, this model has been used to reveal differences in the relative activities of muscarinic agonists and how they are influenced by a hydrolysis-resistant guanine nucleotide.

Arecoline↗

PubMed2002

A mechanism of regulating transmembrane potassium flux through a ligand-mediated conformational switch.

The regulation of cation content is critical for cell growth. However, the molecular mechanisms that gate the systems that control K+ movements remain unclear. KTN is a highly conserved cytoplasmic domain present ubiquitously in a variety of prokaryotic and eukaryotic K+ channels and transporters. Here we report crystal structures for two representative KTN domains that reveal a dimeric hinged assembly. Alternative ligands NAD+ and NADH block or vacate, respectively, the hinge region affecting the dimer's conformational flexibility. Conserved, surface-exposed hydrophobic patches that become coplanar upon hinge closure provide an assembly interface for KTN tetramerization. Mutational analysis using the KefC system demonstrates that this domain directly interacts with its respective transmembrane constituent, coupling ligand-mediated KTN conformational changes to the permease's activity.

Amino Acid Sequence↗

PubMed2001

X-ray structure of sensory rhodopsin II at 2.1-A resolution.

Sensory rhodopsins (SRs) belong to a subfamily of heptahelical transmembrane proteins containing a retinal chromophore. These photoreceptors mediate the cascade of vision in animal eyes and phototaxis in archaebacteria and unicellular flagellated algae. Signal transduction by these photoreceptors occurs by means of transducer proteins. The two archaebacterial sensory rhodopsins SRI and SRII are coupled to the membrane-bound HtrI and HtrII transducer proteins. Activation of these proteins initiates phosphorylation cascades that modulate the flagellar motors, resulting in either attractant (SRI) or repellent (SRII) phototaxis. In addition, transducer-free SRI and SRII were shown to operate as proton pumps, analogous to bacteriorhodopsin. Here, we present the x-ray structure of SRII from Natronobacterium pharaonis (pSRII) at 2.1-A resolution, revealing a unique molecular architecture of the retinal-binding pocket. In particular, the structure of pSRII exhibits a largely unbent conformation of the retinal (as compared with bacteriorhodopsin and halorhodopsin), a hydroxyl group of Thr-204 in the vicinity of the Schiff base, and an outward orientation of the guanidinium group of Arg-72. Furthermore, the structure reveals a putative chloride ion that is coupled to the Schiff base by means of a hydrogen-bond network and a unique, positively charged surface patch for a probable interaction with HtrII. The high-resolution structure of pSRII provides a structural basis to elucidate the mechanisms of phototransduction and color tuning.

Animals↗

PubMed2004

Agonist-induced internalization of leukotriene B(4) receptor 1 requires G-protein-coupled receptor kinase 2 but not arrestins.

The leukotriene B(4) (LTB(4)) receptor (BLT1) becomes desensitized upon repeated agonist stimulation. Little is known, however, about BLT1 internalization, which follows desensitization in most G-protein-coupled receptors (GPCR). In the current study, transiently expressed BLT1 readily internalized, after LTB(4) stimulation, in RBL-2H3 cells that express high levels of endogenous GPCR kinase 2 (GRK2) but did not in COS-7 or human embryonic kidney (HEK) 293 cells, which do not overexpress GRK. The internalization of BLT1 could be blocked in RBL-2H3 cells by coexpressing dominant-negative (DN) GRK2 K220R and could be promoted in HEK293 cells by coexpressing wild-type (WT) GRK2. Coexpression of WT or DN nonvisual arrestins had no effect on BLT1 internalization. Moreover, upon stimulation with LTB(4), BLT1 did not induce arrestin-green fluorescence protein redistribution in either cell type, even in the presence of overexpressed GRK2. Coimmunoprecipitation experiments confirmed that BLT1 could associate with GRK2 but not with arrestins. A C-tail-truncated mutant of BLT1 lost the capacity to internalize and associate with GRK2 upon exposure to LTB(4), suggesting that the C-tail was required for receptor internalization and association with GRK2. Taken together, our results indicate that the C terminus of BLT1 plays a pivotal role in receptor internalization and GRK2 association. Moreover, ligand-induced BLT1 internalization is dependent on GRK2 but independent of arrestins. This may allow differential, cell-type-specific signaling in response to LTB(4), depending on GRK expression levels.

Animals↗

PubMed1996

Modeling plasmalemma ion transport of the aquatic plant Egeria densa.

Fresh-water plants generate extraordinarily high electric potential differences at the plasma membrane. For a deeper understanding of the underlying transport processes a mathematical model of the electrogenic plasmalemma ion transport was developed based on experimental data mainly obtained from Egeria densa. The model uses a general nonlinear network approach and assumes coupling of the transporters via membrane potential. A proton pump, an outward-rectifying K+ channel, an inward-rectifying K+ channel, a Cl- channel and a (2H-Cl)+ symporter are considered to be elements of the system. The model takes into consideration the effects of light, external pH and ionic content of the bath medium on ion transport. As a result it does not only satisfactorily describe the membrane potential as a function of these external physiological factors but also succeeds in simulating the effects of specific inhibitors as well as I-V-curves obtained with the patch-clamp technique in the whole cell mode. The quality of the model was checked by stability and sensitivity analyses.

Cell Membrane↗

PubMed2005

Role of endogenous TRPC6 channels in Ca2+ signal generation in A7r5 smooth muscle cells.

The ubiquitously expressed canonical transient receptor potential (TRPC) ion channels are considered important in Ca2+ signal generation, but their mechanisms of activation and roles remain elusive. Whereas most studies have examined overexpressed TRPC channels, we used molecular, biochemical, and electrophysiological approaches to assess the expression and function of endogenous TRPC channels in A7r5 smooth muscle cells. Real time PCR and Western analyses reveal TRPC6 as the only member of the diacylglycerol-responsive TRPC3/6/7 subfamily of channels expressed at significant levels in A7r5 cells. TRPC1, TRPC4, and TRPC5 were also abundant. An outwardly rectifying, nonselective cation current was activated by phospholipase C-coupled vasopressin receptor activation or by the diacylglycerol analogue, oleoyl-2-acetyl-sn-glycerol (OAG). Introduction of TRPC6 small interfering RNA sequences into A7r5 cells by electroporation led to 90% reduction of TRPC6 transcript and 80% reduction of TRPC6 protein without any detectable compensatory changes in the expression of other TRPC channels. The OAG-activated nonselective cation current was similarly reduced by TRPC6 RNA interference. Intracellular Ca2+ measurements using fura-2 revealed that thapsigargin-induced store-operated Ca2+ entry was unaffected by TRPC6 knockdown, whereas vasopressin-induced Ca2+ entry was suppressed by more than 50%. In contrast, OAG-induced Ca2+ transients were unaffected by TRPC6 knockdown. Nevertheless, OAG-induced Ca2+ entry bore the hallmarks of TRPC6 function; it was inhibited by protein kinase C and blocked by the Src-kinase inhibitor, 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine (PP2). Importantly, OAG-induced Ca2+ entry was blocked by the potent L-type Ca2+ channel inhibitor, *nimodipine. Thus, TRPC6 activation probably results primarily in Na ion entry and depolarization, leading to activation of L-type channels as the mediators of Ca2+ entry. Calculations reveal that even 90% reduction of TRPC6 channels would allow depolarization sufficient to activate L-type channels. This tight coupling between TRPC6 and L-type channels is probably important in mediating smooth muscle cell membrane potential and muscle contraction.

Animals↗