Funktionelle Charakterisierung des TRPM5-Gens

In der vorliegenden Promotionsarbeit wurde der zur TRP (transient receptor potential)-Familie gehörende TRPM5-Kanal funktionell charakterisiert. Elektrophysiologische Analysen TRPM5-überexprimierender HEK 293-Zellen zeigten, dass TRPM5 einen Ca2+-aktivierbaren, nicht-selektiven Kationenkanal darstellt, der monovalente Ionen leitet. Die Aktivierung des TRPM5-Kanals hängt insbesondere von der Geschwindigkeit des intrazellulären Ca2+-Anstiegs ab. Somit stellt TRPM5 eine Komponente der zellulären Signaltransduktionskaskaden dar: Nach Rezeptoraktivierung induziert TRPM5 einen raschen, transienten Kationeneinstrom, der zur Depolarisation der Zellmembran führt. Die Expression der beiden humanen TRPM5-Spleißformen als TRPM5/EGFP-Fusionsproteine in HEK 293-Zellen zeigte eine vorwiegende Lokalisation in der Zellmembran. In elektrophysiologischen Analysen wurde nachgewiesen, dass TRPM5-short als TRPM5-Kanalblocker funktioniert. Für die funktionelle in vivo-Charakterisierung des TRPM5-Kanals wurde ein auf RNAi (RNA interference) basierendes, transgenes Trpm5-knock down-Mausmodell hergestellt. Obwohl in drei der vier etablierten Knock down-Mauslinien eine Trpm5-Herunterregulation in der Leber und/oder in der Zunge nachgewiesen werden konnte, zeigten alle Mäuse einen wildtyp-ähnlichen Phänotyp. Weiterführende Untersuchungen an den von Zhang et al. (Cell, 2003) hergestellten Trpm5-knock out-Mäusen offenbarten, dass Trpm5 für eine geregelte Glukosetoleranz essentiell ist. Insulinsekretionsanalysen mit isolierten Langerhans’schen Inseln dieser Mäuse zeigten, dass ohne Trpm5 eine beeinträchtigte Insulinsekretionskinetik in den pankreatischen Betazellen vorliegt. Somit stellt TRPM5 einen neuen Kandidaten für Erkrankungen wie Diabetes Typ 2 dar, die durch eine Fehlregulation der Insulinsekretion gekennzeichnet sind.

Activity and mechanisms of action of novel organosulfur derivatives of the HDAC inhibitor Valproic Acid in human experimental models of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) represents the leading cause of cancer death worldwide, and 5-year survival is about 16% for patients diagnosed with advanced lung cancer and about 70-90% when the disease is diagnosed and treated at earlier stages. Treatment of NSCLC is changed in the last years with the introduction of targeted agents, such as gefitinib and erlotinib, that have dramatically changed the natural history of NSCLC patients carrying specific mutations in the EGFR gene, or crizotinib, for patients with the EML4-ALK translocation. However, such patients represent only about 15-20% of all NSCLC patients, and for the remaining individuals conventional chemotherapy represents the standard choice yet, but response rate to thise type of treatment is only about 20%. Development of new drugs and new therapeutic approaches are so needed to improve patients outcome. In this project we aimed to analyse the antitumoral activity of two compounds with the ability to inhibit histone deacethylases (ACS 2 and ACS 33), derived from Valproic Acid and conjugated with H2S, in human cancer cell lines derived from NSCLC tissues. We showed that ACS 2 represents the more promising agent. It showed strong antitumoral and pro-apoptotic activities, by inducing membrane depolarization, cytocrome-c release and caspase 3 and 9 activation. It was able to reduce the invasive capacity of cells, through inhibition of metalloproteinases expression, and to induce a reduced chromatin condensation. This last characteristic is probably responsible for the observed high synergistic activity in combination with cisplatin. In conclusion our results highlight the potential role of the ACS 2 compound as new therapeutic option for NSCLC patients, especially in combination with cisplatin. If validated in in vivo models, this compound should be worthy for phase I clinical trials.

Excitability properties of mouse motor axons in the mutant SOD1(G93A) model of amyotrophic lateral sclerosis

Non-invasive excitability studies of motor axons in patients with amyotrophic lateral sclerosis (ALS) have revealed a changing pattern of abnormal membrane properties with disease progression, but the heterogeneity of the changes has made it difficult to relate them to pathophysiology. The SOD1(G93A) mouse model of ALS displays more synchronous motoneuron pathology. Multiple excitability measures of caudal and sciatic nerves in mutant and wild-type mice were compared before onset of signs and during disease progression (4-19 weeks), and they were related to changes in muscle fiber histochemistry. Excitability differences indicated a modest membrane depolarization in SOD1(G93A) axons at about the time of symptom onset (8 weeks), possibly due to deficient energy supply. Previously described excitability changes in ALS patients, suggesting altered sodium and potassium conductances, were not seen in the mice. This suggests that those changes relate to features of the human disease that are not well represented in the animal model.

Nerve excitability changes in critical illness polyneuropathy

Patients in intensive care units frequently suffer muscle weakness and atrophy due to critical illness polyneuropathy (CIP), an axonal neuropathy associated with systemic inflammatory response syndrome and multiple organ failure. CIP is a frequent and serious complication of intensive care that delays weaning from mechanical ventilation and increases mortality. The pathogenesis of CIP is not well understood and no specific therapy is available. The aim of this project was to use nerve excitability testing to investigate the changes in axonal membrane properties occurring in CIP. Ten patients (aged 37-76 years; 7 males, 3 females) were studied with electrophysiologically proven CIP. The median nerve was stimulated at the wrist and compound action potentials were recorded from abductor pollicis brevis muscle. Strength-duration time constant, threshold electrotonus, current-threshold relationship and recovery cycle (refractoriness, superexcitability and late subexcitability) were recorded using a recently described protocol. In eight patients a follow-up investigation was performed. All patients underwent clinical examination and laboratory investigations. Compared with age-matched normal controls (20 subjects; aged 38-79 years; 7 males, 13 females), CIP patients exhibited reduced superexcitability at 7 ms, from -22.3 +/- 1.6% to -7.6 +/- 3.1% (mean +/- SE, P approximately 0.0001) and increased accommodation to depolarizing (P < 0.01) and hyperpolarizing currents (P < 0.01), indicating membrane depolarization. Superexcitability was reduced both in patients with renal failure and without renal failure. In the former, superexcitability correlated with serum potassium (R = 0.88), and late subexcitability was also reduced (as also occurs owing to hyperkalaemia in patients with chronic renal failure). In patients without renal failure, late subexcitability was normal, and the signs of membrane depolarization correlated with raised serum bicarbonate and base excess, indicating compensated respiratory acidosis. It is inferred that motor axons in these CIP patients are depolarized, in part because of raised extracellular potassium, and in part because of hypoperfusion. The chronic membrane depolarization may contribute to the development of neuropathy.

Velocity recovery cycles of human muscle action potentials and their sensitivity to ischemia

This study was undertaken to test whether recovery cycle measurements can provide useful information about the membrane potential of human muscle fibers. Multifiber responses to direct muscle stimulation through needle electrodes were recorded from the brachioradialis of healthy volunteers, and the latency changes measured as conditioning stimuli were applied at interstimulus intervals of 2-1000 ms. In all subjects, the relative refractory period (RRP), which lasted 3.27 +/- 0.45 ms (mean +/- SD, n = 12), was followed by a phase of supernormality, in which the velocity increased by 9.3 +/- 3.4% at 6.1 +/- 1.3 ms, and recovered over 1 s. A broad hump of additional supernormality was seen at around 100 ms. Extra conditioning stimuli had little effect on the early supernormality but increased the later component. The two phases of supernormality resembled early and late afterpotentials, attributable respectively to the passive decay of membrane charge and potassium accumulation in the t-tubules. Five minutes of ischemia progressively prolonged the RRP and reduced supernormality, confirming that these parameters are sensitive to membrane depolarization. Velocity recovery cycles may provide useful information about altered muscle membrane potential and t-tubule function in muscle disease. Muscle Nerve, 2008.

The electrophysiology of glomerular mesangial cells

Glomerular mesangial cells (MC) are renal vascular cells that regulate the surface area of glomerular capillaries and thus, partly control glomerular filtration rate. Clarification of the signal transduction pathways and ionic mechanisms modulating MC tone are critical to understanding the physiology and pathophysiology of these cells, and the integrative role these cells play in fluid and electrolyte homeostasis. The patch clamp technique and an assay of cell concentration were used to electrophysiologically and pharmacologically analyze the ion channels of the plasmalemmal of human glomerular MC maintained in tissue culture. Moreover, the signal transduction pathways modulating channels involved in relaxation were investigated. Three distinct K$\sp+$-selective channels were identified: two low conductance channels (9 and 65pS) maintained MC at rest, while a larger conductance (206pS) K$\sp+$ channel was quiescent at rest. This latter channel was pharmacologically and biophysically similar to the large, Ca$\sp{2+}$-activated K$\sp+$ channel (BK$\rm\sb{Ca}$) identified in smooth muscle. BK$\rm\sb{Ca}$ played an essential role in relaxation of MC. In cell-attached patches, the open probability (P$\rm\sb{o}$) of BK$\rm\sb{Ca}$ increased from a basal level of $<$0.05 to 0.22 in response to AII (100nM)-induced mobilization of cytosolic Ca$\sp{2+}$. Activation in response to contractile signals (membrane depolarization and Ca$\sp{2+}$ mobilization) suggests that BK$\rm\sb{Ca}$ acts as a low gain feedback regulator of contraction. Atrial natriuretic factor (ANF; 1.0$\mu$M) and nitroprusside (NP; 0.1mM), via the second messenger, cGMP, increase the feedback gain of BK$\rm\sb{Ca}$. In cell-attached patches bathed with physiological saline, these agents transiently activated BK$\rm\sb{Ca}$ from a basal $\rm P\sb{o}<0.05$ to peak responses near 0.50. As membrane potential hyperpolarizes towards $\rm E\sb{K}$ (2-3 minutes), BK$\rm\sb{Ca}$ inactivates. Upon depolarizing V$\rm\sb{m}$ with 140 mM KCl, db-cGMP (10$\mu$M) activated BK$\rm\sb{Ca}$ to a sustained P$\rm\sb{o}$ = 0.51. Addition of AII in the presence of cGMP further increased P$\rm\sb{o}$ to 0.82. Activation of BK$\rm\sb{Ca}$ by cGMP occured via an endogenous cGMP-dependent protein kinase (PKG): in excised, inside-out patches, PKG in the presence of Mg-ATP (0.1mM) and cGMP increased P$\rm\sb{o}$ from 0.07 to 0.39. In contrast, neither PKC nor PKA influenced BK$\rm\sb{Ca}$. Endogenous okadaic acid-sensitive protein phosphatase suppressed BK$\rm\sb{Ca}$ activity. Binning the change in P$\rm\sb{o}\ (\Delta P\sb{o}$) of BK$\rm\sb{Ca}$ in response to PKG (n = 69) established two distinct populations of channels: one that responded ($\cong$67%, $\rm\Delta P\sb{o} = 0.45 \pm 0.03$) and one that was unresponsive ($\Delta\rm P\sb{o} = 0.00 \pm 0.01$) to PKG. Activation of BK$\rm\sb{Ca}$ by PKG resulted from a decrease in the Ca$\sp{2+}$- and voltage-activation thresholds independent of sensitivities. In conclusion, mesangial BK$\rm\sb{Ca}$ channels sense both electrical and chemical signals of contraction and act as feedback regulators by repolarizing the plasma membrane. ANF and NO, via cGMP, stimulate endogenous PKG, which subsequently decreases the activation threshold of BK$\rm\sb{Ca}$ to increase the gain of this feedback regulatory signal. ^

Potassium and the excitability properties of normal human motor axons in vivo.

Hyperkalemia is an important cause of membrane depolarization in renal failure. A recent theoretical model of axonal excitability explains the effects of potassium on threshold electrotonus, but predicts changes in superexcitability in the opposite direction to those observed. To resolve this contradiction we assessed the relationship between serum potassium and motor axon excitability properties in 38 volunteers with normal potassium levels. Most threshold electrotonus measures were strongly correlated with potassium, and superexcitability decreased at higher potassium levels (P = 0.016), contrary to the existing model. Improved modelling of potassium effects was achieved by making the potassium currents obey the constant-field theory, and by making the potassium permeabilities proportional to external potassium, as has been observed in vitro. This new model also accounted well for the changes in superexcitability and other excitability measures previously reported in renal failure. These results demonstrate the importance of taking potassium levels into account when assessing axonal membrane dysfunction by excitability testing, and provide evidence that potassium currents are activated by external potassium in vivo.

CXCL14 Displays Antimicrobial Activity against Respiratory Tract Bacteria and Contributes to Clearance of Streptococcus pneumoniae Pulmonary Infection

CXCL14 is a chemokine with an atypical, yet highly conserved, primary structure characterized by a short N terminus and high sequence identity between human and mouse. Although it induces chemotaxis of monocytic cells at high concentrations, its physiological role in leukocyte trafficking remains elusive. In contrast, several studies have demonstrated that CXCL14 is a broad-spectrum antimicrobial peptide that is expressed abundantly and constitutively in epithelial tissues. In this study, we further explored the antimicrobial properties of CXCL14 against respiratory pathogens in vitro and in vivo. We found that CXCL14 potently killed Pseudomonas aeruginosa, Streptococcus mitis, and Streptococcus pneumoniae in a dose-dependent manner in part through membrane depolarization and rupture. By performing structure-activity studies, we found that the activity against Gram-negative bacteria was largely associated with the N-terminal peptide CXCL141-13. Interestingly, the central part of the molecule representing the β-sheet also maintained ∼62% killing activity and was sufficient to induce chemotaxis of THP-1 cells. The C-terminal α-helix of CXCL14 had neither antimicrobial nor chemotactic effect. To investigate a physiological function for CXCL14 in innate immunity in vivo, we infected CXCL14-deficient mice with lung pathogens and we found that CXCL14 contributed to enhanced clearance of Streptococcus pneumoniae, but not Pseudomonas aeruginosa. Our comprehensive studies reflect the complex bactericidal mechanisms of CXCL14, and we propose that different structural features are relevant for the killing of Gram-negative and Gram-positive bacteria. Taken together, our studies show that evolutionary-conserved features of CXCL14 are important for constitutive antimicrobial defenses against pneumonia.

Phentolamine block of KATP channels is mediated by Kir6.2

The ATP-sensitive K+-channel (KATP channel) plays a key role in insulin secretion from pancreatic β cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca2+ entry and insulin release. The β cell KATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K+-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit KATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. KATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac KATP channel.

Molecular mechanism of use-dependent calcium channel block by phenylalkylamines: Role of inactivation

The role of channel inactivation in the molecular mechanism of calcium (Ca2+) channel block by phenylalkylamines (PAA) was analyzed by designing mutant Ca2+ channels that carry the high affinity determinants of the PAA receptor site [Hockerman, G. H., Johnson, B. D., Scheuer, T., and Catterall, W. A. (1995) J. Biol. Chem. 270, 22119–22122] but inactivate at different rates. Use-dependent block by PAAs was studied after expressing the mutant Ca2+ channels in Xenopus oocytes. Substitution of single putative pore-orientated amino acids in segment IIIS6 by alanine (F-1499-A, F-1500-A, F-1510-A, I-1514-A, and F-1515-A) gradually slowed channel inactivation and simultaneously reduced inhibition of barium currents (IBa) by (−)D600 upon depolarization by 100 ms steps at 0.1 Hz. This apparent reduction in drug sensitivity was only evident if test pulses were applied at a low frequency of 0.1 Hz and almost disappeared at the frequency of 1 Hz. (−)D600 slowed IBa recovery after maintained membrane depolarization (1–3 sec) to a comparable extent in all channel constructs. A drug-induced delay in the onset of IBa recovery from inactivation suggests that PAAs promote the transition to a deep inactivated channel conformation. These findings indicate that apparent PAA sensitivity of Ca2+ channels is not only defined by drug interaction with its receptor site but also crucially dependent on intrinsic gating properties of the channel molecule. A molecular model for PAA-Ca2+ channel interaction that accounts for the relationship between drug induced inactivation and channel block by PAA is proposed.

The Arabidopsis CHL1 protein plays a major role in high-affinity nitrate uptake

The CHL1 (NRT1) gene of Arabidopsis encodes a nitrate-inducible nitrate transporter that is thought to be a component of the low-affinity (mechanism II) nitrate-uptake system in plants. A search was performed to find high-affinity (mechanism I) uptake mutants by using chlorate selections on plants containing Tag1 transposable elements. Chlorate-resistant mutants defective in high-affinity nitrate uptake were identified, and one had a Tag1 insertion in chl1, which was responsible for the phenotype. Further analysis showed that chl1 mutants have reduced high-affinity uptake in induced plants and are missing a saturable component of the constitutive, high-affinity uptake system in addition to reduced low-affinity uptake. The contribution of CHL1 to constitutive high-affinity uptake is higher when plants are grown at more acidic pH, conditions that increase the level of CHL1 mRNA. chl1 mutants show reduced membrane depolarization in root epidermal cells in response to low (250 μM) and high (10 mM) concentrations of nitrate. Low levels of nitrate (100 μM) induce a rapid increase in CHL1 mRNA. These results show that CHL1 is an important component of both the high-affinity and the low-affinity nitrate-uptake systems and indicate that CHL1 may be a dual-affinity nitrate transporter.

Voltage-controlled gating in a large conductance Ca2+-sensitive K+channel (hslo)

Large conductance calcium- and voltage-sensitive K+ (MaxiK) channels share properties of voltage- and ligand-gated ion channels. In voltage-gated channels, membrane depolarization promotes the displacement of charged residues contained in the voltage sensor (S4 region) inducing gating currents and pore opening. In MaxiK channels, both voltage and micromolar internal Ca2+ favor pore opening. We demonstrate the presence of voltage sensor rearrangements with voltage (gating currents) whose movement and associated pore opening is triggered by voltage and facilitated by micromolar internal Ca2+ concentration. In contrast to other voltage-gated channels, in MaxiK channels there is charge movement at potentials where the pore is open and the total charge per channel is 4–5 elementary charges.

Caldesmon Inhibits Nonmuscle Cell Contractility and Interferes with the Formation of Focal Adhesions

Caldesmon is known to inhibit the ATPase activity of actomyosin in a Ca2+–calmodulin-regulated manner. Although a nonmuscle isoform of caldesmon is widely expressed, its functional role has not yet been elucidated. We studied the effects of nonmuscle caldesmon on cellular contractility, actin cytoskeletal organization, and the formation of focal adhesions in fibroblasts. Transient transfection of nonmuscle caldesmon prevents myosin II-dependent cell contractility and induces a decrease in the number and size of tyrosine-phosphorylated focal adhesions. Expression of caldesmon interferes with Rho A-V14-mediated formation of focal adhesions and stress fibers as well as with formation of focal adhesions induced by microtubule disruption. This inhibitory effect depends on the actin- and myosin-binding regions of caldesmon, because a truncated variant lacking both of these regions is inactive. The effects of caldesmon are blocked by the ionophore A23187, thapsigargin, and membrane depolarization, presumably because of the ability of Ca2+–calmodulin or Ca2+–S100 proteins to antagonize the inhibitory function of caldesmon on actomyosin contraction. These results indicate a role for nonmuscle caldesmon in the physiological regulation of actomyosin contractility and adhesion-dependent signaling and further demonstrate the involvement of contractility in focal adhesion formation.

Activation of a heterologously expressed octopamine receptor coupled only to adenylyl cyclase produces all the features of presynaptic facilitation in Aplysia sensory neurons

Short-term behavioral sensitization of the gill-withdrawal reflex after tail stimuli in Aplysia leads to an enhancement of the connections between sensory and motor neurons of this reflex. Both behavioral sensitization and enhancement of the connection between sensory and motor neurons are importantly mediated by serotonin. Serotonin activates two types of receptors in the sensory neurons, one of which is coupled to the cAMP/protein kinase A (PKA) pathway and the other to the inositol triphosphate/protein kinase C (PKC) pathway. Here we describe a genetic approach to assessing the isolated contribution of the PKA pathway to short-term facilitation. We have cloned from Aplysia an octopamine receptor gene, Ap oa1, that couples selectively to the cAMP/PKA pathway. We have ectopically expressed this receptor in Aplysia sensory neurons of the pleural ganglia, where it is not normally expressed. Activation of this receptor by octopamine stimulates all four presynaptic events involved in short-term synaptic facilitation that are normally produced by serotonin: (i) membrane depolarization; (ii) increased membrane excitability; (iii) increased spike duration; and (iv) presynaptic facilitation. These results indicate that the cAMP/PKA pathway alone is sufficient to produce all the features of presynaptic facilitation.

Ascorbic acid acts as an inhibitory transmitter in the hypothalamus to inhibit stimulated luteinizing hormone-releasing hormone release by scavenging nitric oxide

Because ascorbic acid (AA) is concentrated in synaptic vesicles containing glutamic acid, we hypothesized that AA might act as a neurotransmitter. Because AA is an antioxidant, it might therefore inhibit nitric oxidergic (NOergic) activation of luteinizing hormone-releasing hormone (LH-RH) release from medial basal hypothalamic explants by chemically reducing NO. Cell membrane depolarization induced by increased potassium concentration [K+] increased medium concentrations of both AA and LH-RH. An inhibitor of NO synthase (NOS), NG-monomethyl-l-arginine (NMMA), prevented the increase in medium concentrations of AA and LH-RH induced by high [K+], suggesting that NO mediates release of both AA and LH-RH. Calcium-free medium blocked not only the increase in AA in the medium but also the release of LH-RH. Sodium nitroprusside, which releases NO, stimulated LH-RH release and decreased the concentration of AA in the incubation medium, presumably because the NO released oxidized AA to dehydro-AA. AA (10−5 to 10−3 M) had no effect on basal LH-RH release but completely blocked high [K+]- and nitroprusside-induced LH-RH release. N-Methyl-d-aspartic acid (NMDA), which mimics the action of the excitatory amino acid neurotransmitter glutamic acid, releases LH-RH by releasing NO. AA (10−5 to 10−3 M) inhibited the LH-RH-releasing action of NMDA. AA may be an inhibitory neurotransmitter that blocks NOergic stimulation of LH-RH release by chemically reducing the NO released by the NOergic neurons.