Chloride absorption and bicarbonate secretion are vital functions of epithelia, as highlighted by cystic fibrosis and diseases associated with mutations in members of the SLC26 chloride-bicarbonate exchangers. Many SLC26 transporters (SLC26T) are expressed in the luminal membrane together with CFTR, which activates electrogenic chloride-bicarbonate exchange by SLC26T. However, the ability of SLC26T to regulate CFTR and the molecular mechanism of their interaction are not known. We report here a reciprocal regulatory interaction between the SLC26T DRA, SLC26A6 and CFTR. DRA markedly activates CFTR by increasing its overall open probablity (NP(o)) sixfold. Activation of CFTR by DRA was facilitated by their PDZ ligands and binding of the SLC26T STAS domain to the CFTR R domain. Binding of the STAS and R domains is regulated by PKA-mediated phosphorylation of the R domain. Notably, CFTR and SLC26T co-localize in the luminal membrane and recombinant STAS domain activates CFTR in native duct cells. These findings provide a new understanding of epithelial chloride and bicarbonate transport and may have important implications for both cystic fibrosis and diseases associated with SLC26T.

PPT1 and PPT2 encode two lysosomal thioesterases that catalyze the hydrolysis of long chain fatty acyl CoAs. In addition to this function, PPT1 (palmitoyl-protein thioesterase 1) hydrolyzes fatty acids from modified cysteine residues in proteins that are undergoing degradation in the lysosome. PPT1 deficiency in humans causes a neurodegenerative disorder, infantile neuronal ceroid lipofuscinosis (also known as infantile Batten disease). In the current work, we engineered disruptions in the PPT1 and PPT2 genes to create ''knockout'' mice that were deficient in either enzyme. Both lines of mice were viable and fertile. However, both lines developed spasticity (a ''clasping'' phenotype) at a median age of 21 wk and 29 wk, respectively. Motor abnormalities progressed in the PPT1 knockout mice, leading to death by 10 mo of age. In contrast, the majority of PPT2 mice were alive at 12 mo. Myoclonic jerking and seizures were prominent in the PPT1 mice. Autofluorescent storage material was striking throughout the brains of both strains of mice. Neuronal loss and apoptosis were particularly prominent in PPT1-deficient brains. These studies provide a mouse model for infantile neuronal ceroid lipofuscinosis and further suggest that PPT2 serves a role in the brain that is not carried out by PPT1.

Mucolipidosis type IV (MLIV) is caused by mutations in the ion channel mucolipin 1 (TRP-ML1). MLIV is typified by accumulation of lipids and membranous materials in intracellular organelles

TRPML3 is an inward rectifying Ca 2+ channel that is regulated by extracytosolic H + . Although gain-of-function mutation in TRPML3 causes the varitint-waddler phenotype, the role of TRPML3 in cellular physiology is not known. In this study, we report that TRPML3 is a prominent regulator of endocytosis, membrane trafficking and autophagy. Gradient fractionation and confocal localization reveal that TRPML3 is expressed in the plasma membrane and multiple intracellular compartments. However, expression of TRPML3 is dynamic, with accumulation of TRPML3 in the plasma membrane upon inhibition of endocytosis, and recruitment of TRPML3 to autophagosomes upon induction of autophagy. Accordingly, overexpression of TRPML3 leads to reduced constitutive and regulated endocytosis, increased autophagy and marked exacerbation of autophagy evoked by various cell stressors with nearly complete recruitment of TRPML3 into the autophagosomes. Importantly, both knockdown of TRPML3 by siRNA and expression of the channel-dead dominant negative TRPML3(D458K) have a reciprocal effect, reducing endocytosis and autophagy. These findings reveal a prominent role for TRPML3 in regulating endocytosis, membrane trafficking and autophagy, perhaps by controlling the Ca 2+ in the vicinity of cellular organelles that is necessary to regulate these cellular events. TRPML3 is a member of the TRPML subfamily of the transient receptor potential (TRP) channel superfamily (1). The TRPML subfamily was established with identification of TRPML1 as the protein that is mutated in the lysosomal storage disease mucolipidosis type IV (MLIV) (2). Subsequently, TRPML3 was found by positional cloning as the channel mutated in the mouse varitint-waddler phenotype (3), and TRPML2 was identified by database searches (1). The varitint-waddler phenotype is caused by the gain-of-function mutation A419P in TRPML3 (4-8).Members of the TRPML subfamily share the same basic structure of six transmembrane domains, a pore domain between transmembrane domains 5 and 6 and a unique large extracytosolic loop between transmembrane domains 1 and 2.TRPML1 is a lysosomal channel (9) that functions to regulate lysosomal pH (10,11) and thereby membrane trafficking (11,12). TRPML1 was suggested to regulate interaction between late endosomes and lysosomes to control delivery of cellular material to lysosomes (12). However, a recent work used knockdown (KD) of TRPML1 to show that absence of TRPML1 causes loss of protein and lipid hydrolytic activity that led to lysosomal dysfunction (11). Lysosomal dysfunction appears to be associated with increased autophagy (13). TRPML1 can function as a Ca 2+ channel (4). A new potential role for TRPML1 is to be the channel activated by the Ca Much less is known about the localization and function of TRPML3. It was suggested that TRPML3 localizes in the endoplasmic reticulum (ER) when expressed alone, but to be escorted to the lysosomes when coexpressed with TRPML1 or TRPML2 (16). However, the native TRPML3 is expressed in intracellular vesicular compar...

Mutations in the gene Mucolipidosis type IV (MLIV)2 is a lipid storage disorder characterized by an abnormal accumulation of membranous lipids in patients' cells (reviewed in Refs. 1 and 2). Clinically, the disease manifests as corneal clouding, degeneration of the retina, and severe psychomotor retardation (1-6). MLIV is associated with mutations in MCOLN1 (TRP-ML1), a member of the TRP (transient receptor potential) family of ion channels (7-9). The TRP family includes several members that are implicated in human diseases, such as TRPP2 (10), TRPM1 (11), and TRPV6 (12). A critical question in MLIV pathogenesis is why do mutations in TRP-ML1 lead to the cellular phenotype of MLIV?Previous work on the ion selectivity and permeation of TRP-ML1 produced conflicting results. Thus, transient expression in Xenopus oocytes and in fibroblasts suggests that TRP-ML1 is targeted to the lysosomes and functions as a Ca 2ϩ -permeable channel that may regulate lysosomal Ca 2ϩ release and consequently agonist-evoked Ca 2ϩ signals (13,14). On the other hand, TRP-ML1 synthesized in cell-free system and reconstituted into planar lipid bilayers behaves as a monovalent cations permeable, outwardly rectifying channel (15). The outward rectification indicates that when present in lysosomes, TRP-ML1 primarily moves ions into the lysosomal lumen. The outward rectification makes it unlikely that in vivo TRP-ML1 would function as a lysosomal Ca 2ϩ release channel, which suggested an alternative role of TRP-ML1 in lysosomal and cellular functions.In the present report we analyzed the expression pattern and channel properties of TRP-ML1 and several disease-associated mutants. We report that TRP-ML1 is an outwardly rectifying monovalent cationpermeable channel that is primarily expressed in the lysosomes. In the lysosomes, TRP-ML1 is inactivated by proteolytic cleavage. These findings suggest a novel mechanism of regulating TRP-ML1 function.

Unexpectedly, deletion of slc26a6 in mice and measurement of fluid and HCO 3 À secretion into sealed intralobular pancreatic ducts revealed that deletion of slc26a6 enhanced spontaneous and decreased stimulated secretion. Remarkably, inhibition of CFTR activity with CFTR inh -172, knock-down of CFTR by siRNA and measurement of CFTR current in WT and slc26a6 À/À duct cells revealed that deletion of slc26a6 resulted in dis-regulation of CFTR activity by removal of tonic inhibition of CFTR by slc26a6. These findings reveal the intricate regulation of CFTR activity by slc26a6 in both the resting and stimulated states and the essential role of slc26a6 in pancreatic HCO 3 À secretion in vivo.

TRPML3 belongs to the TRPML subfamily of the transient receptor potential (TRP) channels. The A419P mutation in TRPML3 causes the varitint-waddler phenotype as a result of gain-of-function mutation (GOF). Regulation of the channels and the mechanism by which the A419P mutation leads to GOF are not known. We report here that TRPML3 is a Ca 2 þ -permeable channel with a unique form of regulation by extracytosolic (luminal) H þ (H þ e-cyto ). Regulation by H þ e-cyto is mediated by a string of three histidines (H252, H273, H283) in the large extracytosolic loop between transmembrane domains (TMD) 1 and 2. Each of the histidines has a unique role, whereby H252 and H273 retard access of H þ e-cyto to the inhibitory H283. Notably, the H283A mutation has the same phenotype as A419P and locks the channel in an open state, whereas the H283R mutation inactivates the channel. Accordingly, A419P eliminates regulation of TRPML3 by H þ e-cyto , and confers full activation to TRPML3(H283R). Activation of TRPML3 and regulation by H þ e-cyto are altered by both the a-helix-destabilizing A419G and the a-helixfavouring A419M and A419K. These findings suggest that regulation of TRPML3 by H þ e-cyto is due to an effect of the large extracytosolic loop on the orientation of fifth TMD and thus pore opening and show that the GOF of TRPML3(A419P) is due to disruption of this communication.

Signalling by G proteins is controlled by the regulator of G-protein signalling (RGS) proteins that accelerate the GTPase activity of Galpha subunits and act in a G-protein-coupled receptor (GPCR)-specific manner. The conserved RGS domain accelerates the G subunit GTPase activity, whereas the variable amino-terminal domain participates in GPCR recognition. How receptor recognition is achieved is not known. Here, we show that the scaffold protein spinophilin (SPL), which binds the third intracellular loop (3iL) of several GPCRs, binds the N-terminal domain of RGS2. SPL also binds RGS1, RGS4, RGS16 and GAIP. When expressed in Xenopus laevis oocytes, SPL markedly increased inhibition of alpha-adrenergic receptor (alphaAR) Ca2+ signalling by RGS2. Notably, the constitutively active mutant alphaAR(A293E) (the mutation being in the 3iL) did not bind SPL and was relatively resistant to inhibition by RGS2. Use of betaAR-alphaAR chimaeras identified the 288REKKAA293 sequence as essential for the binding of SPL and inhibition of Ca2+ signalling by RGS2. Furthermore, alphaAR-evoked Ca2+ signalling is less sensitive to inhibition by SPL in rgs2-/- cells and less sensitive to inhibition by RGS2 in spl-/- cells. These findings provide a general mechanism by which RGS proteins recognize GPCRs to confer signalling specificity.