Search

Cav1.4 L-type Ca2+ channels are predominantly expressed in retinal neurons, particularly at the photoreceptor terminals where they mediate sustained Ca2+ entry needed for continuous neurotransmitter release at their ribbon synapses. Cav1.4 channel gating properties are controlled by accessory subunits, associated regulatory proteins, and also alternative splicing. In humans, mutations in the CACNA1F gene encoding for Cav1.4 channels are associated with X-linked retinal disorders such as congenital stationary night blindness type 2. Mutations in the Cav1.4 protein result in a spectrum of altered functional channel activity. Several mouse models broadened our understanding of the role of Cav1.4 channels not only as Ca2+ source at retinal synapses but also as synaptic organizers. In this review, we highlight different structural and functional phenotypes of Cav1.4 mutations that might also occur in patients with congenital stationary night blindness type 2. A further important yet mostly neglected aspect that we discuss is the influence of alternative splicing on channel dysfunction. We conclude that currently available functional phenotyping strategies should be refined and summarize potential specific therapeutic options for patients carrying Cav1.4 mutations. Importantly, the development of new therapeutic approaches will permit a deeper understanding of not only the disease pathophysiology but also the physiological function of Cav1.4 channels in the retina.

The large-conductance, Ca(2+)- and voltage-activated K(+) (BK) channel is ubiquitously expressed in mammalian tissues and displays diverse biophysical or pharmacological characteristics. This diversity is in part conferred by channel modulation with different regulatory auxiliary subunits. To date, two distinct classes of BK channel auxiliary subunits have been identified: β subunits and γ subunits. Modulation of BK channels by the four auxiliary β (β1-β4) subunits has been well established and intensively investigated over the past two decades. The auxiliary γ subunits, however, were identified only very recently, which adds a new dimension to BK channel regulation and improves our understanding of the physiological functions of BK channels in various tissues and cell types. This chapter will review the current understanding of BK channel modulation by auxiliary β and γ subunits, especially the latest findings.

Ca2+/calmodulin-dependent regulation of voltage-gated CaV1-2 Ca2+ channels shows extraordinary modes of spatial Ca2+ decoding and channel modulation, vital for many biological functions. A single calmodulin (CaM) molecule associates constitutively with the channel's carboxy-terminal tail, and Ca2+ binding to the C-terminal and N-terminal lobes of CaM can each induce distinct channel regulations. As expected from close channel proximity, the C-lobe responds to the roughly 100-microM Ca2+ pulses driven by the associated channel, a behaviour defined as 'local Ca2+ selectivity'. Conversely, all previous observations have indicated that the N-lobe somehow senses the far weaker signals from distant Ca2+ sources. This 'global Ca2+ selectivity' satisfies a general signalling requirement, enabling a resident molecule to remotely sense cellular Ca2+ activity, which would otherwise be overshadowed by Ca2+ entry through the host channel. Here we show that the spatial Ca2+ selectivity of N-lobe CaM regulation is not invariably global but can be switched by a novel Ca2+/CaM-binding site within the amino terminus of channels (NSCaTE, for N-terminal spatial Ca2+ transforming element). Native CaV2.2 channels lack this element and show N-lobe regulation with a global selectivity. On the introduction of NSCaTE into these channels, spatial Ca2+ selectivity transforms from a global to local profile. Given this effect, we examined CaV1.2/CaV1.3 channels, which naturally contain NSCaTE, and found that their N-lobe selectivity is indeed local. Disruption of this element produces a global selectivity, confirming the native function of NSCaTE. Thus, differences in spatial selectivity between advanced CaV1 and CaV2 channel isoforms are explained by the presence or absence of NSCaTE. Beyond functional effects, the position of NSCaTE on the channel's amino terminus indicates that CaM can bridge the amino terminus and carboxy terminus of channels. Finally, the modularity of NSCaTE offers practical means for understanding the basis of global Ca2+ selectivity.

Delta-palutoxins from the spider Paracoelotes luctuosus (Araneae: Amaurobiidae) are 36-37 residue long peptides that show preference for insect sodium channels (NaChs) and modulate their function. Although they slow NaCh inactivation in a fashion similar to that of receptor site 3 modifiers, such as scorpion alpha-toxins, they actually bind with high affinity to the topologically distinct receptor site 4 of scorpion beta-toxins. To resolve this riddle, we scanned by Ala mutagenesis the surface of delta-PaluIT2, a delta-palutoxin variant with the highest affinity for insect NaChs, and compared it to the bioactive surface of a scorpion beta-toxin. We found three regions on the surface of delta-PaluIT2 important for activity: the first consists of Tyr-22 and Tyr-30 (aromatic), Ser-24 and Met-28 (polar), and Arg-8, Arg-26, Arg-32, and Arg-34 (basic) residues; the second is made of Trp-12; and the third is made of Asp-19, whose substitution by Ala uncoupled the binding from toxicity to lepidopteran larvae. Although spider delta-palutoxins and scorpion beta-toxins have developed from different ancestors, they show some commonality in their bioactive surfaces, which may explain their ability to compete for an identical receptor (site 4) on voltage-gated NaChs. Yet, their different mode of channel modulation provides a novel perspective about the structural relatedness of receptor sites 3 and 4, which until now have been considered topologically distinct.

Key elements of tumor development include proliferation, migration, invasiveness, and angiogenesis. Activation of the volume-sensitive organic osmolyte/anion channel (VSOAC) has been suggested to play a role in all of these processes. VSOACs may therefore represent an important therapeutic target in the etiology of cancer. However, pharmacological inhibitors of VSOAC are nonselective and of low potency, highlighting the importance of identifying novel regulators of the channel. The use of electrophysiological methods coupled with techniques such as pull-down assays, yeast 2-hybrid, and functional protein arrays have already proved valuable in studying protein-protein interactions in a variety of systems. Some of these methods have been used to identify small molecules that modulate the function of other types of ion channels. Given that several proteins have already been identified as putative modulators of VSOACs, proteomics technologies may prove useful in elucidating the molecular identity of VSOACs and helpful in identifying novel modulators of channel function. In this paper, we review the involvement of VSOACs in tumor development processes and its regulation by pharmacological agents and cellular proteins. Proteomic approaches to study protein-protein interactions and how such approaches may be used to study VSOACs are also discussed. We speculate on how modulation of protein-protein interactions may result in the identification of a novel class of compounds for modulating VSOACs.

Inhibition of N- (Cav2.2) and P/Q-type (Cav2.1) calcium channels by G-proteins contribute importantly to presynaptic inhibition as well as to the effects of opiates and cannabinoids. Accordingly, elucidating the molecular mechanisms underlying G-protein inhibition of voltage-gated calcium channels has been a major research focus. So far, inhibition is thought to result from the interaction of multiple proposed sites with the Gbetagamma complex (Gbetagamma). Far less is known about the important interaction sites on Gbetagamma itself. Here, we developed a novel electrophysiological paradigm, "compound-state willing-reluctant analysis," to describe Gbetagamma interaction with N- and P/Q-type channels, and to provide a sensitive and efficient screen for changes in modulatory behavior over a broad range of potentials. The analysis confirmed that the apparent (un)binding kinetics of Gbetagamma with N-type are twofold slower than with P/Q-type at the voltage extremes, and emphasized that the kinetic discrepancy increases up to ten-fold in the mid-voltage range. To further investigate apparent differences in modulatory behavior, we screened both channels for the effects of single point alanine mutations within four regions of Gbeta1, at residues known to interact with Galpha. These residues might thereby be expected to interact with channel effectors. Of eight mutations studied, six affected G-protein modulation of both N- and P/Q-type channels to varying degrees, and one had no appreciable effect on either channel. The remaining mutation was remarkable for selective attenuation of effects on P/Q-, but not N-type channels. Surprisingly, this mutation decreased the (un)binding rates without affecting its overall affinity. The latter mutation suggests that the binding surface on Gbetagamma for N- and P/Q-type channels are different. Also, the manner in which this last mutation affected P/Q-type channels suggests that some residues may be important for "steering" or guiding the protein into the binding pocket, whereas others are important for simply binding to the channel.

Voltage-dependent inhibition of N- and P/Q-type calcium channels by G proteins is crucial for presynaptic inhibition of neurotransmitter release, and may contribute importantly to short-term synaptic plasticity. Such calcium-channel modulation could thereby impact significantly the neuro-computational repertoire of neural networks. The differential modulation of N and P/Q channels could even further enrich their impact upon synaptic tuning. Here, we performed in-depth comparison of the G-protein inhibition of recombinant N and P/Q channels, expressed in HEK 293 cells with the m2 muscarinic receptor. While both channel types display classic features of G-protein modulation (kinetic slowing of activation, prepulse facilitation, and voltage dependence of inhibition), we confirmed previously reported quantitative differences, with N channels displaying stronger inhibition and greater relief of inhibition by prepulses. A more fundamental, qualitative difference in the modulation of these two channels was revealed by a modified tail-activation paradigm, as well as by a novel “slope” analysis method comparing time courses of slow activation and prepulse facilitation. The stark contrast in modulatory behavior can be understood within the context of the “willing–reluctant” model, in which binding of G-protein βγ subunits to channels induces a reluctant mode of gating, where stronger depolarization is required for opening. Our experiments suggest that only N channels could be opened in the reluctant mode, at voltages normally spanned by neuronal action potentials. By contrast, P/Q channels appear to remain closed, especially over these physiological voltages. Further, the differential occurrence of reluctant openings is not explained by differences in the rate of G-protein unbinding from the two channels. These two scenarios predict very different effects of G-protein inhibition on the waveform of Ca2+ entry during action potentials, with potentially important consequences for the timing and efficacy of synaptic transmission.

Cyclic nucleotide–gated (CNG) channels are critical components in the visual and olfactory signal transduction pathways, and they primarily gate in response to changes in the cytoplasmic concentration of cyclic nucleotides. We previously found that the ability of the native rod CNG channel to be opened by cGMP was markedly inhibited by analogues of diacylglycerol (DAG) without a phosphorylation reaction (Gordon, S.E., J. Downing-Park, B. Tam, and A.L. Zimmerman. 1995. Biophys. J. 69:409–417). Here, we have studied cloned bovine rod and rat olfactory CNG channels expressed in Xenopus oocytes, and have determined that they are differentially inhibited by DAG. At saturating [cGMP], DAG inhibition of homomultimeric (α subunit only) rod channels was similar to that of the native rod CNG channel, but DAG was much less effective at inhibiting the homomultimeric olfactory channel, producing only partial inhibition even at high [DAG]. However, at low open probability (Po), both channels were more sensitive to DAG, suggesting that DAG is a closed state inhibitor. The Hill coefficients for DAG inhibition were often greater than one, suggesting that more than one DAG molecule is required for effective inhibition of a channel. In single-channel recordings, DAG decreased the Po but not the single-channel conductance. Results with chimeras of rod and olfactory channels suggest that the differences in DAG inhibition correlate more with differences in the transmembrane segments and their attached loops than with differences in the amino and carboxyl termini. Our results are consistent with a model in which multiple DAG molecules stabilize the closed state(s) of a CNG channel by binding directly to the channel and/or by altering bilayer–channel interactions. We speculate that if DAG interacts directly with the channel, it may insert into a putative hydrophobic crevice among the transmembrane domains of each subunit or at the hydrophobic interface between the channel and the bilayer.

Dendritic arbors are critical for the information processing capability of central neurons, but quantitative analysis of their membrane properties has been hampered by their geometrical complexity. Here, we have focused on an important source of Ca 2+ entry in dendrites, the voltage-gated Ca 2+ channels, by applying the whole-cell voltage-clamp technique to isolated dendritic segments ("dendrosomes") from rat hippocampal neurons. We found that low voltage-activated T-type Ca 2+ channels provide a significantly larger fraction of the Ca 2+ influx in dendrites than their counterparts in cell bodies. Surprisingly, 60%–70% of the high voltage-activated Ca 2+ current in dendrosomes was N and P/Q type, and these channels were susceptible to neurotransmitter inhibition, suggesting a novel physiological role for G protein–regulated Ca 2+ channel modulation in controlling dendritic excitability and Ca 2+ signaling.

Calmodulin (CaM) regulation of mammalian CaV channels is both biologically critical and mechanistically rich, rendering this system a central prototype for the decoding of Ca2+ signals and the modulation of channels. This system showcases the remarkable ability of the N- and C-terminal lobes of CaM to function as semiautonomous Ca2+ sensors and effectors, a theme initially recognized in Paramecium (Cell 62:165). In mammalian CaV channels, Ca2+-free CaM (apoCaM) starts off already preassociated with a host channel, and then the C-lobe turns out to respond preferentially to the strong Ca2+ influx through the host channel to induce one form of channel modulation (local selectivity), whereas the N-lobe frequently prefers the far weaker Ca2+ signal emanating from Ca2+ sources at a distance (global selectivity). These striking contrasts in spatial Ca2+ selectivity, crucial to biological Ca2+ signaling, can be simply explained by mechanisms involving the translocation of CaM among an apoCaM preassociation locus and multiple Ca2+/CaM effector sites (Cell 133:1228). However, beyond apoCaM preassociation at an IQ domain on the carboxy-terminus of channels, little has been definitively established regarding the structural identity of Ca2+/CaM effector sites, outside of an NSCaTE element in the channel amino terminus (Nature 451:830). Here, we outline new evidence identifying dominant Ca2+/CaM effector sites outside of the IQ and NSCaTE regions (Ben Johnny et al, Bazzazi et al, this meeting), thereby bolstering a modulatory mechanism wherein CaM departs from an initial IQ preassociation locus, then interacts with structurally distinct effector sites. This intricate translocating dance of CaM complexed with a target molecule may be a general scheme enabling high-order Ca2+ decoding in many signaling complexes throughout biology; indeed, an analogous hypothesis of migratory CaM has been proposed for NaV channels (J. Biol. Chem. 284:6436).

Vertebrate photoreceptors are thought to adapt to light by a change in Ca2+, which is postulated to mediate modulation of (1) excited rhodopsin (Rh*) by Ca2+-dependent binding of recoverin, (2) guanylyl cyclase activity via Ca2+-dependent GCAP proteins, and (3) cyclic nucleotide-gated channels by binding of Ca2+-calmodulin. Previous experiments genetically deleted recoverin and the GCAPs and showed that significant regulation of sensitivity survives removal of (1) and (2). We genetically deleted the channel Ca2+-calmodulin binding site in the mouseMus musculusand found that removal of (3) alters response waveform, but removal of (3) or of (2) and (3) together still leaves much of adaptation intact. These experiments demonstrate that an important additional mechanism is required, which other experiments indicate may be regulation of phosphodiesterase 6 (PDE6). We therefore constructed a kinetic model in which light produces a Ca2+-mediated decrease in PDE6 decay rate, with the novel feature that both spontaneously activated and light-activated PDE6 are modulated. This model, together with Ca2+-dependent acceleration of guanylyl cyclase, can successfully account for changes in sensitivity and response waveform in background light.

The study of channel modulation by regulatory subunits has attracted considerable attention. Evidence indicates a pivotal role for accessory proteins in the channelosome. For instance, these regulatory subunits are necessary to recapitulate in vivo ion currents and to further understand the physiological role of ion channels. KCNEs are a family of regulatory subunits that interact with a wide range of channels. We have described for the first time a molecular interaction between KCNE4 and the voltage-dependent potassium channel K(V)1.3. The association of KCNE4, which alters the biophysical properties, trafficking and membrane localization of K(V)1.3, functions as an endogenous dominantnegative mechanism. Since both proteins are expressed in the immune system, K(V)1.3/KCNE4 channels may contribute to the fine-tuning of the immune response. Therefore, our results point to KCNE4 as a novel target for immunomodulation. KCNE4 is not the only KCNE which is expressed in leukocytes. All KCNEs (KCNE1-5) are present, and some members demonstrate modulation during proliferation and cancer. In summary, regulatory KCNE subunits are expressed in the immune system. In addition, several voltage-dependent K(+) channels, which could interact with KCNEs, are also detected. Therefore, KCNE subunits may play a yet undiscovered role in the physiology of the immune system.

EF-hand Ca2+-binding proteins such as calmodulin and CaBP1 have emerged as important regulatory subunits of voltage-gated Ca2+ channels. Here, we show that caldendrin, a variant of CaBP1 enriched in the brain, interacts with and distinctly modulates Cav1.2 (L-type) voltage-gated Ca2+ channels relative to other Ca2+-binding proteins. Caldendrin binds to the C-terminal IQ-domain of the pore-forming alpha1-subunit of Cav1.2 (alpha(1)1.2) and competitively displaces calmodulin and CaBP1 from this site. Compared with CaBP1, caldendrin causes a more modest suppression of Ca2+-dependent inactivation of Cav1.2 through a different subset of molecular determinants. Caldendrin does not bind to the N-terminal domain of alpha11.2, a site that is critical for functional interactions of the channel with CaBP1. Deletion of the N-terminal domain inhibits CaBP1, but spares caldendrin modulation of Cav1.2 inactivation. In contrast, mutations of the IQ-domain abolish physical and functional interactions of caldendrin and Cav1.2, but do not prevent channel modulation by CaBP1. Using antibodies specific for caldendrin and Cav1.2, we show that caldendrin coimmunoprecipitates with Cav1.2 from the brain and colocalizes with Cav1.2 in somatodendritic puncta of cortical neurons in culture. Our findings reveal functional diversity within related Ca2+-binding proteins, which may enhance the specificity of Ca2+ signaling by Cav1.2 channels in different cellular contexts.

The Drosophila Slowpoke calcium-dependent potassium channel (dSlo) binding protein Slob was discovered by a yeast two-hybrid screen using the carboxy-terminal tail region of dSlo as bait. Slob binds to and modulates the dSlo channel. We have found that there are several Slob proteins, resulting from multiple translational start sites and alternative splicing, and have named them based on their molecular weights (in kD). The larger variants, which are initiated at the first translational start site and are called Slob71 and Slob65, shift the voltage dependence of dSlo activation, measured by the whole cell conductance–voltage relationship, to the left (less depolarized voltages). Slob53 and Slob47, initiated at the third translational start site, also shift the dSlo voltage dependence to the left. In contrast, Slob57 and Slob51, initiated at the second translational start site, shift the conductance–voltage relationship of dSlo substantially to more depolarized voltages, cause an apparent dSlo channel inactivation, and increase the deactivation rate of the channel. These results indicate that the amino-terminal region of Slob plays a critical role in its modulation of dSlo.

Weighing in at about 5 μg, with 302 neurons and 5000 synapses, C. elegans is unlikely to prove theorems, write poetry, or challenge Mike Tyson. Still, remarkable behavioral complexity is packed into this tiny worm. Worms live in a sensory world very different from our own, one dominated by touch, taste, and smell, one in which bacteria are the size of cherries, and Brownian motion and surface tension take the place of wind and gravity. It is difficult for us to intuit what their world is like, but one measure of what the worm cares about is the behavioral and genetic complexity devoted to various sensory modes. By this measure, thermotaxis must matter a lot to the worm. This behavior was first described in 1975 by Hedgecock and Russell, who noted, among other things, two remarkable features. First, worms are capable of moving along curving isothermal lines in their medium with precision, deviating as little as 0.1°C from their preferred temperature. Second, thermal preference is plastic, being conditioned by the presence or absence of food.The plasticity in C. elegans thermotaxis is apt. When a worm experiences prosperity (abundant food) at a given temperature, it acquires a preference for this temperature over others. When the temperature is held steady and food is removed, this preference degrades with time and is replaced by a temperature-independent searching mode of locomotion. When food remains abundant and temperature is changed, the result is a gradual shift in thermal preference toward the new growth temperature. The assay for these changes is simple (Hedgecock and Russell, 1975xHedgecock, E.M. and Russell, R.L. PNAS. 1975; 72: 4061–4065Crossref | PubMedSee all References(Hedgecock and Russell, 1975). A conditioned worm is placed on an agar surface (without food) in which a radial thermal gradient has been formed. As the worm moves, it leaves behind a detectable indentation in the agar, providing a record of its locomotory behavior over time. A happily conditioned worm will spend most of its time making precise circles along its preferred isotherm. In the absence of other cues, the worm may anticipate that food is likely to be found at this temperature or it may simply be physiologically optimized at this temperature. In contrast, a worm conditioned by the absence of food will move in the same assay without regard for temperature as long as it remains within a tolerable range (Wittenburg and Baumeister, 1999xWittenburg, N. and Baumeister, R. PNAS. 1999; 96: 10477–10482Crossref | PubMed | Scopus (79)See all References(Wittenburg and Baumeister, 1999). The main features of the neural circuit controlling thermotaxis are known. A single class of thermosensory neuron (AFD) makes synaptic output to a single class of interneuron (AIY). AIY in turn makes synapses mostly to three other classes of interneurons: AIZ, RIA, and RIB. AIZ makes synapses back to AIY and to various other interneurons. Based on synaptic connections, laser ablation studies, and genetic perturbations, AIY and AIZ are particularly important for thermotaxis, whereas RIA and RIB seem to be integrative interneurons that respond to many sensory cues (Mori and Ohshima 1995xMori, I. and Ohshima, Y. Nature. 1995; 376: 344–348Crossref | PubMedSee all References, White et al. 1986xWhite, J., Southgate, E., Thomson, J.N., and Brenner, S. Phil. Trans. R. Soc. Lond. B. 1986; 314: 1–340Crossref | PubMedSee all References, Hobert et al. 1997xHobert, O., Mori, I., Yamashita, Y., Honda, H., Ohshima, Y., Liu, Y., and Ruvkun, G. Neuron. 1997; 19: 345–357Abstract | Full Text | Full Text PDF | PubMed | Scopus (171)See all References). AIY and AIZ play antagonistic roles in thermotaxis: AIY activity favors migration to high temperatures and AIZ activity favors low temperatures (Mori and Ohshima, 1995xMori, I. and Ohshima, Y. Nature. 1995; 376: 344–348Crossref | PubMedSee all References(Mori and Ohshima, 1995).The paper by Gomez et al. (2001)xGomez, M., De Castro, E., Guarin, E., Sasakura, H., Kuhara, A., Mori, I., Bartfai, T., Bargmann, C.I., and Nef, P. Neuron. 2001; 30: 241–248Abstract | Full Text | Full Text PDF | PubMed | Scopus (161)See all ReferencesGomez et al. (2001) in this issue of Neuron reports a role for a C. elegans neuronal Ca2+ sensor protein (NCS-1) in thermotaxis accuracy and plasticity. NCS-1 belongs to a large family of EF hand containing calcium binding proteins and is highly conserved across species, including yeast, Drosophila, C. elegans, rodents, and humans. They report that NCS-1 is expressed in 13 classes of neurons and one muscle cell. The neurons are mostly sensory, including the thermosensory neuron AFD, but they also include the interneuron AIY. The authors generate a null deletion allele of NCS-1 using reverse genetic methods (Plasterk, 1995xPlasterk, R. Methods Cell Biol. 1995; 48: 59–80Crossref | PubMed | Scopus (43)See all References(Plasterk, 1995). ncs-1(null) mutants develop normally and perform normally in chemotactic odorant responses, suggesting that their fine locomotory and taxis systems are unperturbed. However, their performance in isothermal tracking is substantially degraded, resembling that seen when the thermosensory neuron AFD is removed. Transgenic experiments with altered ncs-1 genes show that its function in thermotaxis depends on its Ca2+ binding sites and on expression specifically in the interneuron AIY.Up to this point, these results are interesting but, one might argue, not particularly exceptional: NCS-1 functions in AIY in its familiar Ca2+ binding role to mediate thermosensory response. However, perhaps inspired by similar approaches to the study of Drosophila learning and memory (Yin et al., 1995xYin, J.C., Del Vecchio, M., Zhou, H., and Tully, T. Cell. 1995; 81: 107–115Abstract | Full Text PDF | PubMed | Scopus (484)See all References(Yin et al., 1995), the authors go on to make a very striking set of observations. Transgenic overexpression of normal NCS-1 protein from its own promoter enhances peak isothermal tracking accuracy, speeds the acquisition of a new thermal preference after temperature shift paired with food, and delays the extinction of thermal preference when food is removed. This is the kind of result every scientist dreams of: simple and compelling. It is hard to escape the conclusion that NCS-1 is a critical component of a process that mediates thermotaxis plasticity and memory. Are worms with more NCS-1 smarter? I doubt this is the right way to think about it. We can presume that the quality of isothermal tracking and the rate of change in thermal preference are adaptive traits. As with sensory attentiveness and long-term memory in humans, it is presumably important for worms to place a selectable weight on particular sensory information and to modify existing associations with appropriate deliberation.Naturally, it is tempting to speculate about the mechanistic role of NCS-1 in plasticity. However, the NCS family of proteins is sizeable, diverse, and largely unexplored. C. elegans appears to have five NCS-related genes and humans have perhaps a dozen (Burgoyne and Weiss, 2001xBurgoyne, R.D. and Weiss, J.L. Biochem. J. 2001; 353: 1–12Crossref | PubMed | Scopus (367)See all References(Burgoyne and Weiss, 2001), with different members implicated in processes as diverse as guanylyl-cyclase regulation (Palczewski et al., 1994xPalczewski, K., Subbaraya, I., Gorczyca, W.A., Helekar, B.S., Ruiz, C.C., Ohguro, H., Huang, J., Zhao, X., Crabb, J.W., Johnson, R.S. et al. Neuron. 1994; 13: 395–404Abstract | Full Text PDF | PubMed | Scopus (270)See all References(Palczewski et al., 1994), K+ channel modulation (An et al., 2000xAn, W.F., Bowlby, M.R., Betty, M., Cao, J., Ling, H.P., Mendoza, G., Hinson, J.W., Mattsson, K.I., Strassle, B.W., Trimmer, J.S., and Rhodes, K.J. Nature. 2000; 403: 553–556Crossref | PubMed | Scopus (698)See all References(An et al., 2000), and protein kinase inhibition (Chen et al., 1995xChen, C.K., Inglese, J., Lefkowitz, R.J., and Hurley, J.B. J. Biol. Chem. 1995; 270: 18060–18066Crossref | PubMedSee all References(Chen et al., 1995). A role in control of synaptic strength (e.g., Pongs et al., 1993xPongs, O., Lindemeier, J., Zhu, X.R., Theil, T., Engelkamp, D., Krah-Jentgens, I., Lambrecht, H.G., Koch, K.W., Schwemer, J. et al. Neuron. 1993; 11: 15–28Abstract | Full Text PDF | PubMed | Scopus (236)See all ReferencesPongs et al., 1993), though mechanistically not yet understood, is the most promising in explaining the current results. This NCS-1 function is likely to be in the interneuron AIY, but whether it functions pre- or postsynaptically, at what synapse, and by what biochemical mechanism are all unknown. The pump has been primed, but much remains to be investigated.

Publisher Summary This chapter provides an overview of the patch clamp techniques. The patch clamp technique provides the experimental means for merging the tools of modern molecular and cellular biology with those of electrophysiology. Using various recording configurations, it is possible to dissect the mechanisms of channel modulation. In cell-attached recording, modulation of channel activity in response to bath-applied agonist generally indicates a second-messenger mechanism. Candidate messengers can be tested directly on excised patches or in whole-cell recording. Current research on signaling pathways seeks to establish the functionally meaningful mechanisms through selective activation or inhibition of a portion of the pathway. A variety of patch clamp configurations, combined with single-channel resolution, provides a powerful experimental approach from the molecular level, in which channel genes are altered and expressed, to the cellular level, in which posttranslational signaling mechanisms are elucidated, to the systems level, in which cellular interactions in intact or slice preparations are revealed.

The capsaicin receptor, TRPV1, contributes to thermal and chemical sensitivity of primary afferent neurons of the pain pathway, but many aspects of its regulation remain elusive. In this issue of Neuron , Lishko et al. describe a high-resolution structure of a TRPV1 domain, providing insight into the molecular basis of channel modulation while revealing new functions for a widely expressed protein interaction fold.