Ionic currents induced by ATP in Leydig cells

Leydig cells are responsible for the production and secretion of testosterone. They are controlled by the Luteinizing Hormone (LH) in a process modulated by several substances. Recently, adenosine triphosphate (ATP) was found to be another modulator, acting through the activation of purinergic receptors. In this paper we used the patch clamp technique in order to detect and characterize the ionic currents induced by extracellular ATP in Leydig cells freshly isolated from mice testis. Addition of ATP to the bathing solution induces an inward current strongly rectifying at hyperpolarizing potentials. Such current is dependent on the doses of ATP and shows an apparent desensitization for ATP concentrations higher than 60 m M. The I-V plot reveals a reversal potential around zero mV, indicating a non-selective cation current. These results confirm, from an electrophysiological point of view, the presence of purinergic receptors in Leydig cells, probably of the P2X type.  As células de Leydig produzem e secretam testosterona, num processo controlado pelo Hormônio Luteinizante (LH) e modulado através de diversos fatores. O trifosfato de adenosina (ATP), no meio extracelular, tem se constituído num novo modulador do processo, atuando via ativação de receptores purinérgicos. Neste trabalho, a técnica de “patch-clamp” foi utilizada para detectar e caracterizar as correntes iônicas, induzidas pela ativação desses receptores, em células de Leydig, isoladas a fresco, de testículos de camundongos. A adição de ATP ao banho levou ao surgimento de uma corrente orientada para dentro, em potenciais hiperpolarizantes. A amplitude da corrente é dependente da dose de ATP, para cada nível de potencial aplicado à célula, e apresenta dessensibilização para doses acima de 60 m M. A relação corrente-voltagem é fortemente retificada na direção de potenciais hiperpolarizantes e apresenta um potencial de reversão próximo de zero mV, indicando uma via de baixa seletividade a cátions. Esses  resultados vêm confirmar a presença de receptores purinérgicos em células de Leydig, muito provavelmente pertencentes à família P2X. &nbsp

AFM imaging reveals the assembly of a P2X receptor complex containing P2X2, P2X4 and P2X6 subunits

Seven P2X purinergic receptor subunits have been identified: P2X1-P2X7. All except P2X6 assemble as homotrimers, and six heteromeric receptors (P2X1/2, P2X1/4, P2X1/5, P2X2/3, P2X2/6 and P2X4/6) have been described. In addition, P2X4 homomers associate with P2X2 or P2X7 homomers as dimers of trimers. The various P2X receptors show individual functional properties, suggesting distinct physiological roles. The overlapping expression of P2X2, P2X4 and P2X6 subunits has been shown in different cell types, and functional analysis of P2X receptors in Leydig cells suggests that the three subunits interact

Mass spectrometry study of N-alkylbenzenesulfonamides with potential antagonist activity to potassium channels

Herein, we report the synthesis and mass spectrometry studies of several N-alkylbenzenesulfonamides structurally related to sulfanilic acid. The compounds were synthesized using a modified Schotten-Baumann reaction coupled with Meisenheimer arylation. Sequential mass spectrometry by negative mode electrospray ionization (ESI(-)-MS/MS) showed the formation of sulfoxylate anion (m/z 65) observed in the mass spectrum of p-chloro-N-alkylbenzenesulfonamides. Investigation of the unexpected loss of two water molecules, as observed by electron ionization mass spectrometry (EI-MS) analysis of p-(N-alkyl)lactam sulfonamides, led to the proposal of corresponding fragmentation pathways. These compounds showed loss of neutral iminosulfane dioxide molecule (M-79) with formation of ions observed at m/z 344 and 377. These ions were formed by rearrangement on ESI(+)-MS/MS analysis. Some of the molecules showed antagonistic activity against Kv3.1 voltage-gated potassium channels.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Allosteric Control of Gating Mechanisms Revisited: The Large Conductance Ca2+-Activated K+ Channel

Large-conductance Ca2+-activated K+ channels (BK) play a fundamental role in modulating membrane potential in many cell types. The gating of BK channels and its modulation by Ca2+ and voltage has been the subject of intensive research over almost three decades, yielding several of the most complicated kinetic mechanisms ever proposed. A large number of open and closed states disposed, respectively, in two planes, named tiers, characterize these mechanisms. Transitions between states in the same plane are cooperative and modulated by Ca2+. Transitions across planes are highly concerted and voltage-dependent. Here we reexamine the validity of the two-tiered hypothesis by restricting attention to the modulation by Ca2+. Large single channel data sets at five Ca2+ concentrations were simultaneously analyzed from a Bayesian perspective by using hidden Markov models and Markov-chain Monte Carlo stochastic integration techniques. Our results support a dramatic reduction in model complexity, favoring a simple mechanism derived from the Monod-Wyman-Changeux allosteric model for homotetramers, able to explain the Ca2+ modulation of the gating process. This model differs from the standard Monod-Wyman-Changeux scheme in that one distinguishes when two Ca2+ ions are bound to adjacent or diagonal subunits of the tetramer

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

1. We investigated the action of TsTX-Kα on cloned Kv1.3 channels of the Shaker subfamily of voltage-gated potassium channels, using the voltage–clamp technique. Highly purified TsTX-Kα was obtained from the venom of the Brazilian scorpion Tityus serrulatus using a new purification protocol. Our results show that TsTX-Kα blocks Kv1.3 with high affinity in two expression systems. 2. TsTX-Kα 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-Kα 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-Kα 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-Kα 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