Paracellular ion channel at the tight junction

Abstract

The tight junction of epithelial cells excludes macromolecules but allows permeation of ions. However, it is not clear whether this ion-conducting property is mediated by aqueous pores or by ion channels. To investigate the permeability properties of the tight junction, we have developed paracellular ion flux assays for four major extracellular ions, Na(+), Cl(-), Ca(2+), and Mg(2+). We found that the tight junction shares biophysical properties with conventional ion channels, including size and charge selectivity, dependency of permeability on ion concentration, competition between permeant molecules, anomalous mole-fraction effects, and sensitivity to pH. Our results support the hypothesis that discrete ion channels are present at the tight junction. Unlike conventional ion channels, which mediate ion transport across lipid bilayers, the tight junction channels must orient parallel to the plane of the plasma membranes to support paracellular ion movements. This new class of paracellular-tight junction channels (PTJC) facilitates the transport of ions between separate extracellular compartments.

FIGURE 1

Paracellular ion permeability. (A) Schematic drawing of the paracellular pathway of an epithelial monolayer. Estimated pore diameter of the paracellular tight junction channel (PTJC) is ∼6 Å. (B) PTJC is inserted at the tight junction permeability barrier.

FIGURE 2

Three epithelial cell lines, MDCK I, MDCK II, and T84, had different paracellular ion and tracer permeabilities. (A) Voltage and current clamps showed ohmic I-V relationships in all three cell types. (B) Paracellular tracer fluxes of ¹⁴C-ethanolamine and ¹⁴C -mannitol in MDCK I, MDCK II, and T84 cells (n = 6).

FIGURE 3

Distinct ion selectivities of the MDCK I, MDCK II, and T84 PTJCs. (A) Schematic protocol for measurements of ion selectivities using chopstick electrodes. (B) Monovalent ion selectivities of MDCK II (three monolayers). (C) Monovalent ion selectivities of MDCK I (one monolayer) and T84 (four monolayers). (D–F) Ca²⁺ sensitivities of MDCK I, MDCK II, and T84 PTJCs (n = 3).

FIGURE 4

Establishing ion flux assays. (A) Schematic protocol for measurements of paracellular ion fluxes. (B) A Na⁺ standard curve generated with sodium potentials. (C) A Ca²⁺ standard curve. (D) A Mg²⁺ standard curve. (E) A Cl⁻ standard curve.

FIGURE 5

Paracellular Na⁺, Ca²⁺, and Mg²⁺ flux in MDCK II and T84 cells. (A) Na⁺ flux in MDCK II and T84 cells, as a function of Na⁺ concentration gradients (n = 3). (B) Na⁺, Ca²⁺, and Mg²⁺ fluxes in MDCK II cells, as functions of concentration gradients. Net sodium flux was calculated by multiplying the concentration of sodium in the final apical solution (molar, M) by its volume (liter, L), divided by the duration of flux (time, h) and the area of the monolayer (cm²), expressed here as μmol/h cm². Permeability (μmol/M h cm²) is defined here as the net flux divided by the chemical driving force, which has the same unit as the conventional cm/s. (C) Na⁺, Ca²⁺, and Mg²⁺ fluxes in T84 cells, as functions of concentration gradients. (D) Permeabilities of Na⁺, Ca²⁺, and Mg²⁺ in MDCK II cells, as functions of ion concentration gradients. (E) Permeabilities of Na⁺, Ca²⁺, and Mg²⁺ in T84 cells, as functions of ion concentration gradients. For each data point, n = 3.

FIGURE 6

Distinct tetraionic permeability profiles in the three epithelial cells. (A) Concurrent fluxes of Na⁺, Cl⁻, Ca²⁺, and Mg²⁺ in MDCK I, MDKC II, and T84 monolayers (n = 3). (B) Comparison of tetraionic permeabilities at 37°C and 4°C for MDCK I, MDKC II, and T84 monolayers (n = 3).

FIGURE 7

Paracellular ion selectivity required the formation of intact tight junctions. (A) Generation of TER and concomitant decline in paracellular ion fluxes upon tight junction formation by the addition of normal calcium to MDCK II cells (t = 0) that had grown in low calcium medium. Y axis on the left showed ion permeabilities from concurrent tetraionic flux, also displayed as fractional fluxes in pie charts. Y axis on the right showed the corresponding transepithelial electrical resistance (TER). Duplicate monolayers were studied for each time point. The graph was generated using a representative measurement for each time point. (B) Immunofluorescence detection of occludin at various time points after calcium induced tight junction formation.

FIGURE 8

Competition between Na⁺ and Ca²⁺ for transit through MDCK II PTJCs. (A) Diminished ¹⁴C-ethanolamine flux in the presence of concurrent Na⁺, Ca²⁺, and Mg²⁺ fluxes (n = 6). (B) Addition of Ca²⁺ did not affect paracellular Na⁺ permeability (n = 3). (C) Monovalent ion selectivity remained unaltered in the presence of 5 mM Ca²⁺. Two cell monolayers are plotted. (D) Increased Ca²⁺ and Mg²⁺ permeabilities in increasing ratios of basal Ca²⁺ to Na⁺ (n = 2). (E) Comparison of tetraionic and triionic (Ca²⁺, and Mg²⁺, and Cl⁻) flux showed increased Ca²⁺ and Mg²⁺ permeabilities in the absence of concurrent Na⁺ flux (n = 3). (F) Anomalous mole-fraction effect between Na⁺ and Ca²⁺ by TER measurements. Two monolayers are plotted.

FIGURE 9

Effects of low pH on paracellular ion selectivity in MDCK II cells. (A) Current-clamp of a cell monolayer showed increased transepithelial electrical resistance at apical pH 4. (B) Monovalent ion selectivity was disrupted at pH 5, which was readily reversible upon replacement to pH 7 solutions (inset). A representative monolayer is shown. (C) Tetraionic permeability profile at apical pH 4 (n = 3). (D) Tracer fluxes of ¹⁴C-mannitol and ¹⁴C-ethanolamine at apical pH 4. For each tracer, n = 4.

FIGURE 10

Working hypothesis of paracellular tight junction channels.