Molecular determination of claudin-15 organization and channel selectivity

Abstract

Tight junctions are macromolecular structures that traverse the space between adjacent cells in epithelia and endothelia. Members of the claudin family are known to determine tight junction permeability in a charge- and size-selective manner. Here, we use molecular dynamics simulations to build and refine an atomic model of claudin-15 channels and study its transport properties. Our simulations indicate that claudin-15 forms well-defined channels for ions and molecules and otherwise "seals" the paracellular space through hydrophobic interactions. Ionic currents, calculated from simulation trajectories of wild-type as well as mutant channels, reflect in vitro measurements. The simulations suggest that the selectivity filter is formed by a cage of four aspartic acid residues (D55), contributed by four claudin-15 molecules, which creates a negative electrostatic potential to favor cation flux over anion flux. Charge reversal or charge ablation mutations of D55 significantly reduce cation permeability in silico and in vitro, whereas mutations of other negatively charged pore amino acid residues have a significantly smaller impact on channel permeability and selectivity. The simulations also indicate that water and small ions can pass through the channel, but larger cations, such as tetramethylammonium, do not traverse the pore. Thus, our model provides an atomic view of claudin channels, their transport function, and a potential three-dimensional organization of its selectivity filter.

Claudin‐15 forms a water channel through the tight junction with distinct function compared to claudin‐2

“…Analysing the water permeability of the channel‐forming proteins claudins‐2, ‐10a, ‐10b and ‐17 revealed that only claudin‐2 is permeable to water, but neither claudin‐10b, although having a similar cation permeability as claudin‐2, nor claudin‐10a or ‐17 are water permeable [for review see]. A molecular dynamics simulation indicated that the claudin‐15 pore is permeable to water . Thus, this study analysed whether or not claudin‐15 acts as a water channel like claudin‐2.…”

“…Claudin‐15 was the first claudin of which a crystal structure was solved which was used as basis for molecular channel models of this and other claudins . In recent molecular dynamics (MD) simulation studies, the model of the claudin‐15 channel was refined. The simulations suggests that claudin‐15 forms channels selective for small cations.…”

Drinking and Water Handling in the Medaka Intestine: A Possible Role of Claudin-15 in Paracellular Absorption?

“…Amino acids are highlighted in red when acidic and in blue when basic. Arrows marks aspartic acids (D55 and D64), which are found to be important for cation and the water pore function of CLDN15 [11,12,15,16]. Mouse CLDN3 has been classified as a barrier protein and included as a reference.…”

“…Cldns are integral membrane proteins with 4 trans-membrane domains and two extracellular loops (ECL). The amino acid residues of the first ECL are critical for the permselectivity of the junction they create in homo-or hetero-dimeric and -tetrameric combinations [13][14][15][16]. There are 27 claudins (CLDNs) paralogs described in mammals [12]; in the teleost lineage, an extensive expansion of the cldn gene family due to gene duplications has led to a higher number with e.g., 56 in Fugu [17] and 54 in zebrafish [18].…”

“…Based on amino acid homology, especially in the first ECL, it is often assumed that fish Cldns give rise to the same permeability properties as mammalian orthologues, but only a few have been investigated thoroughly [21,22]. Mutational analysis and MD simulations [15,16] based on the crystal structure [23] have shown that especially amino acid D55 is critical for CLDN15 pore formation. In support for a similar function in medaka to the mammalian orthologue is the conservation of this amino acid in both medaka Cldn15a and Cldn15b.…”