Patch-clamp

The patch clamp approach allows the recording of ionic currents passing through cell membranes, by controlling and holding the membrane potential while measuring the current (Sakmann and Neher 1984); (Neher and Sakmann 1976). It involves making continuous electric micro-glass pipette filled with an ionic solution of defined composition with the cell membrane. There are four different configurations of patch clamp: whole cell, cell-attached, inside-out and outside-out (Figure 3). Each configuration allows us to obtain different information on the current activated or directly on the channel involved.

During my thesis I used three of these configurations. The two first were at the level of single channel analysis: the cell-attached and the inside-out configurations. The cell-attached allows the direct recording of the activated channels located under the pipette after stimulation (Figure 3A). The advantage of the cell-attached recording is the preservation of the plasma membrane integrity, so the cytosolic factors necessary to channel opening such as second messengers are maintained in the cytoplasm. A channel can be characterized by several parameters. The current voltage (I-V) relationship, determined by recording the current at different voltage, provides much information about a channel. Indeed the shape of the I-V curve (linear, inwardly or outwardly rectified) is one characteristic. The conductance (G) of a channel, which is proportioned to the quantity of current going through it, is given by the slope of the curve:

I = G * ǻV

The I-V relationship also provides information on the ion carrying the current thanks to the reversal potential, corresponding to the membrane potential at which there is no current.

If the reversal potential is at the equilibrium potential of a specific ion this indicates that the

current is carried by this specific ion. The equilibrium potential of a specific ion can be calculated with the Nernst equation:

with E = equilibrium potential

R = universal gas constant (8.314472 JK^í1mol^í1) T = absolute temperature

z = ionic charge

F = Faraday constant (9.64853399×10⁴ Cmol^í1)

The cell attached configuration allows the quantification of the channel activity by calculating the open state probability (Po):

Po = To/Tr

with To = open state time

Tr = recording time

and on the open time, which is the duration of the open state.

The inside-out configuration allows us to characterize the channels under the pipette, in term of ionic selectivity or regulatory mechanisms. The intracellular side of the plasma membrane is in contact with the extracellular solution (figure 3D). So we have an access to the intracellular part of the channel, giving the possibility to study the changes of channels behaviour depending on the ionic changes in the intracellular solution. The selectivity can then be determined by replacing one ion by another and observing the change of the reversal potential on the I-V curve.

The last configuration we used was the perforated patch configuration called "whole cell", which allows us to describe not directly channels, but currents activated during a calcium entry. In fact this configuration, unlike the other two, allows us to record the macroscopic activity of the whole cell currents and is no longer dependent on the presence or absence of channels under the pipette (Figure 3B). We chose the perforated patch rather than the classic whole cell, because this approach maintains the integrity of the plasma membrane and the content of the cell (Ca²⁺, ATP, second messengers). Indeed to have an access inside the cell, a polyene antibiotic is used (nystatin or amphotericin), that creates pores (0.8nm) in

the membranes, permeable to monovalent ions, water and small non-electrolytes (Horn and Marty 1988). The drawback of the perforated patch approach is that we cannot include in the patch pipette substances that modulate the channel behavior. The characterization of current is obtained by the shape of I-V curve and also the selectivity of the current. As in inside-out configuration, the replacement of one ion present in the extracellular solution by another and the observation of the reversal potential allows us to determine the selectivity of the current.

Figure 3: Schematic illustration of the four different methods of patch clamp:

(A) cell-attached configuration; (B) whole cell configuration; (C) outside-out configuration;

(D) inside-out configuration

The three configurations squared in red are those used in this study.

(Modified from (Hamill et al. 1981); http://www.bem.fi/book/)

VI. Publication I

A. Introduction

Ca²⁺ is an important second messenger for the physiology of EC, which use Ca²⁺

elevations in the cytosol to produce and release the factors essential to this functions (Sumpio et al. 2002). The addition of an agonist such as histamine produces a sustained Ca²⁺ entry.

This rise of cytosolic Ca²⁺ is associated with a hyperpolarisation of the membrane, due to Ca²⁺-activated K⁺ channels, increasing the driving force for Ca²⁺ entry. In EA.hy926 cells, the BKCa (Big conductance Ca²⁺-activated K⁺ channels) are the channels involved in this hyperpolarisation (Frieden and Graier 2000).

The two different mechanisms proposed for the Ca²⁺ influxes in EC are the RACE and the SOCE. Many groups were devoted to the study of the SOCE pathway, and these last years, the molecular players, STIM1 and ORAI1, as well as the mechanism of activation have been described (Liou et al. 2005); (Roos et al. 2005); (Feske et al. 2006); (Vig et al. 2006);

(Zhang et al. 2006); (Muik et al. 2008); (Luik et al. 2008). The second messengers and the molecular identity of proteins involved in the RACE pathway seem to be more diverse and totally dependent on the agonist used as well as the cell type (Shuttleworth 2009); (Carter et al. 2006); (Inoue et al. 2001); (Rosker et al. 2004); (Poburko et al. 2007).

Two points make the differentiation of these pathways difficult. The Ca²⁺ response after addition of histamine results from two different events. First a rapid Ca²⁺ release from ER via the IP3 receptor increases transiently the cytosolic Ca²⁺ and this phase is followed by a Ca²⁺ influx, leading to a sustained plateau (Shuttleworth 2004). The SOCE is directly linked to Ca²⁺ filling state of the ER. So it is reasonable to expect a SOCE activation if the level of ER depletion after an agonist activation is sufficient. So the first difficulty is the possible involvement of Ca²⁺ entry due to the depletion of the ER, mixed with a Ca²⁺ entry due to the second messengers produced after agonist stimulation. The second point is the absence of specific inhibitors known for one or the other pathways.

I joined this study that was in progress when I started my thesis. The aim was to investigate the different Ca²⁺ entry pathways present in EC and to study their pharmacology in order to differentiate them and to define the pathway(s) involved after a physiological activation. To do this, the Ca²⁺-imaging approach was used, with two different protocols of Ca²⁺ entry activation. The choice of the protocols was an essential point to separate the different influxes. Indeed most previous studies on the Ca²⁺ entry after agonist stimulation were performed by addition of agonist in absence of extracellular Ca²⁺, followed by a

readdition of Ca²⁺ to the medium. This approach leads not only to the production of second messengers but also to a possible depletion of the stores due to the absence of extracellular Ca²⁺. So, to study agonist-induced Ca²⁺ entry, histamine was applied in presence of Ca²⁺ and its effects, then compared to the store depletion-activated Ca²⁺ entry. To study specifically the Ca²⁺ entry due to the depletion of the intracellular store, without engaging any other second messenger dependent pathway, passive depletion was performed with thapsigargin, in absence of extracellular Ca²⁺. The readdition of extracellular Ca²⁺ leads to a Ca²⁺ entry only due to store depletion. The level of store depletion needs to be measured, to evaluate the ER depletion and thus the involvement of the SOCE in the histamine activated Ca²⁺ entry.

The publication was followed by an erratum regarding the effect of La³⁺. This part is discussed in the last section of the thesis.