Voltage-activated potassium channels play an important part in the control of excitability in nerve and muscle. Different K+ channels are involved in establishing the resting potential, determining the duration of action potentials, modulation of transmitter release, and in rhythmic firing patterns and delayed excitation. Using in vitro transcripts made from a directional complementary DNA library we have isolated, by expression cloning in Xenopus oocytes, a novel K+-channel gene (drk1). Functionally, drk1 encodes channels that are K+ selective and belong to the delayed rectifier class of channels, rather than the A-type class encoded by the Shaker gene of Drosophila. The channels show sigmoidal voltage-dependent activation and do not inactivate within 500 ms. Structurally, drk1 encodes an amino-acid sequence which is more closely related to the Drosophila Shab gene than to the Shaker gene.

Ion channels alternate stochastically between two functional states, open and closed. This gating behavior is controlled by membrane potential or by the binding of neurotransmitters in voltage- and ligand-gated channels, respectively. Although much progress has been made in defining the structure and function of the ligand-binding cores and the voltage sensors, how these domains couple to channel opening remains poorly understood. Here we show that the M3 transmembrane segments of the NMDA receptor allosterically interact with both the ligand-binding cores and the channel gate. It is proposed that M3 functions as a transduction element whose conformational change couples ligand binding with channel opening. Furthermore, amino acid homology between glutamate receptor M3 segments and the equivalent S6 or TM2 segments in K(+) channels suggests that ion channel activation and gating are both structurally and functionally conserved.

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Ion permeation and channel opening are two fundamental properties of ion channels, the molecular bases of which are poorly understood. Channels can exist in two permeability states, open and closed. The relative amount of time a channel spends in the open conformation depends on the state of activation. In voltage-gated ion channels, activation involves movement of a charged voltage sensor, which is required for channel opening. Single-channel recordings of drk1 K channels expressed in Xenopus oocytes suggested that intermediate current levels (sublevels) may be associated with transitions between the closed and open states. Because K channels are formed by four identical subunits, each contributing to the lining of the pore, it was hypothesized that these sublevels resulted from heteromeric pore conformations. A formal model based on this hypothesis predicted that sublevels should be more frequently observed in partially activated channels, in which some but not all subunits have undergone voltage-dependent conformational changes required for channel opening. Experiments using the drk1 K channel, as well as drk1 channels with mutations in the pore and in the voltage sensor, showed that the probability of visiting a sublevel correlated with voltage- and time-dependent changes in activation. A subunit basis is proposed for channel opening and permeation in which these processes are coupled.

Single Ion channels are large integral membrane proteins that form ion-selective pores. An important functional determinant is their mechanism of activation, which defines the two major classes of channels (1). Voltage-gated channels are activated by a change in membrane potential, whereas ligand-gated channels are activated by binding of a neurotransmitter to a receptor domain. Analysis of single ion channel behavior using the patch clamp technique has revealed a common functional characteristic: following activation, channels switch stochastically between an open and a closed pore conformation (2). In fact, the kinetic behavior of different channel types may be so similar that they cannot be easily distinguished (Fig. 1A).Although alternate channel-forming motifs exist [e.g., gramicidin (4)], one simple interpretation of this universal binary open/close behavior is that the pore-forming regions are structurally conserved. Ligand-and voltage-gated channels are composed of subunits or domains surrounding a central ion-conducting pore. Despite this common feature, other aspects of quaternary structure remain distinct (Fig. 1 B-D). Voltage-gated channels consist of four domains or subunits (5), while ligand-gated channels consist of five subunits (6-8). Subunits or domains of voltage-dependent channels consist of six putative a-helicalThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 4882 transmembrane segments, S1-S6. The amino acid sequence linking S5 and S6 contains a highly conserved amphiphatic region (H5 or P region), which forms the narrow part of the pore, including the selectivity filter (9-13). The structural model for ligand-gated channels is based mainly on data obtained for the nicotinic acetylcholine (nACh) receptor (14). Hydrophobicity analysis originally predicted the signal peptide-containing amino terminus and the carboxyl terminus to be extracellular and the four putative a-helical transmembrane segments (M1-M4) to each span the lipid bilayer once. The long segment linking M3 and M4 has a cytoplasmic localization in this model.Although the secondary structure is not known, the tertiary structure of the pore region of voltage-gated K channels is a reentrant loop or hairpin, since charybdotoxin binding is affected by mutations flanking the region, positioning both ends of the pore externally (15, 16). Site-directed mutagenesis has implicated M2 in a pore-forming role for many ligandgated channels (17-19). The originally proposed transmembrane topology of M2 (14) has been supported by experimental evidence in the nACh receptors (20, 21). Thus, the pore of voltage-gated channels is thought to be lined by four hairpins, while the pore of ligand-gated channels seems to be lined by five a-helical transmembrane segments.The ligand-gated channel topology model with four transmembrane segments has recently been challenged for the glutamate ...

A novel imaging-based method is introduced to quantitatively localize Golgi proteins at nanometer resolution. The method reveals different intra-Golgi trafficking of secretory cargoes.

Metastasis is the main cause of cancer mortality. During this process, cancer cells dislodge from a primary tumor, enter the circulation and form secondary tumors in distal organs. It is poorly understood how these cells manage to cross the tight syncytium of endothelial cells that lines the capillaries. Such capillary transmigration would require a drastic change in cell shape. We have therefore developed a microfluidic platform to study the transmigration of cancer cells. The device consists of an array of microchannels mimicking the confined spaces encountered. A thin glass coverslip bottom allows high resolution imaging of cell dynamics. We show that nuclear deformation is a critical and rate-limiting step for transmigration of highly metastatic human breast cancer cells. Transmigration was significantly reduced following the treatment with a protein methyltransferase inhibitor, suggesting that chromatin condensation might play an important role. Since transmigration is critical for cancer metastasis, this new platform may be useful for developing improved cancer therapies.

The fact that Parkinson's disease (PD) can arise from numerous genetic mutations suggests a unifying molecular pathology underlying the various genetic backgrounds. To address this hypothesis, we took an integrated approach utilizing in vitro disease modeling and comprehensive transcriptome profiling to advance our understanding of PD progression and the concordant downstream signaling pathways across divergent genetic predispositions. To model PD in vitro, we generated neurons harboring disease-causing mutations from patient-specific, induced pluripotent stem cells (iPSCs). We observed signs of degeneration in midbrain dopaminergic neurons, reflecting the cardinal feature of PD. Gene expression signatures of PD neurons provided molecular insights into disease phenotypes observed in vitro, including oxidative stress vulnerability and altered neuronal activity. Notably, PD neurons show that elevated RBFOX1, a gene previously linked to neurodevelopmental diseases, underlies a pattern of alternative RNA-processing associated with PD-specific phenotypes.