Voltage-gated channels maintain cellular resting potentials and generate neuronal action potentials by regulating ion flux. Here, we show that Ether-à -go-go (EAG) K ؉ channels also regulate intracellular signaling pathways by a mechanism that is independent of ion flux and depends on the position of the voltage sensor. Regulation of intracellular signaling was initially inferred from changes in proliferation. Specifically, transfection of NIH 3T3 fibroblasts or C2C12 myoblasts with either wild-type or nonconducting (F456A) eag resulted in dramatic increases in cell density and BrdUrd incorporation over vector-and Shaker-transfected controls. The effect of EAG was independent of serum and unaffected by changes in extracellular calcium. Inhibitors of p38 mitogen-activated protein (MAP) kinases, but not p44͞42 MAP kinases (extracellular signal-regulated kinases), blocked the proliferation induced by nonconducting EAG in serum-free media, and EAG increased p38 MAP kinase activity. Importantly, mutations that increased the proportion of channels in the open state inhibited EAG-induced proliferation, and this effect could not be explained by changes in the surface expression of EAG. These results indicate that channel conformation is a switch for the signaling activity of EAG and suggest an alternative mechanism for linking channel activity to the activity of intracellular messengers, a role that previously has been ascribed only to channels that regulate calcium influx.intracellular messenger ͉ mitogen-activated protein kinase ͉ neuromodulation ͉ proliferation ͉ gating V oltage-gated ion channels generate neuronal action potentials, the primary units of information transfer in the brain, by regulating ion f lux (1). Effects of ion channels on synaptic connectivity, transmitter release, plasticity, and other cellular processes are generally assumed to be a secondary consequence of ion f lux. Specifically, changes in membrane potential and action potentials alter Ca 2ϩ inf lux, and Ca 2ϩ regulates multiple intracellular signaling pathways (2-7). Several recent studies, however, have indicated that some voltagegated ion channels are bifunctional proteins (5, 8 -11). These studies show that voltage-gated channels can contribute to transcriptional regulation, protein scaffolding, cell adhesion, and intracellular signaling, and the effects appear largely independent of ion conduction. Recent studies of Ether-á-go-go [EAG (KCNH1)] voltagedependent K ϩ channels suggest that EAG may also be bifunctional. First, a region of Drosophila EAG with similarity to the autoinhibitory domain of Ca 2ϩ ͞calmodulin-dependent protein kinase II can associate with activated, Ca 2ϩ ͞calmodulin-bound Ca 2ϩ ͞calmodulin-dependent protein kinase II. In vitro assays indicate that, once Ca 2ϩ levels decline, EAG-bound kinase retains 5-10% of its maximum Ca 2ϩ -stimulated activity (12). Second, human EAG has been implicated in cell-cycle regulation and cancer: transfection can induce oncogenic transformation, EAG is present in some cancer cell line...

Hegle AP, Nazzari H, Roth A, Angoli D, Accili EA. Evolutionary emergence of N-glycosylation as a variable promoter of HCN channel surface expression. Am J Physiol Cell Physiol 298: C1066 -C1076, 2010. First published February 3, 2010 doi:10.1152/ajpcell.00389.2009.-All four mammalian hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel isoforms have been shown to undergo N-linked glycosylation in the brain. With the mouse HCN2 isoform as a prototype, HCN channels have further been suggested to require N-glycosylation for function, a provocative finding that would make them unique in the voltage-gated potassium channel superfamily. Here, we show that both the HCN1 and HCN2 isoforms are also predominantly N-glycosylated in the embryonic heart, where they are found in significant amounts and where HCN-mediated currents are known to regulate beating frequency. Surprisingly, we find that N-glycosylation is not required for HCN2 function, although its cell surface expression is highly dependent on the presence of N-glycans. Comparatively, disruption of N-glycosylation only modestly impacts cell surface expression of HCN1 and leaves permeation and gating functions almost unchanged. This difference between HCN1 and HCN2 is consistent with evolutionary trajectories that diverged in an isoform-specific manner after gene duplication from a common HCN ancestor that lacked N-glycosylation and was able to localize efficiently to the cell surface. ion channels; hyperpolarization-activated cyclic nucleotide-modulated channel-mediated current; N-linked glycosylation; embryonic heart; molecular evolution AN OUTSTANDING QUESTION in hyperpolarization-activated cyclic nucleotide-modulated (HCN) channel biology, and indeed in the biology of most intrinsic proteins of the plasma membrane, is how expression at the cell surface is regulated to affect functional heterogeneity in health and pathological states. The number of HCN channels on the cell surface is a critical determinant of beating frequency in cardiac conduction tissue. HCN isoforms and HCN-mediated currents (I h ) are upregulated in the embryonic and neonatal ventricle (24,45,53,56), in the neonatal sinoatrial node (1), and in beating embryonic stem cells during development in culture (27,36,40,41). However, the factors that determine HCN channel supply at the cell surface during cardiac development have been studied in only a limited fashion.An important determinant of plasma membrane expression of intrinsic membrane proteins is N-linked glycosylation, which promotes proper folding, stability, and oligomeric assembly in the endoplasmic reticulum (ER) and facilitates transport and targeting to the plasma membrane (14, 15). HCN1 and HCN2 are extensively N-glycosylated in mouse brain (31,39,58) and contain only one Asn-X-Ser/Thr consensus sequon for N-glycosylation in the S5 linker, close to the channel pore (Fig.

Signaling complexes are essential for the modulation of excitability within restricted neuronal compartments. Adaptor proteins are the scaffold around which signaling complexes are organized. Here, we demonstrate that the Camguk (CMG)/CASK adaptor protein functionally modulates Drosophila Ether-á-go-go (EAG) potassium channels. Coexpression of CMG with EAG in Xenopus oocytes results in a more than twofold average increase in EAG whole-cell conductance. This effect depends on EAG-T787, the residue phosphorylated by calcium-and calmodulin-dependent protein kinase II (Wang et al., 2002). CMG coimmunoprecipitates with wild-type and EAG-T787A channels, indicating that T787, although necessary for the effect of CMG on EAG current, is not required for the formation of the EAG-CMG complex. Both CMG and phosphorylation of T787 increase the surface expression of EAG channels, and in COS-7 cells, EAG recruits CMG to the plasma membrane. The interaction of EAG with CMG requires a noncanonical Src homology 3-binding site beginning at position R1037 of the EAG sequence. Mutation of basic residues, but not neighboring prolines, prevents binding and prevents the increase in EAG conductance. Our findings demonstrate that membrane-associated guanylate kinase adaptor proteins can modulate ion channel function; in the case of CMG, this occurs via an increase in the surface expression and phosphorylation of the EAG channel.

Background Cannabis contains Δ 9 -tetrahydrocannabinol (Δ 9 -THC) and cannabidiol (CBD) as the primary constituents responsible for pharmacological activity. However, there are numerous additional chemically-related structures to Δ 9 –THC and CBD that are pharmacologically active and may influence the pharmacological properties of Δ 9 -THC and CBD. This study chemically characterized the cannabinoid constituents in a series of cannabis chemovar extracts and investigated the potential cannabinoid entourage effect in two behavioral assays. Methods Six chemovar extracts were compared to pure Δ 9 -THC, CBD and morphine for effects on the following behavioral assays in mice: hot plate and tail suspension. The battery of behavioral tests was conducted post intravenous administration of cannabis chemovar extract. Cannabinoid profiles of extracts were analyzed using high performance liquid chromatography. Cannabis extracts were administered at equal doses of Δ 9 -THC to investigate the role of their cannabinoid profiles in modulating the effects of Δ 9 -THC. Dose response curves were fit using a log[inhibitor] vs response three parameter model and differences between group means were determined using a one-way ANOVA followed by a post hoc test. Results Cannabis chemovars tested in this study exhibited substantially different cannabinoid profiles. All chemovars produced dose-dependent immobility in the tail suspension assay and dose-dependent antinociception in the hot plate assay. The maximum antinociceptive effect and ED50 was comparable between cannabis chemovars and Δ 9 -THC. Two cannabis chemovars produced significantly greater immobility in the tail suspension test, with no significant differences in ED50. Conclusions Commercially available cannabis chemovars vary widely in cannabinoid content, but when equalized for Δ 9 -THC content, they produce similar behavioral effects with two exceptions. These findings provide only limited support for the entourage hypothesis. Further studies are necessary to characterize the nature of these pharmacological differences between cannabis chemovars and pure Δ 9 -THC.

Hyperpolarization-activated Cyclic Nucleotide (HCN) channels are voltage-gated cation channels and are critical for regulation of membrane potential in electrically active cells. To understand the evolution of these channels at the molecular level, we cloned and examined two of three HCN homologs of the urochordate Ciona intestinalis (ciHCNa and ciHCNb). ciHCNa is like mammalian HCNs in that it possesses similar electrical function and undergoes N-glycosylation of a sequon near the pore. ciHCNb lacks the pore-associated N-glycosylation sequon and is predictably not N-glycosylated, and it also has an unusual gating phenotype in which the channel's voltage-sensitive gate appears to close incompletely. Together with previous findings, the data support an evolutionary trajectory in which an HCN ancestor underwent lineage-specific duplication in Ciona, to yield one HCN with most features that are conserved with the mammalian HCNs and another HCN that has been uniquely altered.