Marcelo Catalán

Basolateral localization of native ClC-2 chloride channels in absorptive intestinal epithelial cells and basolateral sorting encoded by a CBS-2 domain di-leucine motif

The Cl– channel ClC-2 is expressed in transporting epithelia and has been proposed as an alternative route for Cl– efflux that might compensate for the malfunction of CFTR in cystic fibrosis. There is controversy concerning the cellular and membrane location of ClC-2, particularly in intestinal tissue. The aim of this paper is to resolve this controversy by immunolocalization studies using tissues from ClC-2 knockout animals as control, ascertaining the sorting of ClC-2 in model epithelial cells and exploring the possible molecular signals involved in ClC-2 targeting. ClC-2 was exclusively localized at the basolateral membranes of surface colonic cells or villus duodenal enterocytes. ClC-2 was sorted to the basolateral membranes in MDCK, Caco-2 and LLC-PK1-μ1B, but not in LLC-PK1-μ1A cells. Mutating a di-leucine motif (L812L813) to a di-alanine changed the basolateral targeting of ClC-2 to an apical location. The basolateral membrane localization of ClC-2 in absorptive cells of the duodenum and the colon is compatible with an absorptive function for this Cl– channel. Basolateral targeting information is contained in a di-leucine motif (L812L813) within CBS-2 domain at the C-terminus of ClC-2. It is speculated that ClC-2 also contains an apical sorting signal masked by L812L813. The proposal that CBS domains in ClC channels might behave as regulatory sites sensing intracellular signals opens an opportunity for pharmacological modulation of ClC-2 targeting.

The voltage‐dependent ClC‐2 chloride channel has a dual gating mechanism

Functional and structural studies demonstrate that Cl− channels of the ClC family have a dimeric double‐barrelled structure, with each monomer contributing an identical pore. Single protopore gating is a fast process dependent on Cl− interaction within the selectivity filter and in ClC‐0 has a low temperature coefficient over a 10°C range (Q10). A slow gating process closes both protopores simultaneously, has a high Q10, is facilitated by extracellular Zn2+ and Cd2+ and is abolished or markedly reduced by mutation of a cysteine conserved in ClC‐0, ‐1 and ‐2. In order to test the hypothesis that similar slow and fast gates exist in the widely expressed ClC‐2 Cl− channel we have investigated the effects of these manoeuvres on ClC‐2. We find that the time constants of both components of the double‐exponential hyperpolarization‐dependent activation (and deactivation) processes have a high temperature dependence, with Q10 values of about 4–5, suggesting important conformational changes of the channel. Mutating C256 (equivalent to C212 in ClC‐0) to A, led to a significant fraction of constitutively open channels at all potentials. Activation time constants were not affected but deactivation was slower and significantly less temperature dependent in the C256A mutant. Extracellular Cd2+, that inhibits wild‐type (WT) channels almost fully, inhibited C256A only by 50%. In the WT, the time constants for opening were not affected by Cd2+ but deactivation at positive potentials was accelerated by Cd2+. This effect was absent in the C256A mutant. The effect of intracellular Cl− on channel activation was unchanged in the C256A mutant. Collectively our results strongly support the hypothesis that ClC‐2 possesses a common gate and that part of the current increase induced by hyperpolarization represents an opening of the common gate. In contrast to the gating in ClC‐0, the protopore gate and the common gate of ClC‐2 do not appear to be independent.

A conserved pore-lining glutamate as a voltage- and chloride-dependent gate in the ClC-2 chloride channel

ClC‐2 is a ubiquitously expressed, two‐pore homodimeric Cl− channel opened by hyperpolarisation. Little is known about its gating mechanisms. Crystallographic and functional studies in other ClC channels suggest that a conserved glutamate residue carboxylate side‐chain can close protopores by interacting with a Cl−‐binding site in the pore. Competition for this site is thought to provide the molecular basis for gating by extracellular Cl−. We now show that ClC‐2 gating depends upon intra‐ but not extracellular Cl− and that neutralisation of E217, the homologous pore glutamate, leads to loss of sensitivity to intracellular Cl− and voltage. Experiments testing for transient activation by extracellular protons demonstrate that E217 is not available for protonation in the closed channel state but becomes so after opening by hyperpolarisation. The results suggest that E217 is a hyperpolarisation‐dependent protopore gate in ClC‐2 and that access of intracellular Cl− to a site normally occupied by its side‐chain in the pore stabilises the open state. A remaining hyperpolarisation‐dependent gate might correspond to that closing both pores simultaneously in other ClC channels.

Purinergic P2X7 Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP ≫ ATP > ADP ≫ UTP, and removal of external Ca2+ dramatically suppressed the initial ATP-induced fluid secretion (∼85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X7 gene were used to evaluate the role of the P2X7 receptor in nucleotide-evoked fluid secretion. P2X7 receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X7-null mice. Consistent with these observations, the ATP-induced increases in [Ca2+]i were nearly abolished in P2X7–/– submandibular acinar and duct cells. ATP appeared to also act through the P2X7 receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X7 receptor regulates fluid secretion in the mouse submandibular gland.

Removal of gating in voltage-dependent ClC-2 chloride channel by point mutations affecting the pore and C-terminus CBS-2 domain

Functional and structural studies demonstrate that Cl− channels of the ClC family have a dimeric double‐barrelled structure, with each monomer contributing an identical pore. Studies with ClC‐0, the prototype ClC channel, show the presence of independent mechanisms gating the individual pores or both pores simultaneously. A single‐point mutation in the CBS‐2 domain of ClC‐0 has been shown to abolish slow gating. We have taken advantage of the high conservation of CBS domains in ClC channels to test for the presence of a slow gate in ClC‐2 by reproducing this mutation (H811A). ClC‐2‐H811A showed faster opening kinetics and opened at more positive potentials than ClC‐2. There was no difference in [Cl−]i dependence. Additional neutralization of a putative pore gate glutamate side chain (E207V) abolished all gating. Resolving slow and fast gating relaxations, however, revealed that the H811A mutation affected both fast and slow gating processes in ClC‐2. This suggests that slow and fast gating in ClC‐2 are coupled, perhaps with slow gating contributing to the operation of the pore E207 as a protopore gate.

Clcn2 encodes the hyperpolarization-activated chloride channel in the ducts of mouse salivary glands

Transepithelial Cl(-) transport in salivary gland ducts is a major component of the ion reabsorption process, the final stage of saliva production. It was previously demonstrated that a Cl(-) current with the biophysical properties of ClC-2 channels dominates the Cl(-) conductance of unstimulated granular duct cells in the mouse submandibular gland. This inward-rectifying Cl(-) current is activated by hyperpolarization and elevated intracellular Cl(-) concentration. Here we show that ClC-2 immunolocalized to the basolateral region of acinar and duct cells in mouse salivary glands, whereas its expression was most robust in granular and striated duct cells. Consistent with this observation, nearly 10-fold larger ClC-2-like currents were observed in granular duct cells than the acinar cells obtained from submandibular glands. The loss of inward-rectifying Cl(-) current in cells from Clcn2(-/-) mice confirmed the molecular identity of the channel responsible for these currents as ClC-2. Nevertheless, both in vivo and ex vivo fluid secretion assays failed to identify significant changes in the ion composition, osmolality, or salivary flow rate of Clcn2(-/-) mice. Additionally, neither a compensatory increase in Cftr Cl(-) channel protein expression nor in Cftr-like Cl(-) currents were detected in Clcn2 null mice, nor did it appear that ClC-2 was important for blood-organ barrier function. We conclude that ClC-2 is the inward-rectifying Cl(-) channel in duct cells, but its expression is not apparently required for the ion reabsorption or the barrier function of salivary ductal epithelium.

Salivary Gland Secretion

The principal function of mammalian salivary glands is to secrete a protein-rich, watery fluid into the oral cavity. Saliva contains a large repertoire of biologically active molecules that facilitate speech, swallowing, digestion, and taste, and preserve the integrity of the hard and soft tissues. Saliva is secreted by three major pairs of glands (parotid, submandibular, and sublingual) and numerous minor salivary glands located throughout the oral cavity. The fluid secreted by the different salivary glands differs in composition. Parotid glands secrete a watery saliva rich in proteins such as amylase, while the sublingual glands secrete a viscous mucin-containing fluid. The consistency and composition of the fluid secreted by the submandibular glands is intermediate to the fluid secreted by the parotid and sublingual glands. Although the compositions of the major salivary gland secretions differ, the molecular mechanisms by which these glands secrete are conserved. The focus of this chapter is to review the cellular mechanisms involved in fluid, electrolyte, and protein secretion by salivary glands.

The Insensitivity of TASK-3 K2P Channels to External Tetraethylammonium (TEA) Partially Depends on the Cap Structure

Two-pore domain K+ channels (K2P) display a characteristic extracellular cap structure formed by two M1-P1 linkers, the functional role of which is poorly understood. It has been proposed that the presence of the cap explains the insensitivity of K2P channels to several K+ channel blockers including tetraethylammonium (TEA). We have explored this hypothesis using mutagenesis and functional analysis, followed by molecular simulations. Our results show that the deletion of the cap structure of TASK-3 (TWIK-related acid-sensitive K+ channel) generates a TEA-sensitive channel with an IC50 of 11.8 ± 0.4 mM. The enhanced sensitivity to TEA displayed by the cap-less channel is also explained by the presence of an extra tyrosine residue at position 99. These results were corroborated by molecular simulation analysis, which shows an increased stability in the binding of TEA to the cap-less channel when a ring of four tyrosine is present at the external entrance of the permeation pathway. Consistently, Y99A or Y205A single-residue mutants generated in a cap-less channel backbone resulted in TASK-3 channels with low affinity to external TEA.