The melanocortin-4 receptor (MC4R) is involved in energy homeostasis and is an important drug target for syndromic obesity. We report the structure of the antagonist SHU9119-bound human MC4R at 2.8-angstrom resolution. Ca2+ is identified as a cofactor that is complexed with residues from both the receptor and peptide ligand. Extracellular Ca2+ increases the affinity and potency of the endogenous agonist α-melanocyte–stimulating hormone at the MC4R by 37- and 600-fold, respectively. The ability of the MC4R crystallized construct to couple to ion channel Kir7.1, while lacking cyclic adenosine monophosphate stimulation, highlights a heterotrimeric GTP-binding protein (G protein)–independent mechanism for this signaling modality. MC4R is revealed as a structurally divergent G protein–coupled receptor (GPCR), with more similarity to lipidic GPCRs than to the homologous peptidic GPCRs.

Neural M-type (KCNQ/Kv7) K + channels control somatic excitability, bursting and neurotransmitter release throughout the nervous system. Their activity is regulated by multiple signalling pathways. In superior cervical ganglion sympathetic neurons, muscarinic M 1 , angiotensin II AT 1 , bradykinin B 2 and purinergic P2Y agonists suppress M current (I M ). Probes of PLC activity show agonists of all four receptors to induce robust PIP 2 hydrolysis. We have grouped these receptors into two related modes of action. One mode involves depletion of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the membrane, whose interaction with the channels is thought necessary for their function. The other involves IP 3 -mediated intracellular Ca 2+ signals that stimulate PIP 2 synthesis, preventing its depletion, and suppress I M via calmodulin. Carbon-fibre amperometry can evaluate the effect of M channel activity on release of neurotransmitter. Consistent with the dominant role of M current in control of neuronal discharge, M channel openers, or blockers, reduced or augmented the evoked release of noradrenaline neurotransmitter from superior cervical ganglion (SCG) neurons, respectively. We seek to localize the subdomains on the channels critical to their regulation by PIP 2 . Based on single-channel recordings from chimeras between high-PIP 2 affinity KCNQ3 and low-PIP 2 affinity KCNQ4 channels, we focus on a 57-residue domain within the carboxy-terminus that is a possible PIP 2 binding site. Homology modelling of this domain using the published structure of IRK1 channels as a template predicts a structure very similar to an analogous region in IRK1 channels, and shows a cluster of basic residues in the KCNQ2 domain to correspond to those implicated in PIP 2 regulation of Kir channels. We discuss some important issues dealing with these topics.

Voltage-gated K þ channels of the Kv7 family underlie the neuronal M current that regulates action potential firing. Suppression of M current increases excitability and its enhancement can silence neurons. We here show that three of five Kv7 channels undergo strong enhancement of their activity by oxidative modification induced by physiological concentrations of hydrogen peroxide. A triple cysteine pocket in the channel S2-S3 linker is critical for this effect. Oxidation-induced enhancement of M current produced a hyperpolarization and a dramatic reduction of action potential firing frequency in rat sympathetic neurons. As hydrogen peroxide is robustly produced during hypoxia-induced oxidative stress, we used an oxygen/glucose deprivation neurodegeneration model that showed neuronal death to be severely accelerated by M current blockade. Such blockade had no effect on survival of normoxic neurons. This work describes a novel pathway of M-channel regulation and suggests a role for M channels in protective neuronal silencing during oxidative stress.

Members of the KCNQ (Kv7) family of voltage-gated K + channels underlie " M-type " K + currents in many different types of neurons, delayed-rectifi er currents of the heart, and K + transport channels of the inner ear and epithelia ( Jentsch, 2000 ;Robbins, 2001 ). Neuronal M currents play strong roles in regulating excitability and neuronal discharge, and their modulation by several receptors linked to the G q/11 class of G proteins endows them with powerful effects on the function of excitable cells ( Delmas and Brown, 2005 ). As for a plethora of other channels and transporters ( Gamper and Shapiro, 2007 ), M-type channels are very sensitive to the abundance of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the membrane, and PIP 2 depletion is widely accepted as the mechanism of M current suppression by muscarinic receptor stimulation in sympathetic neurons ( Delmas and Brown, 2005 ;Suh et al., 2006 ;Brown et al., 2007 ;Suh and Hille, 2007 ).We have examined the activity of Kv7.2 -7.4 channels at the single-channel level and found these voltage-gated channels to have strikingly differential saturating open probabilities (P o ) in cell-attached patches ( Li et al., 2004 ).Correspondence to Mark S. Shapiro: s h a p i r o m @ u t h s c s a . e d u Abbreviations used in this paper: CaM, calmodulin; C ␣ RMS, carbon ␣ root mean squared; CHO, Chinese hamster ovary; diC8-PIP 2 , dioctanoyl-PIP 2 ; GPMI-P2, l -␣ -glycerophospho-d -myo-inositol 4,5-bisphosphate; Kv7, KCNQ; PIP 2 , phosphatidylinositol 4,5-bisphosphate; P o , open probabilities; RMSD, root mean square distance; wt, wild-type.The online version of this article contains supplemental material.In accord with the role of PIP 2 in regulating gating, channel activity rapidly runs down upon excision as inside-out patches, but activity can be fully restored by adding PIP 2 or an analogue to the cytoplasmically facing bath solution ( Zhang et al., 2003 ;Li et al., 2005 ). For all the channels studied, the P o is a function of the concentration of supplied PIP 2 , but with very different apparent affi nities among the channels. Thus, Kv7.3 homomultimers display a very high apparent affi nity, Kv7.2 and Kv7.4 homomultimers display apparent affi nities some one to two orders of magnitude lower, and Kv7.2/7.3 heteromultimers display an intermediate value, as expected for channels composed of both high-and low-affi nity PIP 2 -binding subunits ( Li et al., 2005 ). Such differential PIP 2 affi nities among the channels are also supported by whole cell experiments in which PIP 2 abundance was either tonically The regulation of M-type (KCNQ [Kv7]) K + channels by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP 2 on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP 2 sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP 2 action....

Epileptic encephalopathies are a devastating group of severe childhood onset epilepsies with medication-resistant seizures and poor developmental outcomes. Many epileptic encephalopathies have a genetic aetiology and are often associated with de novo mutations in genes mediating synaptic transmission, including GABA receptor subunit genes. Recently, we performed next generation sequencing on patients with a spectrum of epileptic encephalopathy phenotypes, and we identified five novel (A106T, I107T, P282S, R323W and F343L) and one known (R323Q) de novo GABRG2 pathogenic variants (mutations) in eight patients. To gain insight into the molecular basis for how these mutations contribute to epileptic encephalopathies, we compared the effects of the mutations on the properties of recombinant α1β2γ2L GABA receptors transiently expressed in HEK293T cells. Using a combination of patch clamp recording, immunoblotting, confocal imaging and structural modelling, we characterized the effects of these GABRG2 mutations on GABA receptor biogenesis and channel function. Compared with wild-type α1β2γ2L receptors, GABA receptors containing a mutant γ2 subunit had reduced cell surface expression with altered subunit stoichiometry or decreased GABA-evoked whole-cell current amplitudes, but with different levels of reduction. While a causal role of these mutations cannot be established directly from these results, the functional analysis together with the genetic information suggests that these GABRG2 variants may be major contributors to the epileptic encephalopathy phenotypes. Our study further expands the GABRG2 phenotypic spectrum and supports growing evidence that defects in GABAergic neurotransmission participate in the pathogenesis of genetic epilepsies including epileptic encephalopathies.