Basal tearing is crucial to maintaining ocular surface wetness. Corneal cold thermoreceptors sense small oscillations in ambient temperature and change their discharge accordingly. Deletion of the cold-transducing ion channel Transient receptor potential cation channel subfamily M member 8 (TRPM8) in mice abrogates cold responsiveness and reduces basal tearing without affecting nociceptor-mediated irritative tearing. Warming of the cornea in humans also decreases tearing rate. These findings indicate that TRPM8-dependent impulse activity in corneal cold receptors contributes to regulating basal tear flow.

Null trk1 trk2 mutants of Saccharomyces cerevisiae exhibit a low-affinity uptake of K ؉ and Rb ؉ . We show that this low-affinity Rb ؉ uptake is mediated by several independent transporters, and that trk1⌬ cells and especially trk1⌬ trk2⌬ cells are highly hyperpolarized. Differences in the membrane potentials were assessed for sensitivity to hygromycin B and by flow cytometric analyses of cellular DiOC 6 (3) fluorescence. On the basis of the latter analyses, it is proposed that Trk1p and Trk2p are involved in the control of the membrane potential, preventing excessive hyperpolarizations. K ؉ starvation and nitrogen starvation hyperpolarize both TRK1 TRK2 and trk1⌬ trk2⌬ cells, thus suggesting that other proteins, in addition to Trk1p and Trk2p, participate in the control of the membrane potential. The HAK1 K ؉ transporter from Schwanniomyces occidentalis suppresses the K ؉ -defective transport of trk1⌬ trk2⌬ cells but not the high hyperpolarization, and the HKT1 K ؉ transporter from wheat suppresses both defects, in the presence of Na ؉ . We discuss the mechanism involved in the control of the membrane potential by Trk1p and Trk2p and the causal relationship between the high membrane potential (negative inside) of trk1⌬ trk2⌬ cells and its ectopic transport of alkali cations.

Molecular determinants of threshold differences among cold thermoreceptors are unknown. Here we show that such differences correlate with the relative expression of I KD , a current dependent on Shaker-like Kv1 channels that acts as an excitability brake, and I TRPM8 , a cold-activated excitatory current. Neurons responding to small temperature changes have high functional expression of TRPM8 (transient receptor potential cation channel, subfamily M, member 8) and low expression of I KD . In contrast, neurons activated by lower temperatures have a lower expression of TRPM8 and a prominent I KD . Otherwise, both subpopulations have nearly identical membrane and firing properties, suggesting that they belong to the same neuronal pool. Blockade of I KD shifts the threshold of cold-sensitive neurons to higher temperatures and augments cold-evoked nocifensive responses in mice. Similar behavioral effects of I KD blockade were observed in TRPA1 Ϫ/Ϫ mice. Moreover, only a small percentage of trigeminal cold-sensitive neurons were activated by TRPA1 agonists, suggesting that TRPA1 does not play a major role in the detection of low temperatures by uninjured somatic cold-specific thermosensory neurons under physiological conditions. Collectively, these findings suggest that innocuous cooling sensations and cold discomfort are signaled by specific low-and high-threshold cold thermoreceptor neurons, differing primarily in their relative expression of two ion channels having antagonistic effects on neuronal excitability. Thus, although TRPM8 appears to function as a critical cold sensor in the majority of peripheral sensory neurons, the expression of Kv1 channels in the same terminals seem to play an important role in the peripheral gating of cold-evoked discomfort and pain.

Transient receptor potential melastatin 8 (TRPM8) is the best molecular candidate for innocuous cold detection by peripheral thermoreceptor terminals. To dissect out the contribution of this cold-and menthol-gated, nonselective cation channel to cold transduction, we identified BCTC [N-(4-tert-butylphenyl)-4-(3-chloropyridin-2-yl)piperazine-1-carboxamide] as a potent and full blocker of recombinant TRPM8 channels. In cold-sensitive trigeminal ganglion neurons of mice and guinea pig, responses to menthol were abolished by BCTC. In contrast, the effect of BCTC on cold-evoked responses was variable but showed a good correlation with the presence or lack of menthol sensitivity in the same neuron, suggesting a specific blocking action of BCTC on TRPM8 channels. The biophysical properties of native cold-gated currents (I cold ), and the currents blocked by BCTC were nearly identical, consistent with a role of this channel in cold sensing at the soma. The temperature activation threshold of native TRPM8 channels was significantly warmer than those reported in previous expression studies. The effect of BCTC on native I cold was characterized by a dose-dependent shift in the temperature threshold of activation.The role of TRPM8 in transduction was further investigated in the guinea pig cornea, a peripheral territory densely innervated with cold thermoreceptors. All cold-sensitive terminals were activated by menthol, suggesting the functional expression of TRPM8 channels in their membrane. However, the spontaneous activity and firing pattern characteristic of cold thermoreceptors was totally immune to TRPM8 channel blockade with BCTC or SKF96365 (1-[2-(4-methoxyphenyl)-2-[3-(4-methoxyphenyl)propoxy]ethyl-1 H-imidazole hydrochloride). Cold-evoked responses in corneal terminals were also essentially unaffected by these drugs, whereas responses to menthol were completely abolished. The minor impairment in the ability to transduce cold stimuli by peripheral corneal thermoreceptors during TRPM8 blockade unveils an overlapping functional role for various thermosensitive mechanisms in these nerve terminals.

human immunodeficiency virus type 1 (HIV-1) Nef interacts with the clathrin-associated AP-1 and AP-3 adaptor complexes, stabilizing their association with endosomal membranes. These findings led us to hypothesize a general impact of this viral protein on the endosomal system. Here, we have shown that Nef specifically disturbs the morphology of the early/recycling compartment, inducing a redistribution of early endosomal markers and a shortening of the tubular recycling endosomal structures. Furthermore, Nef modulates the trafficking of the transferrin receptor (TfR), the prototypical recycling surface protein, indicating that it also disturbs the function of this compartment. Nef reduces the rate of recycling of TfR to the plasma membrane, causing TfR to accumulate in early endosomes and reducing its expression at the cell surface. These effects depend on the leucine-based motif of Nef, which is required for the membrane stabilization of AP-1 and AP-3 complexes. Since we show that this motif is also required for the full infectivity of HIV-1 virions, these results indicate that the positive influence of Nef on viral infectivity may be related to its general effects on early/recycling endosomal compartments.Trafficking of membrane proteins is governed by a regulated machinery that involves the vesicular transport of proteins throughout different intracellular compartments. One major regulatory mechanism is related to the function of the adaptor protein (AP) 1 complexes that assemble on donor membranes of the endocytic pathway to form transport vesicles (for review, see Ref. 1). The sorting of transmembrane proteins into these vesicles requires the recognition by the AP complexes of specific tyrosine-or leucine-based motifs contained within the cytoplasmic domains of cargo proteins (2). Four different types of heterotetrameric AP complexes (AP-1-AP-4) have been identified (3). AP-2 is specifically involved in the formation of clathrincoated vesicles at the plasma membrane, whereas AP-1 and AP-3 mediate the formation of clathrin-coated vesicles at the levels of the trans-Golgi network (TGN) and endosomes. The function of AP-4 is less well documented, but it regulates formation of non-clathrin-coated vesicles at the TGN. The association of the AP-1, AP-3, and AP-4 complexes with TGN and endosomal membranes is regulated by ADP-ribosylation factor 1 (ARF1). The Nef protein of HIV-1 is a 27-kDa protein that associates with the cell membranes through N-terminal myristoylation and is abundantly produced shortly after virus infection (for review, see Refs. 4 and 5). Nef is an essential factor in vivo for efficient viral replication and pathogenesis. In vitro, Nef also facilitates virus replication and enhances the infectivity of virions. Although the positive influence of Nef on viral replication and infectivity may be multifactorial, genetic evidence suggests a relationship between these virological effects and the ability of Nef to modulate the cell surface expression of multiple membrane-associated proteins. In additio...

R.Madrid and S.Le Maout contributed equally to this workAquaporin 4 (AQP4) is the predominant water channel in the brain. It is targeted to speci®c membrane domains of astrocytes and plays a crucial role in cerebral water balance in response to brain edema formation. AQP4 is also speci®cally expressed in the basolateral membranes of epithelial cells. However, the molecular mechanisms involved in its polarized targeting and membrane traf®cking remain largely unknown. Here, we show that two independent C-terminal signals determine AQP4 basolateral membrane targeting in epithelial MDCK cells. One signal involves a tyrosine-based motif; the other is encoded by a di-leucine-like motif. We found that the tyrosinebased basolateral sorting signal also determines AQP4 clathrin-dependent endocytosis through direct interaction with the m subunit of AP2 adaptor complex. Once endocytosed, a regulated switch in m subunit interaction changes AP2 adaptor association to AP3. We found that the stress-induced kinase casein kinase (CK)II phosphorylates the Ser276 immediately preceding the tyrosine motif, increasing AQP4±m3A interaction and enhancing AQP4±lysosomal targeting and degradation. AQP4 phosphorylation by CKII may thus provide a mechanism that regulates AQP4 cell surface expression. Keywords: adaptor protein complex/AQP4/ phosphorylation/protein sorting/regulated membrane traf®cking IntroductionAquaporin water channels are essential for mediating rapid osmotic water transport across cell membranes. The product of at least one of the 10 distinct mammalian aquaporin genes has been identi®ed in nearly all tissues, re¯ecting the fundamental nature of water transport processes. The ubiquitous character of aquaporins is associated with remarkably conserved structural features. Structural and functional analyses have revealed that aquaporins are homomultimeric complexes containing four identical subunits. All the aquaporin subunits identi®ed to date have N-and C-termini facing the cytosol, and contain six transmembrane domains that are connected by ®ve loops (Deen and van Os, 1998;Engel et al., 2000). Multiple aquaporins are often expressed in a single cell on different subcellular membrane domains where they are subjected to distinct regulatory cues. Although the phenomenon underlies a plethora of physiologically relevant water transport processes, little is known about the molecular mechanisms that govern the membrane targeting and expression of aquaporin channels.Consider, for example, the epithelial renal collecting duct principal cell and the cellular basis for the regulation of water balance. These cells express at least three different types of aquaporins in a polarized fashion. The vasopressin-regulated aquaporin 2 (AQP2) is speci®cally targeted to the apical cell domain. The hormone induces insertion of a vesicular pool of AQP2 channels in the apical membranes to allow ef®cient transcellular osmotic water re-absorption to occur in accord with physiological demands (Brown et al., 1988). The two other water channels, AQP3 ...

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. Mutant SOD1-expressing astrocytes release toxic factors that trigger motoneuron death by inducing hyperexcitability.