Local Membrane Deformations Activate Ca2+-Dependent K+ and Anionic Currents in Intact Human Red Blood Cells

BACKGROUND: The mechanical, rheological and shape properties of red blood cells are determined by their cortical cytoskeleton, evolutionarily optimized to provide the dynamic deformability required for flow through capillaries much narrower than the cell's diameter. The shear stress induced by such flow, as well as the local membrane deformations generated in certain pathological conditions, such as sickle cell anemia, have been shown to increase membrane permeability, based largely on experimentation with red cell suspensions. We attempted here the first measurements of membrane currents activated by a local and controlled membrane deformation in single red blood cells under on-cell patch clamp to define the nature of the stretch-activated currents. METHODOLOGY/PRINCIPAL FINDINGS: The cell-attached configuration of the patch-clamp technique was used to allow recordings of single channel activity in intact red blood cells. Gigaohm seal formation was obtained with and without membrane deformation. Deformation was induced by the application of a negative pressure pulse of 10 mmHg for less than 5 s. Currents were only detected when the membrane was seen domed under negative pressure within the patch-pipette. K(+) and Cl(-) currents were strictly dependent on the presence of Ca(2+). The Ca(2+)-dependent currents were transient, with typical decay half-times of about 5-10 min, suggesting the spontaneous inactivation of a stretch-activated Ca(2+) permeability (PCa). These results indicate that local membrane deformations can transiently activate a Ca(2+) permeability pathway leading to increased [Ca(2+)](i), secondary activation of Ca(2+)-sensitive K(+) channels (Gardos channel, IK1, KCa3.1), and hyperpolarization-induced anion currents. CONCLUSIONS/SIGNIFICANCE: The stretch-activated transient PCa observed here under local membrane deformation is a likely contributor to the Ca(2+)-mediated effects observed during the normal aging process of red blood cells, and to the increased Ca(2+) content of red cells in certain hereditary anemias such as thalassemia and sickle cell anemia

Biophysical properties of neuronal nicotinic acetylcholine receptors (nAChRs) in the superior cervical ganglion of mice with deletions of distinct nAChR subunit genes

Neuronale nikotinische AcetylCholin Rezeptoren (nAChR) sind Liganden-aktivierte Ionenkanäle, die im Nervensystem weit verbreitet sind. Rezeptoren im Ganglion Cervicale Superius (SCG) setzen sich aus den Bausteinen ("Untereinheiten") alpha3, alpha5, alpha7, beta2 and beta4 zusammen. Von den insgesamt 12 bekannten Untereinheiten, die zum Aufbau von neuronalen Acetylcholin Rezeptoren beitragen (alpha2-alpha10 and beta2-beta4), wird somit im SCG nur ein Teil angetroffen. Während hetero-pentamere Nikotin-Rezeptoren (bestehend aus sowohl alpha wie beta Untereinheiten) die synaptische Übertragung im SCG vermitteln, ist die Funktion der homo-pentameren alpha7-Rezeptoren weitgehend ungeklärt. Sowohl die pharmakologischen als auch die biophysikalischen Eigenschaften von nikotinischen Acetylcholin Rezeptoren werden von den Untereinheiten bestimmt, aus denen die Rezeptoren aufgebaut sind. In ihrer Zusammensetzung definierte, neuronale Nikotinrezeptoren wurden bisher lediglich in heterologen Expressionssystemen untersucht. Da aber ihre Eigenschaften je nach Expressionssystem variieren, konnten diese Untersuchungen keine definitive Aussagen über Nikotinrezeptoren in Nervenzellen liefern. Ich wählte für meine Untersuchungen daher einen anderen Ansatz: Nervenzellen des SCG von transgenen Mäusen, bei denen sowohl die alpha5 als auch die beta2 Untereinheit ausgeschaltet wurden, exprimieren hetero-oligomere Rezeptoren, die lediglich aus den Untereinheiten alpha3 und beta4 aufgebaut sind. Rezeptoren von Mäusen, denen die alpha5 Untereinheiten fehlt, enthalten zusätzlich die beta2 Untereinheit (alpha3/beta4/beta2 mit 21%), wohingegen in beta2-Knockout Mäusen - neben alpha3 und beta4 - auch (zu 24%) alpha5-hältige Rezeptoren vorkommen (DAVID et al. 2010). Nach meinen Beobachtungen haben alpha3/beta4 Rezeptoren (in alpha5,beta2 doppel- and alpha5,beta2,alpha7 dreifach-KO Nervenzellen) eine dominierende Kanal-Leitfähigkeit von 32.6 0.8 pS und Kanal Öffnungszeiten, die bestmöglich an Exponentialfunktionen mit den Zeitkonstanten tau1 0.79 0.10 und tau2 8.99 0.55 ms angeglichen werden. In den meisten Messungen konnte ich - neben der dominierenden Leitfähigkeit - allerdings auch Kanalöffnungen mit etwas abweichender Amplitude beobachten. Neurone, in denen nur die alpha5 Untereinheit ausgeschaltet war, zeigten hingegen einen Rezeptor mit einer signifikant geringeren Kanal-Leitfähigkeit (13.6 0.5 pS) und geringfügig längeren Kanalöffnungszeiten (tau2: 25.4 3.9 ms). Auch alpha3/beta4/alpha5 Rezeptoren in beta2-KO Neuronen unterschieden sich durch längere Kanalöffnung (tau3 of 56.7 ms) und Bursts signifikant von alpha3/beta4 Rezeptoren. Basierend auf diesen Erkenntnissen konnte ich die 3 Rezeptoren alpha3/beta4, alpha3/beta4/alpha5, sowie alpha3/beta4/beta2 auch in Kontrollmäusen beobachten. Zusammenfassend ist meine Studie die erste dieser Art, in der Eigenschaften neuronaler Nikotinrezeptoren in ihrer natürlichen Umgebung - den Nervenzellen - beschrieben werden.Neuronal nicotinic AcetylCholine Receptors (nAChRs) are ligand-gated ion channels widely expressed in the central and peripheral nervous system. Out of twelve subunit-encoding genes (alpha2-alpha10 and beta2-beta4), autonomic ganglia express only the neuronal nAChR subunits alpha3, alpha5, alpha7, beta2 and beta4. The hetero-pentameric (alpha/beta pairs) receptors mediate fast synaptic transmission, whereas functions of ganglionic homo-pentameric (alpha7) receptors are still enigmatic. The biophysical properties of nAChRs are critically determined by their subunit composition. To date, single-channel properties of distinct neuronal nAChRs have only been studied in heterologous expression systems. However, these data have proven to be unreliable, since properties of recombinant receptors differ depending on the system where they are expressed. Here I used a different approach by taking advantage of transgenic animals. Hence, superior cervical ganglion (SCG) neurons from mice lacking both the alpha5 and the beta2 subunit express pure alpha3/beta4 hetero-oligomeric receptors, whereas single-KO animals missing the subunits alpha5 or beta2 express alpha3/beta4/beta2 (21%) and alpha3/beta4/alpha5 (24%), respectively, alongside with the more numerous (about 75%) alpha3/beta4 hetero-oligomeric receptors (DAVID et al. 2010). I found that alpha3/beta4 receptor channels (in alpha,5beta2 double- and alpha5,beta2,alpha7 triple-KO neurons) had a main unitary conductance of 32.6 0.8 pS and channel open times could be best fitted by a double-exponential function with time constants of 0.79 0.10 and 8.99 0.55 ms. In addition to the main channel conductance I observed secondary conductance levels in most of the patches. In alpha5 single-KO neurons, on the other hand, I characterized a new channel type with a significantly lower unitary conductance of 13.6 0.5 pS and a slightly longer channel open times (1.27 0.59 and 25.4 3.9 ms) than for alpha3/beta4 receptors. I attributed these properties to alpha3/beta4/beta2 receptors. Likewise, in beta2 single-KO patches I recognized channels that differed from alpha3/beta4 receptors by having significantly longer channel open times (indicated by a third time constant tau3 of 56.7 ms) and increased burst duration. Although the channel conductance of these receptors was identical to alpha3/beta4 receptors, these features suggest the presence of alpha3/beta4/alpha5 channels in the patch. After establishing single-channel properties in KO models I then recognized all these receptors in wild type animals as well. In sum, this is the first study revealing properties of distinct neuronal nAChRs in their native environment.submitted by Anna CiuraszkiewiczAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheWien, Med. Univ., Diss., 2013OeBB(VLID)171594

Dynamic compartmentalization of calcium channel signalling in neurons

Calcium fluxes through the neuronal membrane are strictly limited in time due to biophysical properties of voltage-gated and ligand-activated ion channels and receptors. Being embedded into the crowded dynamic environment of biological membranes, Ca2+-permeable receptors and channels undergo perpetual spatial rearrangement, which enables their temporary association and formation of transient signalling complexes. Thus, efficient calcium-mediated signal transduction requires mechanisms to support very precise spatiotemporal alignment of the calcium source and Ca2+-binding lipids and proteins in a highly dynamic environment. The mobility of calcium channels and calcium-sensing proteins themselves can be considered as a physiologically meaningful variable that affects calcium-mediated signalling in neurons. In this review, we will focus on voltage-gated calcium channels (VGCCs) and activity-induced relocation of stromal interaction molecules (STIMs) in the endoplasmic reticulum (ER) to show that particularly in time ranges between milliseconds to minutes, dynamic rearrangement of calcium conducting channels and sensor molecules is of physiological relevance. This article is part of the special issue entitled 'Mobility and trafficking of neuronal membrane proteins'

The role of the nAChR subunits α5, β2, and β4 on synaptic transmission in the mouse superior cervical ganglion

Abstract Our previous immunoprecipitation analysis of nicotinic acetylcholine receptors (nAChRs) in the mouse superior cervical ganglion (SCG) revealed that approximately 55%, 24%, and 21% of receptors are comprised of α3β4, α3β4α5, and α3β4β2 subunits, respectively. Moreover, mice lacking β4 subunits do not express α5‐containing receptors but still express a small number of α3β2 receptors. Here, we investigated how synaptic transmission is affected in the SCG of α5β4‐KO and α5β2‐KO mice. Using an ex vivo SCG preparation, we stimulated the preganglionic cervical sympathetic trunk and measured compound action potentials (CAPs) in the postganglionic internal carotid nerve. We found that CAP amplitude was unaffected in α5β4‐KO and α5β2‐KO ganglia, whereas the stimulation threshold for eliciting CAPs was significantly higher in α5β4‐KO ganglia. Moreover, intracellular recordings in SCG neurons revealed no difference in EPSP amplitude. We also found that the ganglionic blocking agent hexamethonium was the most potent in α5β4‐KO ganglia (IC50: 22.1 μmol/L), followed by α5β2‐KO (IC50: 126.7 μmol/L) and WT ganglia (IC50: 389.2 μmol/L). Based on these data, we estimated an IC50 of 568.6 μmol/L for a receptor population consisting solely of α3β4α5 receptors; and we estimated that α3β4α5 receptors comprise 72% of nAChRs expressed in the mouse SCG. Similarly, by measuring the effects of hexamethonium on ACh‐induced currents in cultured SCG neurons, we found that α3β4α5 receptors comprise 63% of nAChRs. Thus, in contrast to our results obtained using immunoprecipitation, these data indicate that the majority of receptors at the cell surface of SCG neurons consist of α3β4α5

Effect of plasma membrane calcium pump (PMCA) inhibition on current amplitude as a function of time.

<p>Added to the bathing solution 2–3 min before seal formation, vanadate (1 mM) did not modify the initial diversity of current amplitudes, but the declining pattern persisted albeit at a significantly reduced rate. Following seal formation, none of the 25 patches of this series of experiments ceased electrical activity during the first 16 minutes.</p

Diagrammatic representation of hypothesized time course of changes in total cell calcium.

<p>Upon membrane deformation, calcium enters the cell via PCa driven by a steep inward Ca²⁺ gradient. A new pump-leak balance is generated with elevated [Ca²⁺]_i initially set at an arbitrary maximal level of total calcium content (1.0 arbitrary unit). As PCa declines with time, the changing pump-leak balance is with a time-declining pattern in total cell calcium. PMCA <i>V</i>_max declines with RBC age, so that the time course of Ca²⁺ extrusion will be faster in young cells (curve 1) than in old or pump-inhibited cells (curve 2). Dashed line (<b><i>a</i></b>) represents the threshold of Gardos channel activation. Dashed line (<b><i>b</i></b>) represents the calcium level above which Gardos channel remains Ca²⁺-saturated with maximal activity (maximal Po). During the process of Ca²⁺ extrusion with declining PCa, the time spent between levels (<b><i>b</i></b>) and (<b><i>a</i></b>) varies with cell age. During the intervals Δt₁ and Δt₂ the open state probability of the Gardos channels declines progressively to extinction following Ca²⁺ desaturation.</p

Patterns of channel activity decline.

<p>Three fast modalities of current transitions could be discerned during activity decline: Panel <b>A</b> (representative of 6 recordings), fast (3 sec) but progressive transition from a large current (1.4 pA in the present example) to a stable 0.3–0.5 pA current; Panel <b>B</b> and <b>C</b> (representative of 12 recordings), instantaneous transition from large (1.8 pA in the present example) to a stable 0.3–0.5 pA current; Panel <b>D</b> (representative of 9 recordings), instantaneous transition from a large (1.7 pA in the present example) to an intermediary current (0.9 pA) which thereafter followed progressive decline to a stable 0.3–0.5 pA current. In all cases recordings were obtained with solution A (150 mM NaCl, pCa3) in the bathing solutions and solution B (150 mM KCl, pCa3) in the patch pipettes.</p

Analysis of the homeostasis of a red blood cell under cell-attached patch clamp with the use of the Lew-Bookchin model.

<p>The predicted effects of sudden and maximal Gardos channel activation on the membrane potential Em and intracellular K⁺ concentration ([K⁺]) are presented in panels <b>A</b> and <b>B</b>. Electrodiffusional permeability for K⁺ ions (P^G_K) was increased to a value of 10 h⁻¹ at time t = 2 min. The parameter values chosen for this simulation were 5 mM KCl and 150 mM NaCl in external bathing solution. The initial P^G_K value was 0.001651 h⁻¹; for other steady state default parameters refer to Lew and Bookchin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0009447#pone.0009447-Lew7" target="_blank">[34]</a>. Panels C and D present the simulated effects on Em, EK (equilibrium potential for K⁺ ions) and EA (equilibrium potential for anions) of sudden and maximal Gardos channel activation (as above) followed by sudden activation of anionic electrodiffusional permeability (P^G_A). At time t = 7 min, P^G_A was changed from 1.2 h⁻¹ to 10 h⁻¹ (<b>C</b>) or 50 h⁻¹ (<b>D</b>) corresponding to moderate and large activations. To simulate the effects of a gradual increase in anionic electrodiffusional permeability (panel <b>E</b>), P^G_A was changed from 1.2 h⁻¹ to 5 h⁻¹ at t = 3 and incremented by 5 h⁻¹ each min to reach P^G_A = 35 h⁻¹ at t = 18 min). Panel <b>F</b> displays the corresponding evolution of intracellular K⁺ and A⁻ concentrations.</p