Angelika Lampert

Activating Mutation of the Renal Epithelial Chloride Channel ClC-Kb Predisposing to Hypertension

The chloride channel ClC-Kb is expressed in the basolateral cell membrane of the distal nephron and participates in renal NaCl reabsorption. Loss-of-function mutations of ClC-Kb lead to classic Bartter syndrome, a rare salt-wasting disorder. Recently, we identified the ClC-KbT481S polymorphism, which confers a strong gain-of-function effect on the ClC-Kb chloride channel. The present study has been performed to explore the prevalence of the mutation and its functional significance in renal salt handling and blood pressure regulation. As evident from electrophysiological analysis with the 2-electrode voltage-clamp technique, heterologous expression of ClC-KbT481S in Xenopus oocytes gave rise to a current that was 7-fold larger than the current produced by wild-type ClC-Kb. The prevalence of the mutant allele was significantly higher in an African population from Ghana (22%) than in whites (12%). As tested in 1 white population, carriers of ClC-KbT481S were associated with significantly higher systolic (by ≈6.0 mm Hg) and diastolic (by ≈4.2 mm Hg) blood pressures and significantly higher prevalence (45% versus 25%) of hypertensive (≥140/90 mm Hg) blood pressure levels. Individuals carrying ClC-KbT481S had significantly higher plasma Na+ concentrations and significantly decreased glomerular filtration rate. In conclusion, the mutation ClC-KbT481S of the renal epithelial Cl− channel ClC-Kb strongly activates ClC-Kb chloride channel function in vitro and may predispose to the development of essential hypertension in vivo.

Sodium channelopathies and pain

Chronic pain often represents a severe, debilitating condition. Up to 10% of the worldwide population are affected, and many patients are poorly responsive to current treatment strategies. Nociceptors detect noxious conditions to produce the sensation of pain, and this signal is conveyed to the CNS by means of action potentials. The fast upstroke of action potentials is mediated by voltage-gated sodium channels, of which nine pore-forming α-subunits (Nav1.1–1.9) have been identified. Heterogeneous functional properties and distinct expression patterns denote specialized functions of each subunit. The Nav1.7 and Nav1.8 subunits have emerged as key molecules involved in peripheral pain processing and in the development of an increased pain sensitivity associated with inflammation and tissue injury. Several mutations in the SCN9A gene encoding for Nav1.7 have been identified as important cellular substrates for different heritable pain syndromes. This review aims to cover recent progress on our understanding of how biophysical properties of mutant Nav1.7 translate into an aberrant electrogenesis of nociceptors. We also recapitulate the role of Nav1.8 for peripheral pain processing and of additional sodium channelopathies which have been linked to disorders with pain as a significant component.

The Molecular Basis for Species-specific Activation of Human TRPA1 Protein by Protons Involves Poorly Conserved Residues within Transmembrane Domains 5 and 6

Background: Extracellular acidosis mediates pain and inflammation by activating sensory afferent neurons. Results: Protons activate and sensitize human TRPA1 in a strongly species-specific manner encoded by transmembrane domains 5 and 6. Conclusion: Our data identify TRPA1 as an ion channel likely to mediate acid-induced pain in humans. Significance: Protons are the first known endogenous agonists of TRPA1 with species-specificity for human TRPA1. The surveillance of acid-base homeostasis is concerted by diverse mechanisms, including an activation of sensory afferents. Proton-evoked activation of rodent sensory neurons is mainly mediated by the capsaicin receptor TRPV1 and acid-sensing ion channels. In this study, we demonstrate that extracellular acidosis activates and sensitizes the human irritant receptor TRPA1 (hTRPA1). Proton-evoked membrane currents and calcium influx through hTRPA1 occurred at physiological acidic pH values, were concentration-dependent, and were blocked by the selective TRPA1 antagonist HC030031. Both rodent and rhesus monkey TRPA1 failed to respond to extracellular acidosis, and protons even inhibited rodent TRPA1. Accordingly, mouse dorsal root ganglion neurons lacking TRPV1 only responded to protons when hTRPA1 was expressed heterologously. This species-specific activation of hTRPA1 by protons was reversed in both mouse and rhesus monkey TRPA1 by exchange of distinct residues within transmembrane domains 5 and 6. Furthermore, protons seem to interact with an extracellular interaction site to gate TRPA1 and not via a modification of intracellular N-terminal cysteines known as important interaction sites for electrophilic TRPA1 agonists. Our data suggest that hTRPA1 acts as a sensor for extracellular acidosis in human sensory neurons and should thus be taken into account as a yet unrecognized transduction molecule for proton-evoked pain and inflammation. The species specificity of this property is unique among known endogenous TRPA1 agonists, possibly indicating that evolutionary pressure enforced TRPA1 to inherit the role as an acid sensor in human sensory neurons.

Glucocorticoid adrenal steroids and glucocorticoid-inducible kinase isoforms in the regulation of GluR6 expression

Generation of memory is enhanced during stress, an effect attributed to stimulation of neuronal learning by adrenal glucocorticoids. The glucocorticoid‐dependent genes include the serum‐ and glucocorticoid‐inducible kinase SGK1. SGK1 is activated through the phosphatidylinositol 3 kinase (PI3‐kinase) pathway by growth factors such as insulin‐like growth factor‐1 (IGF1) or tumour growth factor β (TGF‐β). Previously, a fourfold higher expression of SGK1 has been observed in fast‐learning rats as compared with slow‐learning rats. The mechanisms linking glucocorticoids or SGK1 with neuronal function have, however, remained elusive. We show here that treatment of mice with the glucocorticoid dexamethasone (238 μg day−1 for 8–20 days) enhances hippocampal expression of GluR6. Immunohistochemistry reveals significantly enhanced GluR6 protein abundance at neurones but not at astrocytes in mice. Immunohistochemistry and patch clamp on hippocampal neurones in primary culture reveal upregulation of GluR6 protein abundance and kainate‐induced currents following treatment with dexamethasone (1 μm) and TGF‐β (1 μm). In Xenopus oocytes expressing rat GluR6, coexpression of SGK1 strongly increases glutamate‐induced current at least partially by increasing the abundance of GluR6 protein in the plasma membrane. The related kinases SGK2 and SGK3 similarly stimulate GluR6, but are less effective than SGK1. The observations point to a novel mechanism regulating GluR6 which contributes to the regulation of neuronal function by glucocorticoids.

Thyrotropin Serum Concentrations in Healthy Volunteers Are Associated with Depression-Related Personality Traits

Background: Alterations of the hypothalamic-pituitary-thyroid system at the hypophyseal level have been described in affective disorders. The NEO Five-Factor Inventory is a widely used instrument, which assesses neuroticism, a strong marker for vulnerability to depression. Methods: Endogenous serum concentrations of thyroid-stimulating hormone (TSH), free T3 and free T4 were measured and the NEO Five-Factor Inventory was performed in 121 healthy unrelated volunteers (51 male, 70 female; median age: 23 years, range from 17 to 71). Results: The TSH serum concentrations showed a median of 1.35 mU/l with a range from 0.1 to 4.0. The free T3 serum concentrations were 5.2 ± 0.7 pmol/l (mean ± SD), and the free T4 concentrations 16.5 ± 2.4 pmol/l (mean ± SD). The power of 0.4 of the TSH serum concentration and the logarithm of the depression-related factor neuroticism had a bivariate normal distribution and were negatively correlated (r = –0.337, p = 0.0002). Conclusion: Low TSH levels in healthy humans might be linked to an increased risk to develop depression.

The distinct effects of lipid emulsions used for "lipid resuscitation" on gating and bupivacaine-induced inhibition of the cardiac sodium channel Nav1.5.

BACKGROUND: Systemic administration of lipid emulsions is an established treatment for local anesthetic intoxication. However, it is unclear by which mechanisms lipids achieve this function. The high cardiac toxicity of the lipophilic local anesthetic bupivacaine probably results from a long-lasting inhibition of the cardiac Na+ channel Nav1.5. In this study, we sought to determine whether lipid emulsions functionally interact with Nav1.5 or counteract inhibition by bupivacaine. METHODS: Human embryonic kidney cells expressing human Nav1.5 were investigated by whole-cell patch clamp. The effects of Intralipid® and Lipofundin® were explored on functional properties and on bupivacaine-induced inhibition. RESULTS: Intralipid and Lipofundin did not affect the voltage dependency of activation, but induced a small hyperpolarizing shift of the steady-state fast inactivation and impaired the recovery from fast inactivation. Lipofundin, but not Intralipid, induced a concentration-dependent but voltage-independent tonic block (42% ± 4% by 3% Lipofundin). The half-maximal inhibitory concentration (IC50) values for tonic block by bupivacaine (50 ± 4 µM) were significantly increased when lipids were coapplied (5% Intralipid: 196 ± 22 µM and 5% Lipofundin: 103 ± 8 µM). Use-dependent block by bupivacaine at 10 Hz was also reduced by both lipid emulsions. Moreover, the recovery of inactivated channels from bupivacaine-induced block was faster in the presence of lipids. CONCLUSIONS: Our data indicate that lipid emulsions reduce rather than increase availability of Nav1.5. However, both Intralipid and Lipofundin partly relieve Nav1.5 from block by bupivacaine. These effects are likely to involve not only a direct interaction of lipids with Nav1.5 but also the ability of lipid emulsions to absorb bupivacaine and thus reduce its effective concentration.

Effect of dexamethasone on voltage-gated K + channels in Jurkat T-lymphocytes

The voltage-gated K+ channel Kv1.3 is an important regulator of lymphocyte function. Activation of lymphocytes is accompanied by stimulation, whereas CD95-induced apoptosis by inhibition, of Kv1.3. The channel serves to maintain cell membrane potential, a prerequisite for signalling through the Ca2+ release-activated Ca2+ channel ICRAC. As glucocorticoids are known to regulate lymphocyte function, the present study addressed the effect of dexamethasone on voltage-gated K+ channels in Jurkat T-lymphocytes. In whole-cell patch-clamp experiments current families evoked by 200-ms potential steps every 15xa0s from −70xa0mV to values from −120 to +100xa0mV revealed the functional expression of voltage-gated K+ channels. Pre-treatment of Jurkat T-lymphocytes for 2–3xa0h with 1xa0µM dexamethasone led to a significant decrease of voltage-gated K+ currents. Fura-2-fluorescence measurements showed that the readdition of Ca2+ to Ca2+-depleted cells led to a rapid increase of cytosolic Ca2+ activity. This increase of Ca2+ activity was blunted by both the K+ channel blocker margatoxin (10xa0nM) and 24 h pre-treatment with dexamethasone (1xa0µM). In conclusion, dexamethasone inhibits voltage-gated K+ channels in Jurkat T-lymphocytes, an effect impeding Ca2+ entry through ICRAC.

Influence of gain of function epithelial chloride channel ClC-Kb mutation on hearing thresholds.

Hearing depends on functional ClC-K-type chloride channels composed of barttin with ClC-Ka or ClC-Kb. Loss-of-function mutations of the barttin gene BSND or of both, the ClC-Ka gene CLNKA and the ClC-Kb gene CLNKB lead to congenital deafness and renal salt wasting. Recently, we identified the gain-of-function mutation ClC-Kb(T481S) which is associated with increased blood pressure. To explore the impact of ClC-Kb(T481S) on hearing, healthy volunteers (n=329) and individuals suffering from tinnitus (n=246) volunteered for hearing tests (n=348) and genetic analysis (n=575). 19.1% of the individuals were heterozygote (ClC-Kb(T481S)/ClC-Kb) and 1.7% homozygote carriers. Pure tone average hearing threshold (PTAt) for air conduction was significantly (p<0.033) lower in ClC-Kb(T481S) carriers (13.2+/-1.2dB) than in wild-type individuals (17.1+/-0.9dB). The prevalence of ClC-Kb(T481S) carriers was significantly increased (29.7%) in individuals with PTAt<15dB (p<0.05) and significantly decreased (13.2%) in individuals with PTAt>30 dB (p<0.017). The difference was largely due to the female population. Bone conduction was less affected pointing to an effect of the mutation on middle ear function. Tinnitus tended to be more frequent in ClC-Kb(T481S) carriers, a difference, however, not statistically significant. In conclusion, hearing thresholds are slightly lower in carriers of ClC-Kb(T481S), i.e., the gain-of-function polymorphism ClC-Kb(T481S) exerts a subtle but significant protective effect against hearing loss.

Deranged Kv channel regulation in fibroblasts from mice lacking the serum and glucocorticoid inducible kinase SGK1.

Coexpression of the serum and glucocorticoid inducible kinase 1 (SGK1) up‐regulates Kv channel activity in Xenopus oocytes and human embryonic kidney cells. To investigate the physiological impact of SGK1 dependent Kv channel regulation, we recorded whole‐cell currents in lung fibroblasts from SGK1 knockout mice (sgk1−/−) and wild‐type littermates (sgk1+/+). Serum‐grown mouse lung fibroblasts (MLF) from both genotypes exhibited voltage‐gated outwardly rectifying K+‐currents with time‐dependent activation (τact∼3 msec), slow inactivation (τinact∼700 msec), use‐dependent inactivation, and (partial) inhibition by K+ channel blockers TEA, 4‐AP, and margatoxin. In serum grown MLF peak Kv current density at +100 mV was significantly lower in sgk1−/− (14u2009±u20092 pA/pF, nu2009=u200913) than in sgk1+/+ (31u2009±u20094 pA/pF, nu2009=u200916). PCR amplification of different Kv1 and Kv3 subunits from mouse fibroblasts demonstrated the expression of Kv1.1‐1.7, Kv3.1, and Kv3.3 mRNA in both sgk1+/+ and sgk1−/− cells. Upon serum deprivation Kv currents almost disappeared in sgk1+/+ (4u2009±u20091 pA/pF, nu2009=u200911) but not in sgk1−/− (10u2009±u20091 pA/pF, nu2009=u20096) MLF. Accordingly, following serum deprivation Kv current density was significantly lower in sgk1+/+ than in sgk1−/−. Stimulation of serum‐depleted cells with dexamethasone (dex) (1 μM, 1 day), IGF‐1 (6.7 μM, 4–6 h) or both, significantly activated Kv currents in sgk1+/+ but not in sgk1−/− MLF. In the presence of both, dex and IGF‐1, the Kv current density was significantly larger in sgk1+/+ (27u2009±u20093 pA/pF, nu2009=u200912) than in sgk1−/− (13u2009±u20093 pA/pF, nu2009=u200910) cells. Similar to MLF, Kv currents were significantly higher in sgk1+/+ mouse tail fibroblasts (MTF). In sgk1+/+ but not sgk1−/− MTF the Kv currents were inhibited upon serum deprivation and reincreased after stimulation of serum deprived MTF with dex (1 μM, 1 day) and afterwards with IGF‐1 (6.7 μM, 4–6 h). According to Fura‐2‐fluorescence capacitative Ca2+ entry was lower in sgk1−/− MTF compared to sgk1+/+ MTF. Upon serum deprivation capacitative Ca2+ entry decreased significantly in sgk1+/+ but not in sgk1−/− MTF. Stimulation of depleted cells with dex (1 μM, 1 day) and afterwards with IGF‐1 (6.7 μM, 4–6 h) reincreased capacitative Ca2+ entry in sgk1+/+ MTF, whereas in sgk1−/− cells it remained unchanged. In conclusion, lack of SGK1 does not abrogate Kv channel activity but abolishes regulation of those channels by serum, glucocorticoids and IGF‐1, an effect influencing capacitative Ca2+ entry.

Sodium channel slow inactivation interferes with open channel block

Mutations in the voltage-gated sodium channel Nav1.7 are linked to inherited pain syndromes such as erythromelalgia (IEM) and paroxysmal extreme pain disorder (PEPD). PEPD mutations impair Nav1.7 fast inactivation and increase persistent currents. PEPD mutations also increase resurgent currents, which involve the voltage-dependent release of an open channel blocker. In contrast, IEM mutations, whenever tested, leave resurgent currents unchanged. Accordingly, the IEM deletion mutation L955 (ΔL955) fails to produce resurgent currents despite enhanced persistent currents, which have hitherto been considered a prerequisite for resurgent currents. Additionally, ΔL955 exhibits a prominent enhancement of slow inactivation (SI). We introduced mutations into Nav1.7 and Nav1.6 that either enhance or impair SI in order to investigate their effects on resurgent currents. Our results show that enhanced SI is accompanied by impaired resurgent currents, which suggests that SI may interfere with open-channel block.