We describe the development of multifunctional nanoparticle probes based on semiconductor quantum dots (QDs) for cancer targeting and imaging in living animals. The structural design involves encapsulating luminescent QDs with an ABC triblock copolymer and linking this amphiphilic polymer to tumor-targeting ligands and drug-delivery functionalities. In vivo targeting studies of human prostate cancer growing in nude mice indicate that the QD probes accumulate at tumors both by the enhanced permeability and retention of tumor sites and by antibody binding to cancer-specific cell surface biomarkers. Using both subcutaneous injection of QD-tagged cancer cells and systemic injection of multifunctional QD probes, we have achieved sensitive and multicolor fluorescence imaging of cancer cells under in vivo conditions. We have also integrated a whole-body macro-illumination system with wavelength-resolved spectral imaging for efficient background removal and precise delineation of weak spectral signatures. These results raise new possibilities for ultrasensitive and multiplexed imaging of molecular targets in vivo.

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Ca 2+-activated Cl − channels (CaCCs) are exceptionally well adapted to subserve diverse physiological roles, from epithelial fluid transport to sensory transduction, because their gating is cooperatively controlled by the interplay between ionotropic and metabotropic signals. A molecular understanding of the dual regulation of CaCCs by voltage and Ca 2+ has recently become possible with the discovery that Ano1 (TMEM16a) − channels (CaCCs) play manifold roles in cell physiology (1, 2), including epithelial secretion (3, 4), sensory transduction and adaptation (5-8), regulation of smooth muscle contraction (9), control of neuronal and cardiac excitability (10), and nociception (11). This myriad of functions has attracted attention for more than 25 years (12, 13), but a lack of consensus regarding their molecular composition has stymied a mechanistic understanding of their gating. Recently, two members of the TMEM16/anoctamin family (Ano1 and Ano2) were identified as CaCC channels (14-16) and shown to be essential for salivary exocrine secretion (14,17,18), gut slow-wave activity (18, 19), tracheal secretion (18,20,21), and olfactory transduction (5-7).Ano1 and Ano2 are well suited for their diverse roles because they are dually gated by voltage (V m ) and intracellular Ca 2+ concentration ([Ca 2+ ] i ), so that their activity is tuned by the interplay between metabotropic and ionotropic inputs (14,16,(22)(23)(24) and depolarization. In the absence of Ca 2+ , no current was evident at V m between −100 mV and +100 mV, but as [Ca 2+ ] i was increased, an outward current was activated by depolarization and deactivated by hyperpolarization (Fig. 1 A-D and F). As [Ca 2+ ] i was increased, outward rectification (Fig. 1F) and the fraction of total current exhibiting time-dependence (Fig. 1E) were reduced. V m -dependent activation of Ano1 was evaluated by plotting normalized conductance versus V m (G/G max vs. V m curves; Fig. 1G). The data were well fit by the Boltzmann equation,where G/G max is normalized conductance; z is the equivalent gating charge associated with voltage-dependent channel opening; V 0.5 is the membrane potential (V m ) where G/G max is halfmaximal and is related to the conformational energy associated with voltage-independent channel opening; and F/RT = 0.039 mV −1 . At 1 μM Ca 2+ , V 0.5 was 64 ± 0.9 mV (Fig. 1G, black squares); doubling [Ca 2+ ] to 2 μM shifted the G/G max vs. V m curve to the left by −145 mV (Fig. 1G, red circles) with no significant effect on z. Because Ca 2+ shifts the G/G max vs. V m curves so dramatically, a complete G/G max vs. V m curve could be recorded for only a narrow range of [Ca 2+ ] i . For these [Ca 2+ ], z was not obviously Ca 2+ -dependent (z = 0.40-0.46). This indicates that Ca 2+ does not change the V m sensitivity (z) of the Ano1 channel, but rather shifts V 0.5 , the energy associated with V m -independent gating.Although these data may imply that Ano1 is a simple ligandgated channel, Ano1 is actually more complicated, because Ca 2+ gating is st...

Rationale Ca2+-activated Cl channels (CaCCs) play pivotal roles in the cardiovascular system: they regulate vascular smooth muscle tone and participate in cardiac action potential repolarization in some species. CaCCs were recently discovered to be encoded by members of the Anoctamin (Ano, also called Tmem16) superfamily, but the mechanisms of Ano1 gating by Ca2+ remain enigmatic. Objective The objective was to identify regions of Ano1 involved in channel gating by Ca2+. Methods and results The Ca2+ sensitivity of Ano1 was estimated from rates of current activation and deactivation in excised patches rapidly switched between zero and high Ca2+ on the cytoplasmic side. Mutation of glutamates E702 and E705 dramatically altered Ca2+ sensitivity. E702 and E705 are predicted to be in an extracellular loop, but antigenic epitopes introduced into this loop are not accessible to extracellular antibodies, suggesting this loop is intracellular. Cytoplasmically-applied membrane-impermeant sulfhydryl reagents alter the Ca2+ sensitivity of Ano1 E702C and E705C, as expected if E702 and E705 are intracellular. Substituted cysteine accessibility mutagenesis of the putative re-entrant loop suggests that E702 and E705 are located adjacent to the Cl conduction pathway. Conclusions We propose an alternative model of Ano1 topology based on mutagenesis, epitope accessibility, and cysteine-scanning accessibility. These data contradict the popular re-entrant loop model by showing that the putative 4th extracellular loop (ECL 4) is intracellular and may contain a Ca2+ binding site. These studies provide new perspectives on regulation of Ano1 by Ca2+.

Temperature sensing is crucial for homeotherms, including human beings, to maintain a stable body core temperature and respond to the ambient environment. A group of exquisitely temperaturesensitive transient receptor potential channels, termed thermoTRPs, serve as cellular temperature sensors. How thermoTRPs convert thermal energy (heat) into protein conformational changes leading to channel opening remains unknown. Here we demonstrate that the pathway for temperature-dependent activation is distinct from those for ligand-and voltage-dependent activation and involves the pore turret. We found that mutant channels with an artificial pore turret sequence lose temperature sensitivity but maintain normal ligand responses. Using site-directed fluorescence recordings we observed that temperature change induces a significant rearrangement of TRPV1 pore turret that is coupled to channel opening. This movement is specifically associated to temperature-dependent activation and is not observed during ligand-and voltage-dependent channel activation. These observations suggest that the turret is part of the temperature-sensing apparatus in thermoTRP channels, and its conformational change may give rise to the large entropy that defines high temperature sensitivity.conformational change | fluorescence resonance energy transfer | temperature sensing | thermodynamics T emperature-sensitive transient receptor potential channels, or thermoTRPs, include four heat-activated channels (TRPV1-4) and two cold-activated channels (TRPM8 and TRPA1) that exhibit nicely spaced activation temperatures covering the physiological temperature range (1-4). These are expressed in dorsal root ganglion sensory neurons, keratinocytes, and other cells (1). Temperature changes cause rapid, reversible activation of thermoTRP channels in both native cells (3) and expression systems ( Fig. 1 A and B). Depolarizing currents through these nonselective, cationpermeable channels lead to the generation of action potentials (5) or the release of messenger molecules (6, 7) that encodes temperature information. Structurally, thermoTRPs resemble voltagedependent potassium channels, with four subunits surrounding a central ion permeation pore (4). Each subunit contains six transmembrane segments (S1-S6) and long intracellular N and C termini. The channel pore is formed by S6 and a P-loop that in most thermoTRPs is noticeably longer than those of KcsA and voltagegated potassium channels (8, 9). Unique TRP channel structural elements, such as TRP Box immediately after S6 and N-terminal ankyrin repeats, are found in most thermoTRPs.Despite extensive research, the channel structure bestowing high temperature sensitivity on thermoTRPs remains elusive. ThermoTRP channels are polymodal sensors responsive to a wide range of physical and chemical stimuli, such as transmembrane voltage, ligands, and pH. It has been proposed that heat might control thermoTRP activation by shifting the channel's response to these stimuli (10, 11). Alternatively, synergistic activation by mul...