Benzenesulfonamides act as open-channel blockers on KV3.1 potassium channel.

KV3.1 blockers can serve as modulators of the rate of action potential firing in neurons with high rates of firing such as those of the auditory system. We studied the effects of several bioisosteres of N-alkylbenzenesulfonamides, and molecules derived from sulfanilic acid on KV3.1 channels, heterologously expressed in L-929 cells, using the whole-cell patch-clamp technique. Only the N-alkyl-benzenesulfonamides acted as open-channel blockers on KV3.1, while molecules analogous to PABA (p-aminobenzoic acid) and derived from sulfanilic acids did not block the channel. The IC50 of six N-alkyl-benzenesulfonamides ranged from 9 to 55 µM; and the Hill coefficient suggests the binding of two molecules to block KV3.1. Also, the effects of all molecules on KV3.1 were fully reversible. We look for similar features amongst the molecules that effectively blocked the channel and used them to model a blocker prototype. We found that bulkier groups and amino-lactams decreased the effectiveness of the blockage, while the presence of NO2 increased the effectiveness of the blockage. Thus, we propose N-alkylbenzenesulfonamides as a new class of KV3.1 channel blockers.

Correction to: 4-Chloro-3-nitro-N-butylbenzenesulfonamide acts on KV3.1 channels by an open-channel blocker mechanism.

Unfortunately, grey trace on Figure 2c was missing in the original publication of the article.

Nitric Oxide Modulates HCN Channels in Magnocellular Neurons of the Supraoptic Nucleus of Rats by an S-Nitrosylation-Dependent Mechanism.

The control of the excitability in magnocellular neurosecretory cells (MNCs) of the supraoptic nucleus has been attributed mainly to synaptic inputs from circunventricular organs. However, nitric oxide (NO), a gaseous messenger produced in this nucleus during isotonic and short-term hypertonic conditions, is an example of a modulator that can act directly on MNCs to modulate their firing rate. NO inhibits the electrical excitability of MNCs, leading to a decrease in the release of vasopressin and oxytocin. Although the effects of NO on MNCs are well established, the mechanism by which this gas produces its effect is, so far, unknown. Because NO acts independently of synaptic inputs, we hypothesized that ion channels present in MNCs are the targets of NO. To investigate this hypothesis, we used the patch-clamp technique in vitro and in situ to measure currents carried by hyperpolarization-activated and nucleotide-gated cation (HCN) channels and establish their role in determining the electrical excitability of MNCs in rats. Our results show that blockade of HCN channels by ZD7288 decreases MNC firing rate with significant consequences on the release of OT and VP, measured by radioimmunoassay. NO induced a significant reduction in HCN currents by binding to cysteine residues and forming S-nitrosothiol complexes. These findings shed new light on the mechanisms that control the electrical excitability of MNCs via the nitrergic system and strengthen the importance of HCN channels in the control of hydroelectrolyte homeostasis. SIGNIFICANCE STATEMENT: Cells in our organism live in a liquid environment whose composition and osmolality are maintained within tight limits. Magnocellular neurons (MNCs) of the supra optic nucleus can sense osmolality and control the synthesis and secretion of vasopressin (VP) and oxytocin (OT) by the neurohypophysis. OT and VP act on the kidneys controlling the excretion of water and sodium to maintain homeostasis. Here we combined electrophysiology, molecular biology, and radioimmunoassay to show that the electrical activity of MNCs can be controlled by nitric oxide (NO), a gaseous messenger. NO reacts with cysteine residues (S-nitrosylation) on hyperpolarization-activated and nucleotide-gated cation channels decreasing the firing rate of MNCs and the consequent secretion of VP and OT.

4-Chloro-3-nitro-N-butylbenzenesulfonamide acts on KV3.1 channels by an open-channel blocker mechanism.

The effects of 4-chloro-3-nitro-N-butylbenzenesulfonamide (SMD2) on KV3.1 channels, heterologous expressed in L-929 cells, were studied with the whole cell patch-clamp technique. SMD2 blocks KV3.1 in a reversible and use-dependent manner, with IC50 around 10 µM, and a Hill coefficient around 2. Although the conductance vs. voltage relationship in control condition can be described by a single Boltzmann function, two terms are necessary to describe the data in the presence of SMD2. The activation and deactivation time constants are weakly voltage dependent both for control and in the presence of SMD2. SMD2 does not change the channel selectivity and tail currents show a typical crossover phenomenon. The time course of inactivation has a fast and a slow component, and SMD2 significantly decreased their values. Steady-state inactivation is best described by a Boltzmann equation with V 1/2 (the voltage where the probability to find the channels in the inactivated state is 50%) and K (slope factor) equals to -22.9 ± 1.5 mV and 5.3 ± 0.9 mV for control, and -30.3 ± 1.3 mV and 6 ± 0.8 mV for SMD2, respectively. The action of SMD2 is enhanced by high frequency stimulation, and by the time the channel stays open. Taken together, our results suggest that SMD2 blocks the open conformation of KV3.1. From a pharmacological and therapeutic point of view, N-alkylsulfonamides may constitute a new class of pharmacological modulators of KV3.1.

Osmoregulation and the Hypothalamic Supraoptic Nucleus: From Genes to Functions.

Due to the relatively high permeability to water of the plasma membrane, water tends to equilibrate its chemical potential gradient between the intra and extracellular compartments. Because of this, changes in osmolality of the extracellular fluid are accompanied by changes in the cell volume. Therefore, osmoregulatory mechanisms have evolved to keep the tonicity of the extracellular compartment within strict limits. This review focuses on the following aspects of osmoregulation: 1) the general problems in adjusting the "milieu interieur" to challenges imposed by water imbalance, with emphasis on conceptual aspects of osmosis and cell volume regulation; 2) osmosensation and the hypothalamic supraoptic nucleus (SON), starting with analysis of the electrophysiological responses of the magnocellular neurosecretory cells (MNCs) involved in the osmoreception phenomenon; 3) transcriptomic plasticity of SON during sustained hyperosmolality, to pinpoint the genes coding membrane channels and transporters already shown to participate in the osmosensation and new candidates that may have their role further investigated in this process, with emphasis on those expressed in the MNCs, discussing the relationships of hydration state, gene expression, and MNCs electrical activity; and 4) somatodendritic release of neuropeptides in relation to osmoregulation. Finally, we expect that by stressing the relationship between gene expression and the electrical activity of MNCs, studies about the newly discovered plastic-regulated genes that code channels and transporters in the SON may emerge.

A calcium-induced calcium release mechanism supports luteinizing hormone-induced testosterone secretion in mouse Leydig cells.

Leydig cells are responsible for the synthesis and secretion of testosterone, processes controlled by luteinizing hormone (LH). Binding of LH to a G protein-coupled receptor in the plasma membrane results in an increase in cAMP and in intracellular Ca(2+) concentration ([Ca(2+)](i)). Here we show, using immunofluorescence, that Leydig cells express ryanodine receptors (RyRs) and inositol 1,4,5-trisphosphate receptors (IP(3)Rs). Measurements of intracellular calcium changes using the fluorescent calcium-sensitive dye fluo-3 and confocal microscopy show that both types of receptors are involved in a calcium-induced calcium release (CICR) mechanism, which amplifies the initial Ca(2+) influx through plasma membrane T-type calcium channels (Ca(V)3). The RyRs and IP(3)Rs are functional, as judged from both their activation by caffeine and IP(3) and block by ryanodine and 2-aminoethoxydiphenyl borate (2-APB), respectively. RyRs are the principal players involved in the release of Ca(2+) from the endoplasmic reticulum, as evidenced by the fact that global Ca(2+) changes evoked by LH are readily blocked by 100 muM ryanodine but not by 2-APB or xestospongin C. Finally, steroid production by Leydig cells is inhibited by ryanodine but not by 2-APB. These results not only broaden our understanding of the role played by calcium in Leydig cells but also show, for the first time, that RyRs have an important role in determining testosterone secretion by the testis.

Neuroendocrine Regulation of Hydromineral Homeostasis.

Since the crucial evolutionary change from an aqueous to a terrestrial environment, all living organisms address the primordial task of equilibrating the ingestion/production of water and electrolytes (primarily sodium) with their excretion. In mammals, the final route for the excretion of these elements is mainly through the kidneys, which can eliminate concentrated or diluted urine according to the requirements. Despite their primary role in homeostasis, the kidneys are not able to recover water and solutes lost through other systems. Therefore, the selective stimulation or inhibition of motivational and locomotor behavior becomes essential to initiate the search and acquisition of water and/or sodium from the environment. Indeed, imbalances affecting the osmolality and volume of body fluids are dramatic challenges to the maintenance of hydromineral homeostasis. In addition to behavioral changes, which are integrated in the central nervous system, most of the systemic responses recruited to restore hydroelectrolytic balance are accomplished by coordinated actions of the cardiovascular, autonomic and endocrine systems, which determine the appropriate renal responses. The activation of sequential and redundant mechanisms (involving local and systemic factors) produces accurate and self-limited effector responses. From a physiological point of view, understanding the mechanisms underlying water and sodium balance is intriguing and of great interest for the biomedical sciences. Therefore, the present review will address the biophysical, evolutionary and historical perspectives concerning the integrative neuroendocrine control of hydromineral balance, focusing on the major neural and endocrine systems implicated in the control of water and sodium balance.

Tityustoxin-K(alpha) blockade of the voltage-gated potassium channel Kv1.3.

1. We investigated the action of TsTX-Kalpha on cloned Kv1.3 channels of the Shaker subfamily of voltage-gated potassium channels, using the voltage-clamp technique. Highly purified TsTX-Kalpha was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX-Kalpha blocks Kv1.3 with high affinity in two expression systems. 2. TsTX-Kalpha blockade of Kv1.3 channels expressed in Xenopus oocytes was found to be completely reversible and to exhibit a pH dependence. The K(D) was 3.9 nM at pH 7.5, 9.5 nM at pH 7.0 and 94.5 nM at pH 6.5. 3. The blocking properties of TsTX-Kalpha in a mammalian cell line (L929), stably transfected to express Kv1.3, were studied using the patch-clamp technique. In this preparation, the toxin had a K(D) of 19.8 nM at pH 7.4. 4. TsTX-Kalpha was found to affect neither the voltage-dependence of activation, nor the activation and deactivation time constants. The block appeared to be independent of the transmembrane voltage and the toxin did not interfere with the C-type inactivation process. 5. Taken as a whole, our findings indicate that TsTX-Kalpha acts as a simple blocker of Kv1.3 channels. It is concluded that this toxin is a useful tool for probing not only the physiological roles of Kv1.2, but also those mediated by Kv1.3 channels.

Luteinizing hormone (LH) acts through PKA and PKC to modulate T-type calcium currents and intracellular calcium transients in mice Leydig cells.

LH increases the intracellular Ca(2+) concentration ([Ca(2+)](i)) in mice Leydig cells, in a process triggered by calcium influx through T-type Ca(2+) channels. Here we show that LH modulates both T-type Ca(2+) currents and [Ca(2+)](i) transients through the effects of PKA and PKC. LH increases the peak calcium current (at -20mV) by 40%. A similar effect is seen with PMA. The effect of LH is completely blocked by the PKA inhibitors H89 and a synthetic inhibitory peptide (IP-20), but only partially by chelerythrine (PKC inhibitor). LH and the blockers induced only minor changes in the voltage dependence of activation, inactivation or deactivation of the currents. Staurosporine (blocker of PKA and PKC) impaired the [Ca(2+)](i) changes induced by LH. A similar effect was seen with H89. Although PMA slowly increased the [Ca(2+)](i) the subsequent addition of LH still triggered the typical transients in [Ca(2+)](i). Chelerythrine also does not avoid the Ca(2+) transients, showing that blockage of PKC is not sufficient to inhibit the LH induced [Ca(2+)](i) rise. In summary, these two kinases are not only directly involved in promoting testosterone synthesis but also act on the overall calcium dynamics in Leydig cells, mostly through the activation of PKA by LH.

Mouse Leydig cells express multiple P2X receptor subunits.

ATP acts on cellular membranes by interacting with P2X (ionotropic) and P2Y (metabotropic) receptors. Seven homomeric P2X receptors (P2X(1)-P2X(7)) and seven heteromeric receptors (P2X(1/2), P2X(1/4), P2X(1/5), P2X(2/3), P2X(2/6), P2X(4/6), P2X(4/7)) have been described. ATP treatment of Leydig cells leads to an increase in [Ca(2+)](i) and testosterone secretion, supporting the hypothesis that Ca(2+) signaling through purinergic receptors contributes to the process of testosterone secretion in these cells. Mouse Leydig cells have P2X receptors with a pharmacological and biophysical profile resembling P2X(2). In this work, we describe the presence of several P2X receptor subunits in mouse Leydig cells. Western blot experiments showed the presence of P2X(2), P2X(4), P2X(6), and P2X(7) subunits. These results were confirmed by immunofluorescence. Functional results support the hypothesis that heteromeric receptors are present in these cells since 0.5 muM ivermectin induced an increase (131.2 +/- 5.9%) and 3 muM ivermectin a decrease (64.2 +/- 4.8%) in the whole-cell currents evoked by ATP. These results indicate the presence of functional P2X(4) subunits. P2X(7) receptors were also present, but they were non-functional under the present conditions because dye uptake experiments with Lucifer yellow and ethidium bromide were negative. We conclude that a heteromeric channel, possibly P2X(2/4/6), is present in Leydig cells, but with an electrophysiological and pharmacological phenotype characteristic of the P2X(2) subunit.

Volume-activated chloride channels in mice Leydig cells.

Production and secretion of testosterone in Leydig cells are mainly controlled by the luteinizing hormone (LH). Biochemical evidences suggest that the activity of Cl(-) ions can modulate the steroidogenic process, but the specific ion channels involved are not known. Here, we extend the characterization of Cl(-) channels in mice Leydig cells (50-60 days old) by describing volume-activated Cl(-) currents (I(Cl,swell)). The amplitude of I(Cl,swell) is dependent on the osmotic gradient across the cell membrane, with an apparent EC(50) of approximately 75 mOsm. These currents display the typical biophysical signature of volume-activated anion channels (VRAC): dependence on intracellular ATP, outward rectification, inactivation at positive potentials, and selectivity sequence (I(- )> Cl(- )> F(-)). Staurosporine (200 nM) did not block the activation of I(Cl,swell). The block induced by 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB; 128 microM), SITS (200 microM), ATP (500 microM), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS; 100 miccroM), and Suramin (10 microM) were described by the permeant blocker model with apparent dissociation constant at 0 mV K(do) and fractional distance of the binding site (delta) of 334 microM and 47 %, 880 microM and 35 %, 2,100 microM and 49%, 188 microM and 27%, and 66.5 microM and 49%, respectively. These numbers were derived from the peak value of the currents. We conclude that I(Cl,swell) in Leydig cells are activated independently of purinergic stimulation, that Suramin and PPADS block these currents by a direct interaction with VRAC and that ATP is able to permeate this channel.

Intracellular calcium changes in mice Leydig cells are dependent on calcium entry through T-type calcium channels.

Luteinizing hormone (LH) regulates testosterone synthesis in Leydig cells by inducing an intracellular increase in cAMP concentration. LH also increases the intracellular calcium concentration ([Ca2+]i), dependent on the presence of Ca2+ in the extracellular medium ([Ca2+]e) for its effect. Despite these evidences, the identity of a pathway for calcium entry has remained elusive and the relationship between cAMP and [Ca2+]i has been questioned. Here we show that mice Leydig cells do have an inward Ca2+ current carried by T-type Ca2+ channels. In 10 mm [Ca2+]e, the currents start to be activated at -60 mV, reaching maximal amplitude of 1.8 +/- 0.3 pA pF(-1) at -20 mV. Currents were not modified by Ba2+ or Sr2+, were suppressed in Ca2+-free external solution, and were blocked by 100 microm nickel or 100 microm cadmium. The Ki for Ni2+ is 2.6 microm and concentrations of Cd2+ smaller than 50 microm have a very small effect on the currents. The calcium currents displayed a window centred at -40 mV. The half-voltage (V0.5) of activation is -30.3 mV, whereas the half-voltage steady-state inactivation is -51.1 mV. The deactivation time constant (taudeactivation) is around 3 ms at -35 mV. Confocal microscopy experiments with Fluo-3 loaded cells reveal that both LH and dibutyryl-cAMP (db-cAMP) increase [Ca2+]i. The db-cAMP induced calcium increase was dependent on Ca2+ influx since it was abolished by removal of extracellular Ca2+ and by 400 microm Ni2+. [Ca2+]i increases in regions close to the plasma membrane and in the cell nucleus. Similar effects are seen when Leydig cells are depolarized by withdrawing K+ from the extracellular solution. Altogether, our studies show that Ca2+ influx through T-type Ca2+ channels in the plasma membrane of Leydig cells plays a crucial role in the intracellular calcium concentration changes that follow binding of LH to its receptor.

P2X receptors in mouse Leydig cells.

ATP-activated currents were studied in Leydig cells of mice with the patch-clamp technique. Whole cell currents were rapidly activating and slowly desensitizing (55% decrement from the peak value on exposure to 100 microM ATP for 60 s), requiring 3 min of washout to recover 100% of the response. The concentration-response relationships for ATP, adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), and 2-methylthio-ATP (2-MeS-ATP) were described by the Hill equation with a concentration evoking 50% of maximal ATP response (K(d)) of 44, 110, and 637 microM, respectively, and a Hill coefficient of 2. The order of efficacy of agonists was ATP >or= ATPgammaS > 2-MeS-ATP > 2',3'-O-(4-benzoylbenzoyl)-ATP (BzATP). alphabeta-Methylene-ATP (alphabeta-MeATP), GTP, UTP, cAMP, and adenosine were ineffective. Suramin and pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the responses in a concentration-dependent manner. The ATP-activated currents were dependent on extracellular pH, being maximal at pH 6.5 and decreasing with both acidification and alkalinization (apparent dissociation constant (pK(a)) of 5.9 and 7.4, respectively). The whole cell current-voltage relationship showed inward rectification and reversed near 0 mV. Experiments performed in bi-ionic conditions for measurement of reversal potentials showed that this channel is highly permeable to calcium [permeability (P)(Ca)/P(Na) = 5.32], but not to chloride (P(Cl)/P(Na) = 0.03) or N-methyl-D-glucamine (NMDG) (P(NMDG)/P(Na) = 0.09). Unitary currents recorded in outside-out patches had a chord conductance of 27 pS (between -90 and -50 mV) and were inward rectifying. The average current passing through the excised patch decreased with time [time constant (tau) = 13 s], resembling desensitization of the macroscopic current. These findings indicate that the ATP receptor present in Leydig cells shows properties most similar to those of cloned homomeric P2X(2).

Glycine binding site of the synaptic NMDA receptor in subpostremal NTS neurons.

The nucleus of the tractus solitarius (NTS) plays an important role in the control of several autonomic reflex functions and has glutamate and GABA as main neurotransmitters. In this work, we used patch-clamp recordings in transverse slice preparations from rats to study whether the glycine binding site of the N-methyl-D-aspartate (NMDA) receptor is saturated or not in neurons of the subpostremal NTS. Except at hyperpolarized voltages and close to the reversal potential, glycine potentiated the NMDA responses in a concentration-dependent manner. The total charge transferred by glutamatergic currents was enhanced by glycine (500 microM; from 28 +/- 13 to 42 +/- 18 pC at +50 mV, n = 7, P < 0.05). Glycine increased the conductance of the postsynaptic membrane, without altering its reversal potential, both in the presence (from 2.4 +/- 0.06 to 3.4 +/- 0.09 nS; n = 7) and absence (from 3.1 +/- 0.06 to 4.4 +/- 0.10 nS; n = 8) of Mg2+ in the bathing solution. d-serine, in the presence of strychnine, also increased the amplitude of the NMDA component (by 68 +/- 19%, P < 0.05, n = 5). The membrane potential was hyperpolarized (16 +/- 6 mV, n = 8) by glycine, suggesting the presence of inhibitory glycinergic receptors. Our results indicate that the glycine site of the NMDA receptor in neurons of the subpostremal NTS is not saturated and that glycine may act as a modulator of the NMDA transmission in this nucleus.

Sera from patients with idiopathic dilated cardiomyopathy decrease ICa in cardiomyocytes isolated from rabbits.

Autoantibodies against muscarinic and adrenergic receptors have been found in the sera of patients with idiopathic dilated cardiomyopathy (IDC) and Chagas disease, but it is still unclear whether they can functionally interact with their respective receptors to modulate cardiac functions. In this study, our goal was to detect the presence of those antibodies in the sera of patients with IDC and characterize their electrophysiological effects on cardiomyocytes from rabbits. By using ELISA immunoassays, we detected high titers of antibodies against muscarinic M2 receptors in the sera of all IDC patients, whereas the detection of antibodies against the beta1-receptor occurred in 50% of them. Electrophysiological experiments using the whole cell configuration of the patch-clamp technique showed that sera from 43% of IDC patients induced a significant decrease (approximately 26%) in isoproterenol-stimulated L-type Ca2+ currents in rabbit ventricular myocytes, whereas the sera from healthy blood donors failed to do so. As expected, IDC sera also decreased the action potential duration (by 10.5%) due to a shortening of the plateau phase. Sera that reduced isoproterenol-stimulated L-type Ca2+ currents did not cause any effect on K+ currents. We conclude that sera from IDC patients have autoantibodies, which interact with muscarinic M2 receptors of rabbit cardiomyocytes, acting in an agonist-like fashion. This action results in changes in electrogenesis, which, as often observed in patients with IDC, could initiate ventricular arrhythmias that lead to sudden death.