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The Journal of Neuroscience, October 18, 2006, 26(42):10621-10622; doi:10.1523/JNEUROSCI.3599-06.2006

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Journal Club

Editor's Note: These short reviews of a recent paper in the Journal, written exclusively by graduate students or postdoctoral fellows, are intended to mimic the journal clubs that exist in your own departments or institutions. For more information on the format and purpose of the Journal Club, please see http://www.jneurosci.org/misc/ifa_features.shtml.

Novel Probes for G-Protein-Coupled Receptor Signaling

Jill B. Jensen¹ and Jane E. Lauckner^1,2

¹Department of Physiology and Biophysics and ²Neurobiology and Behavior Graduate Program, University of Washington School of Medicine, Seattle, Washington 98195

Review of Robbins et al. (http://www.jneurosci.org/cgi/content/full/26/30/7950)

G-protein-coupled receptors (GPCRs) constitute the largest known protein family. They are activated by a wide range of ligands to transduce an extracellular signal into an intracellular one that evokes a variety of physiological responses, including regulation of ion channels by neurotransmitters and hormones. One such example is modulation of M-type potassium current by G_q/11 G-proteins. This plays an important role in regulating neuronal excitability. M current is encoded by Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3) channel subunits. Multiple mutations in these subunits cause neuronal hyperexcitability and underlie a mild epileptic syndrome, benign familial neonatal convulsions. The membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP₂) is the principal determinant of M current activity, and its cleavage on activation of G_q/11 G-proteins underlies channel closure and slow membrane depolarization (Fig. 1). Many ion channels are modulated by PIP₂, including voltage-gated calcium channels, inward rectifier potassium channels, and some transient receptor potential ion channels (Suh and Hille, 2005). Given our increasing understanding of the role of G-proteins and PIP₂ in cellular signaling, it is important to develop tools to examine GPCR–G-protein and PIP₂–channel interactions.

View larger version (34K): [in this window] [in a new window]   Figure 1. Schematic illustration of muscarinic modulation of M current. Activation of the M1 muscarinic receptor couples through Gq and phospholipase C (PLC) to catalyze the degradation of the membrane phospholipid PIP2 with concomitant inhibition of M-type potassium current. ACh, Acetylcholine; IP3, inositol 1,4,5-trisphosphate; DAG, diacylglycerol.

 
Several approaches have been used to study GPCR-mediated pathways. The more successful of these involve selectively attenuating the function of candidate signaling molecules and determining how this affects the end result of GPCR activation. One such technique is the use of anti-G-protein antibodies. The antibodies inhibit GPCR signaling (Caulfield et al., 1994) but can lack specificity, are slow acting and irreversible, and are difficult to use in intact cells. Another approach has been to use dominant-negative proteins or RNA interference to disrupt protein function or expression. The irreversibility of these methods and the time required for expression precludes their use in many experiments. The ideal technique will be highly specific, fast, and reversible, and act in an intact cell. Membrane-targeted molecular interference tools, such as specially designed peptides, come closer to meeting these criteria than several existing techniques. Covic et al. (2002) designed palmitoylated peptides, termed "pepducins," that were directed against the third intracellular loop (one putative G-protein binding region) of GPCRs. These pepducins penetrated the cell membrane and activated G-proteins in the manner of the GPCRs they were based on.

The recent Journal of Neuroscience paper by Robbins et al. (2006) introduced novel applications of this palmitoylated peptide approach. Their technique, modeled after the pepducins designed by Covic et al. (2002), used palmitoyl-tagged peptides (10-mer "palpeptides") that mimicked the binding site for the target molecule and were cell permeant. This method allowed the researchers to prevent activation of specific G-proteins or to sequester PIP₂.

The G_q/11 palpeptide, which contained the receptor-binding region from the C terminus of G_q, effectively blocked coupling between the M₁ muscarinic acetylcholine receptor (M₁R) and downstream inhibition of both M current [Robbins et al., their Fig. 1 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F1)] and calcium current. G_q/11 palpeptide action was dose dependent, fast, and G-protein subtype specific, because it did not interfere with calcium channel inhibition via G_o-coupled adrenergic receptors [Robbins et al., their Fig. 2 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F2)]. Likewise, the G_o palpeptide selectively blocked norepinephrine (NE)-induced calcium current inhibition, but not inhibition by the muscarinic agonist oxotremorine-M (Oxo-M) [Robbins et al., their Fig. 3 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F3)] acting via M₁R. However, both G-protein palpeptides appear to have enhanced calcium channel inhibition nonspecifically. A scrambled version of the G_o palpeptide substantially augmented NE-evoked inhibition relative to palpeptide-free controls [Robbins et al., their Figs. 2B (http://www.jneurosci.org/cgi/content/full/26/30/7950/F2) and 3A (http://www.jneurosci.org/cgi/content/full/26/30/7950/F3)]. The authors showed that palmitoyl tethering prevented a Shaker inactivation ball from blocking the pore of a Kv1.2 channel [Robbins et al., their Fig. 11 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F11)] but did not rule out the possibility of G palpeptide occlusion of calcium channels.

The second set of palpeptides was directed against PIP₂, using a region of the Kv7.2 C terminus shown to be important in regulating channel affinity for PIP₂. PIP₂ palpeptide inhibition of M current was dose dependent, rapid, M₁R independent, and specific because it only inhibited PIP₂-independent potassium currents to a small extent. It was only slightly reversible on washout [Robbins et al., their Fig. 5 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F5)]. The PIP₂ palpeptide acted in a manner consistent with a reduction in the available pool of PIP₂, because its inhibitory action on M current was rescued by addition of the PIP₂ analog, diC₈PIP₂ [Robbins et al., their Fig. 8 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F8)]. Furthermore, the PIP₂ palpeptide enhanced M₁R suppression of M current [Robbins et al., their Fig. 9 (http://www.jneurosci.org/cgi/content/full/26/30/7950/F9)]. Importantly, the authors found a loose relationship between the structure and function of PIP₂ palpeptides; both a scrambled palpeptide and a palpeptide formed exclusively of lysine residues had higher affinities for PIP₂ than the potential PIP₂ binding sequence from Kv7.2 [Robbins et al., their Table 1 (http://www.jneurosci.org/cgi/content/full/26/30/7950/T1)]. This suggests either the absence of a highly conserved PIP₂ binding domain, or the inability of the palpeptides to recreate the binding motif of Kv7.2. Sequence and functional analysis from many channels supports the former; electrostatic attraction to basic residues may constitute the mechanism of PIP₂ binding. Palpeptides based on other channel domains could help to further characterize this mechanism.

Robbins and colleagues motivated their experiments by suggesting that membrane-targeted peptide probes could be used to delineate multiple contributions to regulation of M current. Their results suggest that PIP₂ depletion accounts for >95% of M current inhibition [Robbins et al., their Fig. 5B,D (http://www.jneurosci.org/cgi/content/full/26/30/7950/F5)], making the maximal effect of PIP₂ palpeptides virtually indistinguishable from that of saturating concentrations of Oxo-M. However, because PIP₂ palpeptides may interfere with hydrolysis of bound PIP₂, both release of calcium stores and activation of protein kinase C could be attenuated in their presence. Consequently, it remains difficult to rule out additional roles of other G_q/11 effectors such as calcium/calmodulin or protein kinase C.

Despite these caveats, Robbins et al. (2006) have succeeded in developing a useful and exciting new tool to add to the molecular repertoire for studying GPCR signaling and its many manifestations. This technique may also prove to be useful in the design of new therapeutics that can specifically and rapidly target components of GPCR signaling pathways.

Received Aug. 18, 2006; revised Aug. 25, 2006; accepted Aug. 25, 2006.

Correspondence should be addressed to Jill B. Jensen, Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, WA 98195. Email: jbodily{at}u.washington.edu

Copyright © 2006 Society for Neuroscience 0270-6474/06/2610621-02$15.00/0

References

Caulfield MP, Jones S, Vallis Y, Buckley NJ, Kim GD, Milligan G, Brown DA (1994) Muscarinic M-current inhibition via G_q/11 and -adrenoceptor inhibition of Ca²⁺ current via G_o in rat sympathetic neurones. J Physiol (Lond) 477:415–422.[ISI][Medline]

Covic L, Gresser AL, Talavera J, Swift S, Kuliopulos A (2002) Activation and inhibition of G protein-coupled receptors by cell-penetrating membrane-tethered peptides. Proc Natl Acad Sci USA 99:643–648.[Abstract/Free Full Text]

Robbins J, Marsh SJ, Brown DA (2006) Probing the regulation of M (Kv7) potassium channels in intact neurons with membrane-targeted peptides. J Neurosci 26:7950–7961.[Abstract/Free Full Text]

Suh BC, Hille B (2005) Regulation of ion channels by phosphatidylinositol 4,5-bisphosphate. Curr Opin Neurobiol 15:370–378.[CrossRef][ISI][Medline]

Related articles in J. Neurosci.:

Probing the Regulation of M (Kv7) Potassium Channels in Intact Neurons with Membrane-Targeted Peptides

Jon Robbins, Stephen J. Marsh, and David A. Brown
J. Neurosci. 2006 26: 7950-7961. [Abstract] [Full Text]  

This Article Full Text (PDF) Author Response Submit an eLetter Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Services Email this article to a friend Related articles in J. Neurosci. Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Jensen, J. B. Articles by Lauckner, J. E. Search for Related Content PubMed PubMed Citation Articles by Jensen, J. B. Articles by Lauckner, J. E.

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