植物质膜钾离子转运体研究进展

近年,随着分子生物学技术的不断发展和广泛应用,有关植物质膜钾离子转运体的研究取得重要进展。目前已经克隆到多种质膜钾离子转运体基因并对钾离子转运体生化特性以及结构功能进行了广泛研究。研究认为,质膜钾离子转运体可分为钾离子载体和钾离子通道。钾离子通道又可分为内向性K+通道α亚基、K+通道β亚基及外向性K+通道等三类。本文对上述质膜钾离子转运体的生化特性以及结构功能研究的进展进行了综述。     

High‐resolution structures of transient receptor potential vanilloid channels: Unveiling a functionally diverse group of ion channels

Transient receptor potential vanilloid (TRPV) channels are part of the superfamily of TRP ion channels and play important roles in widespread physiological processes including both neuronal and non‐neuronal pathways. Various diseases such as skeletal abnormalities, chronic pain, and cancer are associated with dysfunction of a TRPV channel. In order to obtain full understanding of disease pathogenesis and create opportunities for therapeutic intervention, it is essential to unravel how these channels function at a molecular level. In the past decade, incredible progress has been made in biochemical sample preparation of large membrane proteins and structural biology techniques, including cryo‐electron microscopy. This has resulted in high resolution structures of all TRPV channels, which has provided novel insights into the molecular mechanisms of channel gating and regulation that will be summarized in this review.     

Recent advances in ion channel research

The field of ion channels has entered into a rapid phase of development in the last few years, partly due to the breakthroughs in determination of the crystal structures of membrane proteins and advances in computer simulations of biomolecules. These advances have finally enabled the long-dreamed goal of relating function of a channel to its underlying molecular structure. Here we present simplified accounts of the competing permeation theories and then discuss their application to the potassium, gramicidin A and calcium channels.     

Recent advances in ion channel research

The field of ion channels has entered into a rapid phase of development in the last few years, partly due to the breakthroughs in determination of the crystal structures of membrane proteins and advances in computer simulations of biomolecules. These advances have finally enabled the long-dreamed goal of relating function of a channel to its underlying molecular structure. Here we present simplified accounts of the competing permeation theories and then discuss their application to the potassium, gramicidin A and calcium channels.     

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research.     

Patch-Clamp Fluorometry: Electrophysiology meets Fluorescence

Ion channels and transporters are membrane proteins whose functions are driven by conformational changes. Classical biophysical techniques provide insight into either the structure or the function of these proteins, but a full understanding of their behavior requires a correlation of both these aspects in time. Patch-clamp and voltage-clamp fluorometry combine spectroscopic and electrophysiological techniques to simultaneously detect conformational changes and ionic currents across the membrane. Since its introduction, patch-clamp fluorometry has been responsible for invaluable advances in our knowledge of ion channel biophysics. Over the years, the technique has been applied to many different ion channel families to address several biophysical questions with a variety of spectroscopic approaches and electrophysiological configurations. This review illustrates the strength and the flexibility of patch-clamp fluorometry, demonstrating its potential as a tool for future research.     

钙离子通道蛋白的研究进展

钙离子是最广泛存在的细胞内信使,调控着几乎所有生命过程。最近的结构生物学研究解析了很多不同种类的钙离子通道在不同开放-关闭状态下的近原子分辨率结构。有关进展揭示了这些通道的分子组成、动态活动、生理功能、调控修饰的分子基础,为阐明钙信号转导和相关疾病的微观机制提供了理论基础.     

The structure and function of Na+ channels.

The past year has seen major advances in our understanding of voltage-gated ion channels through a powerful combination of patch-clamp and molecular biological techniques. These approaches have identified regions (in some cases single amino acid residues) that are essential for voltage-dependent activation and inactivation, lining of the pore, and regulation of channel function.     

Phylogenetic analyses of potassium channel auxiliary subunits

Results of recent genome-sequencing projects together with advances in biochemical, molecular genetic and physiological experimentation have allowed discovery of many transport auxiliary subunits. These subunits facilitate the proper movement of substrates across cell membranes. Mutations of any of these subunits can cause catastrophic effects to the transport mechanism and cause certain genetic diseases. Auxiliary subunits of ion channels are of particular interest because of their potential to diversify the transport properties of the principal subunits. Furthermore, ion channel auxiliary subunits may function in the capacity of enhancing surface expression, allowing gating, and providing chaperone-like activities. As a result of their evolutionary histories, these proteins can be grouped exclusively by phylogenetic techniques. Many of these families are found to be restricted to a single kingdom of life while others seem to be ubiquitous. Here we report the results of systematic analyses of three families of ion channel auxiliary subunits. Some subunits contain one or more transmembrane segments while others exist only in the cytoplasm. We have also observed potential horizontal transfer across kingdoms with these auxiliary subunits. In this report, we present tabulated results of homology searches, partial multiple alignments, secondary structure analyses, and phylogenetic trees for these families.     

The dipole moment of membrane proteins: potassium channel protein and beta-subunit.

The mechanism of ion channel opening is one of the most fascinating problems in membrane biology. Based on phenomenological studies, early researchers suggested that the elementary process of ion channel opening may be the intramembrane charge movement or the orientation of dipolar proteins in the channel. In spite of the far reaching significance of these hypotheses, it has not been possible to formulate a comprehensive molecular theory for the mechanism of channel opening. This is because of the lack of the detailed knowledge on the structure of channel proteins. In recent years, however, the research on the structure of channel proteins made marked advances and, at present, we are beginning to have sufficient information on the structure of some of the channel proteins, e.g. potassium-channel protein and beta-subunits. With these new information, we are now ready to have another look at the old hypothesis, in particular, the dipole moment of channel proteins being the voltage sensor for the opening and closing of ion channels. In this paper, the dipole moments of potassium channel protein and beta-subunit, are calculated using X-ray diffraction data. A large dipole moment was found for beta-subunits while the dipole moment of K-channel protein was found to be considerably smaller than that of beta-subunits. These calculations were conducted as a preliminary study of the comprehensive research on the dipolar structure of channel proteins in excitable membranes, above all, sodium channel proteins.     

Transferring knowledge towards understanding the pore stabilizing variations in K+ channels

Recent advances in structural biology underlying mechanisms of channel gating have strengthened our knowledge about how K⁺ channels can be inter-convertible between conductive and non-conductive states. We have reviewed and combined mutagenesis with biochemical, biophysical and structural information in order to understand the critical roles of the pore residues in stabilizing the pore structure and channel open state. We also discuss how the latest knowledge on the K⁺ channel KcsA may provide a step towards better understanding of distinct pore stabilizing differences among diversified K⁺ channels.     

The purification of ion channels from excitable cells

Conclusion It should be emphasized that the problem of isolating a gene for which the gene product has been defined solely by genetic criteria is not trivial, but this approach is potentially very powerful. Not only does it provide a means for determining the sequence and structure of ion channels for which there are no biochemical probes, but it also provides a system in which specific mutations in the channel gene can be correlated with changes in channel function. Although this approach is limited to organisms that are readily manipulated by genetic methods, the results of these studies should be widely applicable. The biophysical properties of ion channels are generally similar from one species to another, suggesting that the structures of the channels have been highly conserved. The combination of biochemical, biophysical and genetic techniques will undoubtedly be exploited more fully in future studies of ion channel structure and function.     

Ion channel regulation by Ras, Rho, and Rab small GTPases

Regulation of ion channels by heterotrimeric guanosine triphosphatases (GTPases), activated by heptathelical membrane receptors, has been the focus of several recent reviews. In comparison, regulation of ion channels by small monomeric G proteins, activated by cytoplasmic guanine nucleotide exchange factors, has been less well reviewed. Small G proteins, molecular switches that control the activity of cellular and membrane proteins, regulate a wide variety of cell functions. Many upstream regulators and downstream effectors of small G proteins now have been isolated. Their modes of activation and action are understood. Recently, ion channels were recognized as physiologically important effectors of small GTPases. Recent advances in understanding how small G proteins regulate the intracellular trafficking and activity of ion channels are discussed here. We aim to provide critical insight into physiological control of ion channel function and the biological consequences of regulation of these important proteins by small, monomeric G proteins.     

Pharmacology of voltage-gated and calcium-activated potassium channels.

Several important new findings have furthered the development of voltage-gated and calcium-activated potassium channel pharmacology. The molecular constituents of several members of these large ion channel families were identified. Small-molecule modulators of some of these channels were reported, including correolide, the first potent, small-molecule, natural product inhibitor of the Shaker family of voltage-gated potassium channels to be disclosed. The initial crystal structure of a bacterial potassium channel was determined; this work gives a physical basis for understanding the mechanisms of ion selectivity and ion conduction. With the recent molecular characterization of a potassium channel structure and the discovery of new templates for channel modulatory agents, the ability to rationally identify and develop potassium channel agonists and antagonists may become a reality in the near future.     

Tethering chemistry and K+ channels

Voltage-gated K(+) channels are dynamic macromolecular machines that open and close in response to changes in membrane potential. These multisubunit membrane-embedded proteins are responsible for governing neuronal excitability, maintaining cardiac rhythmicity, and regulating epithelial electrolyte homeostasis. High resolution crystal structures have provided snapshots of K(+) channels caught in different states with incriminating molecular detail. Nonetheless, the connection between these static images and the specific trajectories of K(+) channel movements is still being resolved by biochemical experimentation. Electrophysiological recordings in the presence of chemical modifying reagents have been a staple in ion channel structure/function studies during both the pre- and post-crystal structure eras. Small molecule tethering agents (chemoselective electrophiles linked to ligands) have proven to be particularly useful tools for defining the architecture and motions of K(+) channels. This Minireview examines the synthesis and utilization of chemical tethering agents to probe and manipulate the assembly, structure, function, and molecular movements of voltage-gated K(+) channel protein complexes.     

Optical switches and triggers for the manipulation of ion channels and pores

Like fluorescence sensing techniques, methods to manipulate proteins with light have produced great advances in recent years. Ion channels have been one of the principal protein targets of photoswitched manipulation. In combination with fluorescence detection of cell signaling, this has enabled non-invasive, all-optical experiments on cell and tissue function, both in vitro and in vivo. Optical manipulation of channels has also provided insights into the mechanism of channel function. Optical control elements can be classified according to their molecular reversibility as non-reversible phototriggers where light breaks a chemical bond (e.g. caged ligands) and as photoswitches that reversibly photoisomerize. Synthetic photoswitches constitute nanoscale actuators that can alter channel function using three different strategies. These include (1) nanotoggles, which are tethered photoswitchable ligands that either activate channels (agonists) or inhibit them (blockers or antagonists), (2) nanokeys, which are untethered (freely diffusing) photoswitchable ligands, and (3) nanotweezers, which are photoswitchable crosslinkers. The properties of such photoswitches are discussed here, with a focus on tethered photoswitchable ligands. The recent literature on optical manipulation of ion channels is reviewed for the different channel families, with special emphasis on the understanding of ligand binding and gating processes, applications in nanobiotechnology, and with attention to future prospects in the field.     

Short-range molecular rearrangements in ion channels detected by tryptophan quenching of bimane fluorescence

Ion channels are allosteric membrane proteins that open and close an ion-permeable pore in response to various stimuli. This gating process provides the regulation that underlies electrical signaling events such as action potentials, postsynaptic potentials, and sensory receptor potentials. Recently, the molecular structures of a number of ion channels and channel domains have been solved by x-ray crystallography. These structures have highlighted a gap in our understanding of the relationship between a channel's function and its structure. Here we introduce a new technique to fill this gap by simultaneously measuring the channel function with the inside-out patch-clamp technique and the channel structure with fluorescence spectroscopy. The structure and dynamics of short-range interactions in the channel can be measured by the presence of quenching of a covalently attached bimane fluorophore by a nearby tryptophan residue in the channel. This approach was applied to study the gating rearrangements in the bovine rod cyclic nucleotide-gated ion channel CNGA1 where it was found that C481 moves towards A461 during the opening allosteric transition induced by cyclic nucleotide. The approach offers new hope for elucidating the gating rearrangements in channels of known structure.