A proposed route to independent measurements of tight junction conductance at discrete cell junctions

Abstract

Direct recording of tight junction permeability is of pivotal importance to many biologic fields. Previous approaches bear an intrinsic disadvantage due to the difficulty of separating tight junction conductance from nearby membrane conductance. Here, we propose the design of Double whole-cell Voltage Clamp - Ion Conductance Microscopy (DVC-ICM) based on previously demonstrated potentiometric scanning of local conductive pathways. As proposed, DVC-ICM utilizes two coordinated whole-cell patch-clamps to neutralize the apical membrane current during potentiometric scanning, which in models described here will profoundly enhance the specificity of tight junction recording. Several potential pitfalls are considered, evaluated and addressed with alternative countermeasures.

Keywords: SICM; conductance; ion channel; patch clamp; tight junction.

Figure 1.

Illustration of the DVC-ICM setup. Double barrel pipet for topographical mapping (PE) and local potential measurement (UE) is attached to a piezo positioner. Reference electrode (RE), counter electrode (CE) and working electrode (WE) are controlled with home-built electrode controller to apply transmembrane potential across the epithelial monolayer. Two balance electrodes (BE1 and BE2) are used to control the intracellular potentials of 2 patched cells and to record the current flowing from or to BE1 or BE2 (i_BE1 or i_BE2) respectively. Upon the application of transmembrane potential, ion current flows across the epithelial cell layer through both paracellular pathways (i_para) and transcellular pathways (i_trans1 and i_trans2), and both i_trans are undesired background signal for the measurement of  paracellular conductance.

Figure 2.

(A) Equivalent circuit of the DVC-ICM system. Here, tight junction conductance (reciprocal of tight junction resistance, R_TJ) of the junction formed by cell 1 and cell 2 is being measured with P-SICM (electrodes not shown). Cell 1 and cell 2 are voltage-clamped with two patch clamp pipets controlled with an individual patch clamp amplifier respectively. The intracellular potential of cell 1 (cell 2) is held at V₁’ through BE1 (BE2), where the holding voltage by patch clamp amplifier is V₁ (V₂). Series resistance R_S1 (R_S2) consists the resistances of BE and broken membrane patch from cell 1 (cell 2). Apical membrane resistance and basolateral membrane resistance are indicated separately with R_ma1 and R_mb1 for cell 1 and R_ma2 and R_mb2 for cell 2. R_GJ refers to the gap junction resistance through which ion current can pass between cell 1 and 2. (B) Ion current passing each resistor in (A) is labeled with the same subscript.

Figure 3.

Intracellular potentials (V₁’ and V₂’) for both cell 1 and cell 2 are held at their E_rest to yield zero transmembrane current at the apical membranes (i_ma1 and i_ma2) during the application of transmembrane potential V_TE. Majority of V_TE induced basolateral membrane current (i_mb1 and i_mb2) is drawn through BE1 and BE2 (red arrows). Note: R_GJ is omitted from this circuit because V₁’ and V₂’ are always kept the same and there is no current through R_GJ.

Figure 4.

(A) Illustration of possible current flow between the two cells involved in DVC-ICM measurements (green cells in the middle) and their neighboring cells (gray cells in the periphery). Gap junctional currents flow within the epithelial cell layer (green arrow) and paracellular currents flow through tight junctions perpendicular to the cell layer (red arrow). (B) Lateral distribution of relative potential field (normalized to the potential amplitude over nanopore center) at 200 nm above a nanopore for nanopores with inner radius of 1, 10, 100 and 500 nm. The pore resistance (R_pore) of a cylindrical nanopore can be calculated by the following equation: ${\text{R}}_{pore}=p·\frac{4h}{\pi ·d^2}$, in which p represents the solution resistivity, d represents the pore diameter, and h represents the pore length. (C) Lateral distribution of relative potential field (same as in (B)) at different heights above a radius 100 nm nanopore (D_ps = 0, 20, 100, 200, and 500 nm).

Figure 5.

A more complete equivalent circuit of DVC-ICM system. C_S1 and C_S2 represent the capacitance of the patch clamp pipets glass wall. R_L1 and R_L2 are the “gigaohm seal” resistances at the sealed spaces between patch pipets and cell membranes. The cell membrane capacitances are indicated with C_ma1, C_ma2 (for apical membrane) and C_mb1, C_mb2 (for basolateral membrane).