Electrophysiology

To study the properties of the currents mediated by mutated ORAI1 channels, we used the electrophysiological patch–clamp technique, developed by Erwin Neher and Bert Sakmann who received the Nobel prize in 1991 (Neher and Sakmann, 1976). Patch–clamp allows the measurement of ionic currents through the membranes of excitable or non–excitable cells, by establishing an electrical circuit between the intracellular and extracellular side of the respective membrane. To do so, an electrode (ground electrode) is placed in the solution were the cell bathes, and the cell is approached with a thin glass micropipette containing a second electrode (Fig. 19). The bath solution may correspond to the physiological extracellular environment, but ions concentration or pH can vary, and drugs can be added, according to the current property tested, as long as the osmolality is maintained. The pipette ionic solution respects the cytosolic content or the bath composition depending on the recording configuration (whole–cell versus cell–attached).

In the “voltage–clamp” configuration, the membrane voltage is imposed, and currents are recorded. In the “current–clamp” configuration, the current is controlled in order to measure action potentials. With voltage–clamp, the difference between the cell membrane potential and the imposed voltage are transmitted to an amplifier that delivers the required current to counterbalance the voltage difference and “clamp” the membrane at the desired voltage. This feedback current is the exact opposite of the ionic current. Therefore, inward cation (positive) currents have negative current–voltage (I/V) relationships (Ypey et al.).

I/V relationship is a current parameter corresponding to the “channel signature” that expresses the density of a current depending on the voltage (see Fig. 5 for the CRAC I/V relationship). The current reversal potential (Erev) indicates the voltage where the net (in or out) ion current is null. For very selective channels, the Erev matches the equilibrium potential (E) of the ions carried by this specific channel and can be calculated with the Nernst equation:

[X] = concentration of the ion of interest (inside and outside) R = ideal gas constant (8.3145 J K⁻¹ mol⁻¹)

T = temperature (in Kelvin) z = ionic charge

F = Faraday constant (96 485.332 89 C mol⁻¹)

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Therefore, for Ca²⁺ selective channels such as ORAI1, the ICRAC Erev is close to the Ca²⁺

equilibrium potential.

In addition to voltage, the current depends on the channel conductance (G, measured in Siemens) which represents how easily an ion crosses the channel. Following Ohm’s law:

𝐼 =

𝑅

𝑅 =

𝐺

𝐼 = 𝐺 × 𝑉

with R = resistance (measured in ohm, Ω).

Following the establishment of a high resistance seal (GΩ) between the cell membrane and the micropipette glass, different configurations allow current recording at the single–cell or single–channel level (Fig. 20):

a) Cell–attached configuration allows to study single–channel ion currents in the isolated membrane patch. The main advantage of this technique is the conservation of an intact membrane and physiologic cytosolic content.

Figure 19. The patch clamp setup. Modified from Bioelectrolab (University of Florence).

The external electrode (ground) is placed in the bath solution, and the internal electrode in a glass micropipette containing a conducting solution. The establishment of a high resistance seal (GΩ) allows to isolate a membrane patch and reduce the signal noise. The electrodes are connected to an amplifier that measures the difference between a defined holding potential and the cell membrane potential, and delivers feedback currents to clamp the membrane at the desired voltage.

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b) In perforated patch–clamp, the addition of pore–forming antibiotics (e.g. amphotericin B) to the patch pipette offers an access to the intracellular compartment. These very small pores in the membrane patch prevent the escape of large molecules and limits cytosolic dialysis with the pipette content (equilibration of small monovalent ions only).

c) Whole–cell configuration allows to measure all ionic currents across the entire cell membrane, by the rupture of the membrane patch and the establishment of an electrical continuity between the pipette glass and the whole lipid bilayer. In this technique, the cytosolic content equilibrates with the pipette solution.

d) Inside–out configuration is achieved by the suction of the membrane patch in the pipette hole and its detachment from the cell, exposing the intracellular side of the membrane to the bath solution. It is used to record the current behavior of single–

channels upon modifications of the intracellular environment.

e) Outside–in configuration: the opposite allows the exposure of the extracellular side of the membrane to the bath. To achieve this configuration, the patch membrane is first disrupted, and slowly removing the pipette pulls a portion of membrane that further reforms a convex lipid bilayer at the pipette tip.

Figure 20. Patch clamp configurations. From Pr. Stuart Mangel (The Ohio State University).

Cell-attached configuration (A) preserves the membrane integrity and cytosolic content. The addition of pore-forming antibiotics to the pipette solution allows to access the intracellular compartment (perforated patch, B). Disruption of the patch membrane leads to whole-cell configuration (C). Inside-out (D) and outside-in (E) are achieved by pulling a portion of membrane with the pipette and allow the access and modification of the intracellular or extracellular contents respectively.

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The endogenous ICRAC is a very small current (pA range) that is difficult to record with single–

channel configurations. Therefore, the voltage–clamp whole–cell configuration was used to assess the current mediated by the TAM mutated ORAI1 channels overexpressed in HEK–293T cells. The holding potential was set at 0 mV, and voltage ramps from –120 to +70 mV were imposed to measure the ICRAC I/V relationship. ORAI1 is a non–voltage gated channel. To follow the current development over time, the internal pipette solution contained 10 mM EGTA and Tg to allow the passive depletion of ER stores and the activation of SOCE. In addition, cesium methanesulfonate was added to the intracellular solution to block potassium channel currents.

The external bath contained a high concentration of CaCl2 (10 mM) to facilitate the recording of Ca²⁺ currents. In this project, the pH sensitivity of the currents mediated by the ORAI1 mutants was assessed by progressively decreasing the bath pH; the channel permeation evaluated in a Ca²⁺ containing and a divalent–free (DVF) solution; and the Ca²⁺ selectivity by replacing Na⁺ with the large non–permeant molecule NMGD. To measure FCDI, negative voltage steps were imposed. Voltage ramps allowed the evaluation of SCDI in cells dialyzed with low amounts of EGTA (1.2 mM).

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