Abstract
Claudins form paracellular pores at the tight junction in epithelial cells. Profound depletion of extracellular calcium is well known to cause loosening of the tight junction with loss of transepithelial resistance. However, moderate variations in calcium concentrations within the physiological range can also regulate transepithelial permeability. To investigate the underlying molecular mechanisms, we studied the effects of calcium on the permeability of claudin-2, expressed in an inducible MDCK I cell line. We found that in the physiological range, calcium acts as a reversible inhibitor of the total conductance and Na(+) permeability of claudin-2, without causing changes in tight junction structure. The effect of calcium is enhanced at low Na(+) concentrations, consistent with a competitive effect. Furthermore, mutation of an intrapore negatively charged binding site, Asp-65, to asparagine partially abrogated the inhibitory effect of calcium. This suggests that calcium competes with Na(+) for binding to Asp-65. Other polyvalent cations had similar effects, including La(3+), which caused severe and irreversible inhibition of conductance. Brownian dynamics simulations demonstrated that such inhibition can be explained if Asp-65 has a relatively high charge density, thus favoring binding of Ca(2+) over that of Na(+), reducing Ca(2+) permeation by inhibiting its dissociation from this site, and decreasing Na(+) conductance through repulsive electrostatic interaction with Ca(2+). These findings may explain why hypercalcemia inhibits Na(+) reabsorption in the proximal tubule of the kidney.
Highlights
Claudins are four transmembrane domain proteins located at the tight junction between epithelial cells [12, 13]
We show that variations in extracellular calcium concentration within the physiological range regulate claudin-2 conductance and Naϩ permeability
Brownian Dynamics Modeling—Recently we developed a Brownian dynamics (BD) model to investigate the ion permeation characteristics and charge selectivity of different monovalent alkali solutions in claudin-2
Summary
Tissue Culture and Electrophysiological Studies—The generation and maintenance of MDCK I TetOff cells stably expressing the wild type and the D65N mutant of claudin-2 were as reported previously [19]. For measurements of Naϩ permeability (Fig. 2), the 2:1 NaCl dilution potential was measured by exchanging the basolateral solution with Ringer solution containing 70 mM NaCl (with mannitol added to balance the osmolality). This was corrected for the difference in liquid junction potentials between the basolateral and apical pipettes using the method described previously (see Ref. 19, supplemental material therein). Bulk solutions with different Naϩ and Ca2ϩ concentrations were treated in the BD simulations by distributing fixed numbers of ions within the boundary buffer regions at each Monte Carlo cycle. A transmembrane potential of Ϫ60 mV was applied to calculate the channel conductance and mobile ion density profiles
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