giant-liposome-preparation-for-imaging-patch-clamp

Giant Liposome Preparation for Imaging and Patch-Clamp Electrophysiology

The reconstitution of ion channels into chemically defined lipid membranes for electrophysiological recording has been a powerful technique to identify ...

In cyclic nucleotide-gated (CNG) ion channels, binding of cGMP or cAMP drives a conformational change that leads to opening of an ion-conducting pore. One region implicated in the coupling of ligand binding to opening of the pore is the C linker region. Here, we used crosslinking of endogenous cysteines to study interregion proximity. We demonstrate that an individual amino acid--C481--in the C linker region of each of two neighboring subunits can form a disulfide bond. Further, using tandem dimers, we show that a disulfide bond between C35 in the N-terminal region and C481 in the C linker region can form either within a subunit or between subunits. From our data on proximity between individual amino acids and previous studies, a picture emerges of the C linker as a potential dimerization interface.

Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels.

The capsaicin (vanilloid) receptor, VR1, is an agonist-activated ion channel expressed by sensory neurons that serves as a detector of chemical and thermal noxious stimuli.

Subunit modification and association in VR1 ion channels.

Cyclic nucleotide-gated (CNG) channels have been shown to be blocked by diltiazem, tetracaine, polyamines, toxins, divalent cations, and other compounds. Dequalinium is an organic divalent cation which suppresses the rat small conductance Ca(2+)-activated K(+) channel 2 (rSK2) and the activity of protein kinase C. In this study, we have tested the ability of dequalinium to block CNGA1 channels and heteromeric CNGA1+CNGB1 channels. When applied to the intracellular side of inside-out excised patches from Xenopus oocytes, dequalinium blocks CNGA1 channels with a K(1/2) approximately 190 nM and CNGA1+CNGB1 channels with a K(1/2) approximately 385 nM, at 0 mV. This block occurs in a state-independent fashion, and is voltage dependent with a zdelta approximately 1. Our data also demonstrate that dequalinium interacts with the permeant ion probably because it occupies a binding site in the ion conducting pathway. Dequalinium applied to the extracellular surface also produced block, but with a voltage dependence that suggests it crosses the membrane to block from the inside. We also show that at the single-channel level, dequalinium is a slow blocker that does not change the unitary conductance of CNGA1 channels. Thus, dequalinium should be a useful tool for studying permeation and gating properties of CNG channels.

Dequalinium: a novel, high-affinity blocker of CNGA1 channels.

TRPV1 ion channels mediate the response to painful heat, extracellular acidosis, and capsaicin, the pungent extract from plants in the Capsicum family (hot chili peppers) (Szallasi, A., and P.M. Blumberg. 1999. Pharmacol. Rev. 51:159-212; Caterina, M.J., and D. Julius. 2001. Annu. Rev. Neurosci. 24:487-517). The convergence of these stimuli on TRPV1 channels expressed in peripheral sensory nerves underlies the common perceptual experience of pain due to hot temperatures, tissue damage and exposure to capsaicin. TRPV1 channels are nonselective cation channels (Caterina, M.J., M.A. Schumacher, M. Tominaga, T.A. Rosen, J.D. Levine, and D. Julius. 1997. Nature. 389:816-824). When activated, they produce depolarization through the influx of Na+, but their high Ca2+ permeability is also important for mediating the response to pain. In particular, Ca2+ influx is thought to be required for the desensitization to painful sensations over time (Cholewinski, A., G.M. Burgess, and S. Bevan. 1993. Neuroscience. 55:1015-1023; Koplas, P.A., R.L. Rosenberg, and G.S. Oxford. 1997. J. Neurosci. 17:3525-3537). Here we show that in inside-out excised patches from TRPV1 expressed in Xenopus oocytes and HEK 293 cells, Ca2+/calmodulin decreased the capsaicin-activated current. This inhibition was not mimicked by Mg2+, reflected a decrease in open probability, and was slowly reversible. Furthermore, increasing the calmodulin concentration in our patches by coexpression of wild-type calmodulin with TRPV1 produced inhibition by Ca2+ alone. In contrast, patches excised from cells coexpressing TRPV1 with a mutant calmodulin did not respond to Ca2+. Using an in vitro calmodulin-binding assay, we found that TRPV1 in oocyte lysates bound calmodulin, although in a Ca2+-independent manner. Experiments with GST-fusion proteins corresponding to regions of the channel NH2-terminal domain demonstrated that a stretch of approximately 30 amino acids adjacent to the first ankyrin repeat bound calmodulin in a Ca2+-dependent manner. The physiological response to pain involves an influx of Ca2+ through TRPV1. Our results indicate that this Ca2+ influx may feed back on the channels, inhibiting their gating. This type of feedback inhibition could play a role in the desensitization produced by capsaicin.

Ca2+/calmodulin modulates TRPV1 activation by capsaicin.

Cyclic nucleotide-gated (CNG) ion channels are nonselective cation channels with a high permeability for Ca(2+). Not surprisingly, they are blocked by a number of Ca(2+) channel blockers including tetracaine, pimozide, and diltiazem. We studied the effects of dequalinium, an extracellular blocker of the small conductance Ca(2+)-activated K(+) channel. We previously noted that dequalinium is a high-affinity blocker of CNGA1 channels from the intracellular side, with little or no state dependence at 0 mV. Here we examined block by dequalinium at a broad range of voltages in both CNGA1 and CNGA2 channels. We found that dequalinium block was mildly state dependent for both channels, with the affinity for closed channels 3-5 times higher than that for open channels. Mutations in the S4-S5 linker did not alter the affinity of open channels for dequalinium, but increased the affinity of closed channels by 10-20-fold. The state-specific effect of these mutations raises the question of whether/how the S4-S5 linker alters the binding of a blocker within the ion permeation pathway.

State-dependent block of CNG channels by dequalinium.

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels contribute to a wide range of physiological functions and regulate cardiac and neuronal pacemaker activity. Here, we review recent advances in three areas: the polarity of the S4 movement with hyperpolarization, the location of the activation gate, and the structure of the C-terminal C linker and CNBD.

Quickening the pace: looking into the heart of HCN channels.

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels that are activated by the direct binding of the cyclic nucleotides cAMP and cGMP. The region linking the last membrane-spanning region (S6) to the cyclic nucleotide binding domain in the COOH terminus, termed the C-linker, has been shown to play an important role in coupling cyclic nucleotide binding to opening of the pore. In this study, we explored the intersubunit proximity between the A' helices of the C-linker regions of CNGA1 in functional channels using site-specific cysteine substitution. We found that intersubunit disulfide bonds can be formed between the A' helices in open channels, and that inducing disulfide bonds in most of the studied constructs resulted in potentiation of channel activation. This suggests that the A' helices of the C-linker regions are in close proximity when the channel is in the open state. Our finding is not compatible with a homology model of the CNGA1 C-linker made from the recently published X-ray crystallographic structure of the hyperpolarization-activated, cyclic nucleotide-modulated (HCN) channel COOH terminus, and leads us to suggest that the C-linker region depicted in the crystal structure may represent the structure of the closed state. The opening conformational change would then involve a movement of the A' helices from a position parallel to the axis of the membrane to one perpendicular to the axis of the membrane.

Functional interactions between A' helices in the C-linker of open CNG channels.

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85beta subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85beta coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane.

Sensibilisierung des Schmerz-transducing Ionenkanal TRPV1 unterliegt thermischen Hyperalgesie durch Proalgesic Mittel wie Nerv Wachstumfaktor (NGF). Das heute akzeptierte Modell ist, dass der NGF-vermittelten Anstieg der TRPV1-Funktion w&auml;hrend der Hyperalgesie Aktivierung der Phospholipase C (PLC), PIP2, vorgeschlagen, tonisch TRPV1 hemmen cleave nutzt. In dieser Studie wir getestet das PLC-Modell und zwei Linien von beweisen, die seine G&uuml;ltigkeit direkt herauszufordern: (1) Polylysin, eine kationische Phosphoinositid absondern Agent, gehemmt TRPV1 statt potentiating es, und (2) direkte Anwendung von PIP2 Inside-Out-excised Patches potenzierte dramatisch TRPV1. Dar&uuml;ber hinaus zeigen wir vier Arten von Experimenten, die darauf hinweist, dass PI3K physisch und funktionell TRPV1 gekoppelt ist: (1) die p85beta-Untereinheit des PI3K interagiert mit der N-terminalen Region TRPV1 Hefe-2-Hybrid-Experimente, (2) PI3K-p85beta-Coimmunoprecipitated mit TRPV1 von HEK293 Zellen und Spinalganglien Ganglien (DRG) Neuronen, (3) TRPV1 interagiert mit rekombinantem PI3K-p85 in-vitro- und Wortmannin (4), einen spezifischen Inhibitor der PI3K, vollst&auml;ndig abgeschafft NGF-vermittelte Sensibilisierung in akut getrennten DRG-Neuronen. Gleichzeitige elektrophysiologische und total internal Reflection Fluorescence (TIRF) Mikroskopie Aufnahmen zeigen schlie&szlig;lich, dass NGF die Anzahl der Kan&auml;le in der Plasmamembran erh&ouml;ht. Wir schlagen ein neues Modell f&uuml;r NGF-vermittelte Hyperalgesie in welche physikalische Kopplung von TRPV1 und PI3K in ein Signal Transduktion komplexe erleichtert Handel mit TRPV1, der Plasmamembran.

Phosphoinositid-3-Kinase bindet an TRPV1 und vermittelt NGF-stimuliert TRPV1 Menschenhandel an der Plasmamembran.

Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.

A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic.

Once thought of as simply an oily barrier that maintains cellular integrity, lipids are now known to play an active role in a large variety of cellular processes. Phosphoinositides are of particular interest because of their remarkable ability to affect many signaling pathways. Ion channels and transporters are an important target of phosphoinositide signaling, but identification of the specific phosphoinositides involved has proven elusive. TRPV1 is a good example; although phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P(2)) can potently regulate its activation, we show that phosphatidylinositol (4)-phosphate (PI(4)P) and phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P(3)) can as well. To determine the identity of the endogenous phosphoinositide regulating TRPV1, we applied recombinant pleckstrin homology domains to inside-out excised patches. Although a PI(4,5)P(2)-specific pleckstrin homology domain inhibited TRPV1, a PI(3,4,5)P(3)-specific pleckstrin homology domain had no effect. Simultaneous confocal imaging and electrophysiological recording of whole cells expressing a rapamycin-inducible lipid phosphatase also demonstrates that depletion of PI(4,5)P(2) inhibits capsaicin-activated TRPV1 current; the PI(4)P generated by the phosphatases was not sufficient to support TRPV1 function. We conclude that PI(4,5)P(2), and not other phosphoinositides or other lipids, is the endogenous phosphoinositide regulating TRPV1 channels.

Determinants of molecular specificity in phosphoinositide regulation. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the endogenous lipid regulating TRPV1.

The 30+ members of the family of TRP channels are diverse in their physiological roles, yet the mechanisms that regulate their gating may be conserved. In particular, all TRP channels show an activity-dependent inhibition which is mediated by Ca(2+). The mechanism by which Ca(2+) inhibits TRP channels is currently a matter of intense debate, with Ca(2+)-regulated kinases, phosphatases, phospholipases and calmodulin all proposed to be involved. In this review, we will discuss different mechanisms for Ca(2+)-dependent desensitization in TRP channels. We will conclude with a model that focuses on Ca(2+)-dependent activation of phospholipase C and Ca(2+) binding to calmodulin and propose that the phospholipase C and calmodulin pathways are structurally and functionally coupled.

Mechanism of Ca(2+)-dependent desensitization in TRP channels.

Perspectives on: Local calcium signaling.

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca(2+)-dependent desensitization, similar to the other TRPVs, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or on coexpression of mutant calmodulin but was sensitive to changes in membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)), suggesting a role of membrane PIP(2) in TRPV2 desensitization. Simultaneous confocal imaging and electrophysiological recording of cells expressing TRPV2 and a fluorescent PIP(2)-binding probe demonstrated that TRPV2 desensitization was concomitant with depletion of PIP(2). We conclude that the decrease in PIP(2) levels on channel activation underlies a major component of Ca(2+)-dependent desensitization of TRPV2 and may play a similar role in other TRP channels.

Ca2+-dependent desensitization of TRPV2 channels is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate.

Although a large number of ion channels are now believed to be regulated by phosphoinositides, particularly phosphoinositide 4,5-bisphosphate (PIP2), the mechanisms involved in phosphoinositide regulation are unclear. For the TRP superfamily of ion channels, the role and mechanism of PIP2 modulation has been especially difficult to resolve. Outstanding questions include: is PIP2 the endogenous regulatory lipid; does PIP2 potentiate all TRPs or are some TRPs inhibited by PIP2; where does PIP2 interact with TRP channels; and is the mechanism of modulation conserved among disparate subfamilies? We first addressed whether the PIP2 sensor resides within the primary sequence of the channel itself, or, as recently proposed, within an accessory integral membrane protein called Pirt. Here we show that Pirt does not alter the phosphoinositide sensitivity of TRPV1 in HEK-293 cells, that there is no FRET between TRPV1 and Pirt, and that dissociated dorsal root ganglion neurons from Pirt knock-out mice have an apparent affinity for PIP2 indistinguishable from that of their wild-type littermates. We followed by focusing on the role of the C terminus of TRPV1 in sensing PIP2. Here, we show that the distal C-terminal region is not required for PIP2 regulation, as PIP2 activation remains intact in channels in which the distal C-terminal has been truncated. Furthermore, we used a novel in vitro binding assay to demonstrate that the proximal C-terminal region of TRPV1 is sufficient for PIP2 binding. Together, our data suggest that the proximal C-terminal region of TRPV1 can interact directly with PIP2 and may play a key role in PIP2 regulation of the channel.

Localization of the PIP2 sensor of TRPV1 ion channels.

It has been hypothesized that cytoskeletal tension prevents large-scale phase separation within cell plasma membranes. Here, we microaspirate giant unilamellar vesicles to determine the effect of mechanical stress on the liquid/liquid miscibility temperature of a membrane composed of a ternary lipid mixture. An increase in tension of 0.1Â mN/m induces a decrease in miscibility temperature on the order of a few tenths of a degree K, which validates recent theoretical predictions.

Increasing membrane tension decreases miscibility temperatures; an experimental demonstration via micropipette aspiration.

The ability to detect hot temperatures is critical to maintaining body temperature and avoiding injury in diverse animals from insects to mammals. Zebrafish embryos, when given a choice, actively avoid hot temperatures and display an increase in locomotion similar to that seen when they are exposed to noxious compounds such as mustard oil. Phylogenetic analysis suggests that the single zebrafish ortholog of TRPV1/2 may have arisen from an evolutionary precursor of the mammalian TRPV1 and TRPV2. As opposed to TRPV2, mammalian TRPV1 is essential for environmentally relevant heat sensation. In the present study, we provide evidence that the zebrafish TRPV1 ion channel is also required for the sensation of heat. Contrary to development in mammals, zebrafish TRPV1(+) neurons arise during the first wave of somatosensory neuron development, suggesting a vital importance of thermal sensation in early larval survival. In vitro analysis showed that zebrafish TRPV1 acts as a molecular sensor of environmental heat (â‰¥25Â°C) that is distinctly lower than the sensitivity of the mammalian form (â‰¥42Â°C) but consistent with thresholds measured in behavioral assays. Using in vivo calcium imaging with the genetically encoded calcium sensor GCaMP3, we show that TRPV1-expressing trigeminal neurons are activated by heat at behaviorally relevant temperatures. Using knock-down studies, we also show that TRPV1 is required for normal heat-induced locomotion. Our results demonstrate for the first time an ancient role for TRPV1 in the direct sensation of environmental heat and show that heat sensation is adapted to reflect species-dependent requirements in response to environmental stimuli.

Article	Year
Dissecting intersubunit contacts in cyclic nucleotide-gated ion channels. Neuron\| PubMed ID: 11879648	2002
Subunit modification and association in VR1 ion channels. BMC neuroscience\| PubMed ID: 11914134	2002
Dequalinium: a novel, high-affinity blocker of CNGA1 channels. The Journal of general physiology\| PubMed ID: 12508052	2003
Ca2+/calmodulin modulates TRPV1 activation by capsaicin. The Journal of general physiology\| PubMed ID: 14699077	2004
State-dependent block of CNG channels by dequalinium. The Journal of general physiology\| PubMed ID: 14981138	2004
Quickening the pace: looking into the heart of HCN channels. Neuron\| PubMed ID: 15091336	2004
Functional interactions between A' helices in the C-linker of open CNG channels. The Journal of general physiology\| PubMed ID: 15738051	2005
Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane. The Journal of general physiology\| PubMed ID: 17074976	2006
A single N-terminal cysteine in TRPV1 determines activation by pungent compounds from onion and garlic. Nature neuroscience\| PubMed ID: 18297068	2008
Determinants of molecular specificity in phosphoinositide regulation. Phosphatidylinositol (4,5)-bisphosphate (PI(4,5)P2) is the endogenous lipid regulating TRPV1. The Journal of biological chemistry\| PubMed ID: 18574245	2008
Mechanism of Ca(2+)-dependent desensitization in TRP channels. Channels (Austin, Tex.)\| PubMed ID: 18849652	2008
Perspectives on: Local calcium signaling. The Journal of general physiology\| PubMed ID: 20660653	2010
Ca2+-dependent desensitization of TRPV2 channels is mediated by hydrolysis of phosphatidylinositol 4,5-bisphosphate. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 20926660	2010
Localization of the PIP2 sensor of TRPV1 ion channels. The Journal of biological chemistry\| PubMed ID: 21224382	2011
Increasing membrane tension decreases miscibility temperatures; an experimental demonstration via micropipette aspiration. Biophysical journal\| PubMed ID: 23083725	2012
The zebrafish ortholog of TRPV1 is required for heat-induced locomotion. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 23516290	2013

Sharona E. Gordon

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