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W2983302145 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods References Decision letter Author response Article and author information Metrics Abstract Intercellular tight junctions form selectively permeable barriers that seal the paracellular space. Trans-tight junction flux has been measured across large epithelial surfaces, but conductance across individual channels has never been measured. We report a novel trans-tight junction patch clamp technique that detects flux across individual claudin-2 channels within the tight junction of cultured canine renal tubule or human intestinal epithelial monolayers. In both cells, claudin-2 channels display conductances of ~90 pS. The channels are gated, strictly dependent on claudin-2 expression, and display size- and charge-selectivity typical of claudin-2. Kinetic analyses indicate one open and two distinct closed states. Conductance is symmetrical and reversible, characteristic of a passive, paracellular process, and blocked by reduced temperature or site-directed mutagenesis and chemical derivatization of the claudin-2 pore. We conclude that claudin-2 forms gated paracellular channels and speculate that modulation of tight junction channel gating kinetics may be an unappreciated mechanism of barrier regulation. https://doi.org/10.7554/eLife.09906.001 eLife digest Epithelial cells form layers that line the inner surface of the gut, lungs and other organs. They act as barriers to control the movement of water, ions and small molecules between internal compartments within the body and the external environment. Some substances are transported across these barriers by passing through individual epithelial cells, but others pass through the spaces between adjacent cells. These spaces are sealed by tight junctions. If the tight junctions do not work properly, it can cause problems with regulating the movement of molecules across epithelial-lined surfaces. This in turn can contribute to diseases in humans, including inflammatory bowel disease and chronic kidney disease. Proteins called claudins form channels that only allow certain molecules to pass through tight junctions. One member of this family, called claudin-2, allows sodium ions and other small positively charged ions to cross between adjacent cells. However, it is not clear how these channels work, largely due to the absence of appropriate tools to study this process. Here, Weber et al. adapted a technique called patch clamping to study the behavior of individual claudin-2 channels in the tight junctions between mammalian epithelial cells. Weber et al. found that claudin-2 allows positively charged ions to move across a tight junction in short bursts rather than in a steady stream as had been suggested by previous work. These bursts typically begin and end in less than a millisecond. Further experiments revealed that claudin-2 channels have several states; in one state the channel is fully open, in another the channel is firmly closed, and in the third state the channel is temporarily closed but primed to open. Further experiments show that mutations in the gene that encodes claudin-2 or drugs that inhibit claudin-2's function alter the open and closed behaviors of these trans-tight junction channels. The technique developed by Weber et al. will enable researchers to understand how channel proteins at tight junctions assemble and operate. Such studies may lead to the development of drugs that can alter the activity of these channels to treat particular diseases. https://doi.org/10.7554/eLife.09906.002 Introduction Epithelial barriers are essential for the survival of multicellular organisms and allow compartmentalization and controlled interactions between distinct environments (Marchiando et al., 2010, Turner, 2009). While transcellular transport is mediated by proteins that span the plasma membrane, molecular details of ions, water, and solute transport across the tight junction, i.e. the paracellular path, are less well-defined. This, in part, reflects the absence of tools able to detect single channel events at the tight junction and, therefore, a reliance on methods that measure paracellular flux over large multicellular surfaces (Shen et al., 2011). The limited spatial and temporal resolution of these techniques has contributed to the widely held view of tight junction channels as constitutively open and has limited biophysical characterization of these paracellular channels. Nevertheless, it is clear that paracellular transport is critical to the function of transporting epithelia in many organs (Bagnat et al., 2007, Simon et al., 1999, Wada et al., 2013) and can be regulated by physiological and pathophysiological stimuli (Heller et al., 2005, Marchiando et al., 2010, Suzuki et al., 2011, Turner et al., 1997, Weber et al., 2010). Intestinal epithelial expression of the tight junction protein claudin-2, which increases paracellular Na+ conductance (Amasheh et al., 2002, Wada et al., 2013, Weber et al., 2010), is downregulated after the neonatal period (Holmes et al., 2006) but markedly upregulated in inflammatory and infectious enterocolitis and by several cytokines, including IL-13 (Heller et al., 2005). This is essential for IL-13-induced increases in paracellular Na+ permeability, as conductance changes are prevented by siRNA-mediated inhibition of claudin-2 upregulation (Weber et al., 2010). Thus, claudin-2 expression is regulated during development and disease. Detailed functional analysis of claudin-2-based channels, in their own right and as models for all paracellular claudin channels, is therefore critical to understanding fundamental mechanisms of development and disease. Claudin-2 expression induces large increases in paracellular flux of small cations (Amasheh et al., 2002, Furuse et al., 2001, Weber et al., 2010). Inducible claudin-2 expression in MDCKI monolayers, which lack endogenous claudin-2 expression, is therefore an ideal experimental model in which to define paracellular, trans-tight junction channels (Angelow and Yu, 2009). We analyzed claudin-2 channel function using a novel, trans-tight junction patch clamp technique. Here, we show that this approach can detect discreet conductance events, define these in biophysical terms, perform extensive characterization that demonstrates that the currents detected reflect the activity of trans-tight junction channels, and excludes the possibility that these events are due to apical membrane conductances or other artifacts. The data show that these tight junction channels are gated and behave in a manner reminiscent of traditional transmembrane ion channels despite radical differences in orientation and function. Nevertheless, these similarities, the efficacy of pharmacological effectors of transmembrane ion channel function, and the frequency of epithelial barrier defects in disease suggest that it may be possible to develop agents that positively- or negatively-regulate tight junction channel gating for therapeutic benefit. Results As a reductionist system, we expressed claudin-2 under the control of a tet-off regulated system in polarized MDCKI monolayers (Angelow and Yu, 2009). Western blot demonstrated that, relative to the parental MDCKI line, small amounts of functional claudin-2 were present even when expression was repressed, i.e. doxycyline was present, consistent with the known minor leakiness of such regulated expression systems (Figure 1A). As expected based on previous comparisons of MDCKI and MDCKII cells (Stevenson et al., 1988), which differ primarily in their expression of claudin-2, induction of claudin-2 expression did not affect tight junction ultrastructure (Figure 1B). When expression was induced, claudin-2 concentrated at tight junctions and, to a limited extent, in cytoplasmic vesicles (Figure 1C). Induction of claudin-2 expression also reduced transepithelial electrical resistance (TER; Figure 1D) and increased cationic charge selectivity (Figure 1E) with strict size-selectivity (Figure 1F). Claudin-2 expression therefore induces charge- and size-selective increases in paracellular, trans-tight junction conductance. Figure 1 Download asset Open asset Claudin-2 expression enhances tight junction permeability to small cations. (A) Transgenic MDCKI monolayers were developed to express claudin-2 (+Cldn-2) in the absence of doxycycline. Limited claudin-2 expression was detected in the absence of induction and none was detectable in the parental MDCKI line. (B) Induction of claudin-2 expression had no effect on tight junction ultrastructure (Bar = 500 nm). (C) Tight junction claudin-2 was not detectible by immunofluorescence staining after suppression of claudin-2 expression (Bar = 10 µm). (D) Claudin-2 expression reduced TER (E) and increased relative permeability of sodium to chloride (PNa+/PCl-) was increased. (F) Biionic potential analyses show that the reduction in TER was mainly due to increased paracellular permeability to small cations. https://doi.org/10.7554/eLife.09906.003 Claudin-2 expression induces conductance events that can be detected by trans-tight junction patch clamp Measurements of paracellular permeability, such as those above, typically assess relatively large epithelial surfaces and, therefore, reflect global averages rather than local, site-specific conductances. Scanning and impedance approaches have been used in an effort to overcome these limitations (Chen et al., 2013, Gitter et al., 1997, Krug et al., 2009), but these lack the spatial and temporal resolution needed for identification of single channel events. Overall, the greatest obstacle to single channel analyses of tight junction channels has been the orientation of trans-tight junction channels between lateral surfaces of two adjacent cells, i.e. parallel to plasma membranes (Figure 2A). This orientation is orthogonal to traditional ion channels and gap junctions, which cross plasma membranes, and renders most patch clamp techniques unsuitable for measuring trans-tight junction ion flux. Figure 2 Download asset Open asset Claudin-2 expression correlates with the frequency of local tight junction channel openings in MDCKI monolayers. (A) Tight junctions are distinct from plasma membrane ion channels and differ from gap junctions in their ability to define conductance between two extracellular compartments. (B) Trans-tight junction patch clamp placement. Yellow arrowheads show intercellular junction (Bar = 10 μm). (C) Conductance events detected at −100 mV when claudin-2 was expressed (+Cldn-2). (D) In the absence of induced claudin-2 expression (–Cldn-2), the frequency of similar sized conductance events was dramatically reduced. (E) Small claudin-2 independent events were present in parental MDCKI monolayers (F) Conductance events were present at +100 mV when claudin-2 was expressed (+Cldn-2). (G) Events were infrequent in the absence of induced claudin-2 expression (–Cldn-2). (H) NPo was reduced by 87% ± 4% (at –100 mV) and 88% ± 6% (at +100 mV) after suppression of claudin-2 expression. Events were rare in recordings from parental tight junctions. (I) Representative recording of voltage ramp in claudin-2-expresing MDCKI monolayers showing linear current voltage relationship and reversal potential close to 0 mV. (J) Average current voltage relationships (n = 8 to 32 per condition) reveals that average channel conductance was ~90 pS regardless of whether claudin-2 expression was induced (green line) or not (black line). https://doi.org/10.7554/eLife.09906.004 To overcome these challenges, we developed an approach to seal an apical patch pipette across a region of the bicellular tight junction. This required several technical advances, including development of low profile chambers that allowed apical tight junction access using a 50°–60° approach angle while simultaneously viewing cell profiles from below the monolayer. Successful patching of tight junctions also required optimization of cell growth to afford clear morphological delineation of tight junctions while minimizing accumulation of cellular debris that could interfere with pipette sealing. Electrode configuration was also modified so that the pipette was just large enough to span the tight junction, while not so small that it would slip off of the tight junction. This allowed us to achieve a gigaseal with an ~5% success rate. Once a high resistance gigaseal was achieved, it was then possible to measure current through the paracellular pathway in response to an externally applied voltage relative to a basal reference electrode (Figure 2B). The approach above allowed detection of bursts of sub-millisecond duration, flicker-like openings and closings when using holding potentials of −100 or +100 mV (Vapical− Vbasal) in monolayers with claudin-2 expression (Figure 2C,F,H). Such events were infrequent in monolayers without induction of claudin-2 expression, i.e. with low level claudin-2 expression (Figure 2D,G,H), and were rare in parental MDCKI monolayers that completely lacked claudin-2 expression (Figure 2E,H). All-points histograms, fitted to Gaussian distributions, show the claudin-2 dependent conductance centered at ~9 pA, and ranged from ~5 to >10 pA at –100 mV. A separate class of smaller conductance values centered at ~4.3 pA was present in all lines, regardless of claudin-2 expression. To focus on claudin-2-dependent channels, thresholding was used to exclude the small, claudin-2-independent conductances. These analyses showed that opening probability (NPo) of claudin-2-dependent channels was similar at +100 or –100 mV in claudin-2 expressing monolayers, but was reduced by 87 ± 4% (at -100 mV) and 88% ± 6% (at +100 mV) in the absence of claudin-2 induction (Figure 2H). In contrast, amplitude was similar at high and low levels of claudin-2 expression. Thus, the NPo, but not the amplitude, of these openings with conductances of ~92 pS is a function of claudin-2 expression. This further suggests that the number of channels, but not the open probability of individual channels, is a function of claudin-2 expression. To further characterize the voltage dependence of claudin-2-dependent conductances we performed voltage ramps beginning at a holding potential of –100 mV. Current-voltage (I-V) relationship plots (Figure 2I,J) showed that the reversal potential (Vrev) of these events was close to 0 mV, but slightly negative, and followed a linear function of voltage, consistent with a passive process. This result argues strongly that the conductance events cannot be apical cation or anion, e.g. K+, Na+, or Cl- channels, since the extracellular:intracellular gradients of these ions would necessitate equilibrium potentials much different than 0 mV. Notably, this analysis also shows that, despite there being far fewer events when claudin-2 expression was suppressed, individual conductance events were quantitatively similar, in both amplitude and duration, when claudin-2 expression was low (Figure 2J). Therefore, the ~9 pA single channel conductances measured by trans-tight junction patch clamp are non-vectorial, as expected for passive paracellular channels, and is unlikely be due to the activity of apical transmembrane ion channels. Kinetic analysis shows that claudin-2 channels have one open and two closed states The observation that claudin-2-dependent conductance events occur in bursts was somewhat surprising, as tight junction conductance has been assumed to be uniform over time. This widely-held belief was based on stable measurements of paracellular conductance across large epithelial surfaces. However, conductance of individual channels is averaged over space in these measurements, which lack both temporal and spatial resolution of the trans-tight junction patch clamp approach. To better characterize the opening and closing behaviors of claudin-2-dependent channels, histograms of all events were generated. Opening duration histograms of tight junction patch clamp data from cells with high or low claudin-2 expression at holding potentials of +100 or -100 mV. These revealed a single population of openings with τopen< 1 ms (Figure 3A–D). In contrast, closed duration histograms under the same conditions revealed two populations of closings, corresponding to closed states between and within event clusters. Interburst closings were prolonged with τ closed(stable) >1 s while intraburst closings occurred with millisecond kinetics, i.e. τ closed(transient) < 2 ms (Figure 3E–H). One possibility is that the prolonged state (closedstable) could represent channel disassembly, while the shorter closed state (closedtransient) results from regulation of assembled channels. However, given the absence of a significant vesicular claudin-2 pool and the relatively long ~9 hr half-life of claudin-2 protein in MDCK monolayers (Van Itallie et al., 2004), we consider it highly unlikely that vesicular traffic or protein turnover could be responsible for the observed opening and closing events. In contrast, although the pool of claudin-2 at the tight junction is largely immobile (Raleigh et al., 2011), the limited intramembranous diffusion that does occur (Shen et al., 2008) has kinetics within the range of the longer closed state (closedstable) and we cannot exclude this as a possible regulatory mechanism. Figure 3 Download asset Open asset Patch clamp recordings reveal a single open state and two closed states. (A–D) A single population of fast openings was observed in the presence (green) or absence (white) of induced claudin-2 expression at –100 and +100 mV (a-d total recording times: 40 s, 225 s, 31 s, 52 s.). (E–H) Corresponding closed duration histograms from the same representative recordings reveal two distinct closed states. (I) Opening and closing time constants were voltage independent and were similar with and without claudin-2 induction (n=7 to 35 recordings for each condition). (J) Kinetic analysis demonstrates the presence of both stable (cstable) and transient (ctransient) closed states and one and open (o) state. https://doi.org/10.7554/eLife.09906.005 Similar to amplitude, open and closed state kinetics were similar under high or low claudin-2 expression. Claudin-2 channel gating is thus independent of expression level, i.e. is non-cooperative. This also indicates that changes in NPo that occur as a function of claudin-2 expression reflect differences in channel number rather than open probability of individual channels. Further, because properties were similar at +100 and -100 mV, we can conclude that claudin-2-dependent channels are not gated by voltage, unlike many transmembrane ion channels. Overall these data show that claudin-2-dependent channels can exist in a highly dynamic opening state (o) as well as stable (cstable) and transient (ctransient) closed states (Figure 3J). Claudin-2-dependent openings detected by trans-tight junction patch clamp are resistant to traditional ion channel inhibitors Despite the voltage ramp results (Figure 2I,J), we re-considered the possibility that detected events represented transmembrane conductances of apical ion channels within apical membrane captured by the patch pipette. The ~9 pA claudin-2-dependent openings were, however, never observed when electrodes were sealed off of the tight junction, i.e. over apical membranes away from the tight junction. In place of the ~9 pA openings, small conductances could sometimes be detected when electrodes were sealed over apical membranes, but only when the data were low-pass filtered at 500 Hz (Figure 4A). These events differed distinctly from the claudin-2-dependent conducances, as the former were more common at holding potentials of +100 mV, relative to -100 mV, and had amplitudes of less than 2 pA, well below those of claudin-2-dependent events. These data provide spatial evidence that the conductances detected by trans-tight junction patch clamp are not traditional, transmembrane apical ion channels. Figure 4 Download asset Open asset Large and small tight junction currents are not due to transmembrane ion channels. (A) Small (<2 pA) transmembrane ion channel openings were detectable in off-tight junction recordings after applying a 500 Hz low pass filter. (Bar = 10 μm). (B) Events detected by trans-tight junction patch clamp were not blocked by three different ion channel inhibitor cocktails (+Cldn-2; representative of n = 3 to 8 per condition). (C) Monolayers were cooled while recording from trans-tight junction patch clamp. The number of events detected was reduced at 15°C, relative to 37°C, but event amplitude was unaffected (+Cldn-2; representative of n = 4). (D) NPo and Na+ permeability measured across a 0.33 cm2 monolayer were similarly reduced at 15°C (+Cldn-2). (E) ~4 pA events remained detectable after chilling monolayers to 10°C (MDCKI parental monolayers; representative of n = 4). (F) ~4 pA events were not blocked by three different ion channel inhibitor cocktails (MDCKI parental monolayers; representative of n = 3 to 5 per condition). https://doi.org/10.7554/eLife.09906.006 The data above, including the gigaohm seals achieved, symmetrical behavior, near 0 mV reversal potential, detection only at tight junctions, and claudin-2-dependence, suggest that the ~9 pA events detected by trans-tight junction patch clamp cannot be due to artifacts, such as transmembrane ion channels in apical membrane domains sealed within the patch pipette or pipette leak. We nevertheless took a pharmacological approach to further examine the hypothesis that these conductance events represented activity of transmembrane ion channels. Three different inhibitor cocktails were added to the patch pipette (Figure 4B). The first cocktail contained 10 mM 4-aminopyridine and 10 mM TEA-Cl to inhibit voltage-activated K+ channels. The second cocktail contained 100 nM charybdotoxin and 2 μM apamin to inhibit small conductance Ca2+ activated K+ channels. The third cocktail contained 100 μM amiloride, 20 μM CFTR inhibitor-172 (inh-172), and 250 μM 4,4'-Diisothiocyano-2,2'-stilbenedisulfonic acid (DIDS) to block epithelial Na+ channels, CFTR, and anion exchangers respectively. None of these affected frequency or amplitude of the claudin-2-dependent ~9 pA events detected by trans-tight junction patch clamp. We therefore conclude, on the basis of biophysical, molecular, and pharmacological data, that the ~9 pA events detected represent single-channel conductances across the tight junction. Temperature sensitivity of claudin-2 channels is similar whether measured by trans-tight junction patch clamp or traditional, global approaches Previous studies have shown that overall transepithelial conductance as well as claudin-2-dependent Na+ conductance decrease when temperature is reduced (Gonzalez-Mariscal et al., 1984, Martinez-Palomo et al., 1980, Shen et al., 2008, Yu et al., 2009). We therefore assessed temperature sensitivity of the single channel events measured by trans-tight junction patch clamp. These studies were complicated by the technical challenge of cooling the monolayer without creating excessive electrical noise that prevented analysis and limited cooling to ~20°C while recording from the trans-tight junction patch clamp. When monolayers of claudin-2-expressing MDCKI were cooled from 37°C to 15°C while recording, the number of claudin-2-dependent (~9 pA) events fell by 37% (Figure 4C). This was closely paralleled by a 41% decrease in Na+ permeability measured across monolayers using traditional approaches (Figure 4D), providing more support for the conclusion that the ~9 pA conductance events reflect activity of paracellular claudin-2 channels. We also assessed the claudin-2-independent, ~4 pA events using the parental MDCKI cells that completely lacked claudin-2 expression. We did this because these claudin-2-independent currents can be obscured by larger events (e.g. Figure 2). In contrast to the ~9 pA events, the ~4 pA events were resistant to cold (p = 0.35), even when chilled to 10°C (Figure 4E). The ~4 pA events were also resistant to all of the ion channel inhibitor cocktails (Figure 4F). Claudin-2 channel behavior is consistent across different types of epithelia The MDCK cell line is derived from epithelia of the distal convoluted tubule, which function effectively to absorb Na+ and Cl- in the apical-to-basal direction and secrete K+ (Gekle et al., 1994). Cell lines derived from epithelia within other parts of the body have distinct specialized functions. For example, Caco-2 cells are a human colon epithelial cancer cell line that differentiate as absorptive enterocytes and express brush border enzymes and transporters typical of this cell type (Peterson and Mooseker, 1992, Pinto et al., 1983, Turner and Black, 2001, Turner et al., 1996, Turner et al., 1997). While both MDCK and Caco-2 cell lines are both commonly used to study polarized epithelial cell function, their distinct phenotypes are reflected by divergence in both function and protein expression. Nevertheless, tight junction ultrastructure is similar in MDCK and Caco-2 cells, and both are composed of three to five strands (Figure 5A), which express claudin-2 abundantly (Figure 5B,C). We took advantage the availability of a Caco-2BBe line in which claudin-2 expression was stably knocked down (Raleigh et al., 2011) to assess the effects of claudin-2 depletion, rather than addition, on paracellular channel function. This also allowed direct comparison of canine renal epithelia and human intestinal epithelia. Figure 5 Download asset Open asset Global conductance and trans-tight junction patch clamp event frequency correlate with claudin-2 expression in Caco-2BBe intestinal epithelial monolayers. (A) Freeze fracture electron microscopy demonstrating that mature tight junctions in Caco-2BBe monolayers are composed of 3–5 strands (Shen et al., 2006), similar to MDCKI. (B) Western blot confirms >99% knockdown of claudin-2 in Caco-2BBe monolayers. (C) Claudin-2 is not detectable by immunofluorescence microscopy of knockdown Caco-2BBe monolayers (Bar = 10 µm). (D) Biionic potential analyses show that claudin-2 knockdown reduces small cation permeability. (E) Trans-tight junction patch clamp recordings of Caco-2BBe cells detected events at −100 mV (n=5 per condition). Representative traces of trans-tight junction patch clamp data from control and claudin-2 knockdown Caco-2BBe monolayers. (F) All points histogram analysis of patch clamp data from Caco-2BBe monolayers shows a specific reduction in ~9 pA events with no change in frequency of ~4 pA events after claudin-2 knockdown. (G) Average opening conductances was unaffected by the levels of claudin-2 expression. (H) Channel activity (NPo) was reduced by claudin-2 knockdown (n = 5 to 7 per condition). (I) Neither ~9 pA nor ~4 pA events were not detectable when the pipette was sealed away from the tight junction in Caco-2BBe monolayers (n = 12). https://doi.org/10.7554/eLife.09906.007 Paracellular Na+ conductance across Caco-2BBe monolayers was similar to that of MDCKI monolayers with induced claudin-2 expression (Figure 5D vs Figure 1F). When claudin-2 expression was stably suppressed by shRNA-mediated knockdown, Na+ conductance across Caco-2BBe monolayers was greatly diminished (Figure 5D) and fell to a level similar to the claudin-2-deficient MDCKI parental line (Figure 1F). We next analyzed monolayers of Caco-2BBe human intestinal epithelia by trans-tight junction patch clamp (Figure 5E). It was substantially more difficult to achieve a gigaohm seal in these monolayers relative to MDCK, likely due to the well-developed brush border of Caco-2BBe cells. As with MDCK monolayers, all points histograms demonstrate conductance values centered around ~8 pA, i.e. ~82 pS, in claudin-2-expressing monolayers (Figure 5F), and there was a specific reduction in this class of events after claudin-2 knockdown (Figure 5G). We therefore conclude that claudin-2 depletion in human intestinal epithelia eliminates events similar to those generated by claudin-2 expression in canine renal epithelia. While NPo was reduced, conductance of the claudin-2-dependent events was not affected by claudin-2-knockdown (Figure 5H), demonstrating that single channel conductance was not a function of claudin-2 concentration. Similar to the data from MDCK monolayers, this indicates that gating of claudin-2-dependent channels is not cooperative. A class of smaller conductances that were not affected by claudin-2 knockdown was also detected, similar to the claudin-2-independent events detected in MDCK monolayers. Finally, as in MDCK cells, the claudin-2-dependent openings were not detectable when the patch pipette was sealed away from the tight junction in Caco-2BBe monolayers (Figure 5I). Thus, these data demonstrate the tight junction opening events are detectable in two very different epithelia derived from different organ systems, and, in both cases, the events were claudin-2 dependent. Further, these data exclude the possibility that events could be due to off-target effects induced by the tet transactivator used to drive claudin-2 expression in MDCK cells. More importantly, the similar conductance values of these events, despite markedly different NPo values, supports the conclusion that the events are mediated by biophysically similar claudin-2-based paracellular channels, rather than some other paracellular channel that might be expected to differ" @default.
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W2983302145 title "Author response: Claudin-2-dependent paracellular channels are dynamically gated" @default.
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