HCN2 ion channels play a central role in inflammatory and neuropathic pain

The rate of action potential firing in nociceptors is a major determinant of the intensity of pain. Possible modulators of action potential firing include the HCN ion channels, which generate an inward current, I(h), after hyperpolarization of the membrane. We found that genetic deletion of HCN2 removed the cyclic adenosine monophosphate (cAMP)-sensitive component of I(h) and abolished action potential firing caused by an elevation of cAMP in nociceptors. Mice in which HCN2 was specifically deleted in nociceptors expressing Na(V)1.8 had normal pain thresholds, but inflammation did not cause hyperalgesia to heat stimuli. After a nerve lesion, these mice showed no neuropathic pain in response to thermal or mechanical stimuli. Neuropathic pain is therefore initiated by HCN2-driven action potential firing in Na(V)1.8-expressing nociceptors.     

Ion selectivity in a semisynthetic K+ channel locked in the conductive conformation

Potassium channels are K+-selective protein pores in cell membrane. The selectivity filter is the functional unit that allows K+ channels to distinguish potassium (K+) and sodium (Na+) ions. The filter's structure depends on whether K+ or Na+ ions are bound inside it. We synthesized a K+ channel containing the d-enantiomer of alanine in place of a conserved glycine and found by x-ray crystallography that its filter maintains the K+ (conductive) structure in the presence of Na+ and very low concentrations of K+. This channel conducts Na+ in the absence of K+ but not in the presence of K+. These findings demonstrate that the ability of the channel to adapt its structure differently to K+ and Na+ is a fundamental aspect of ion selectivity, as is the ability of multiple K+ ions to compete effectively with Na+ for the conductive filter.     

Molecular basis of gating charge immobilization in Shaker potassium channels

Voltage-dependent ion channels respond to changes in the membrane potential by means of charged voltage sensors intrinsic to the channel protein. Changes in transmembrane potential cause movement of these charged residues, which results in conformational changes in the channel. Movements of the charged sensors can be detected as currents known as gating currents. Measurement of the gating currents of the Drosophila Shaker potassium channel indicates that the charge on the voltage sensor of the channels is progressively immobilized by prolonged depolarizations. The charge is not immobilized in a mutant of the channel that lacks inactivation. These results show that the region of the molecule responsible for inactivation interacts, directly or indirectly, with the voltage sensor to prevent the return of the charge to its original position. The gating transitions between closed states of the channel appear not to be independent, suggesting that the channel subunits interact during activation.     

Crystal structure of the human K2P TRAAK, a lipid- and mechano-sensitive K+ ion channel

TRAAK channels, members of the two-pore domain K(+) (potassium ion) channel family K2P, are expressed almost exclusively in the nervous system and control the resting membrane potential. Their gating is sensitive to polyunsaturated fatty acids, mechanical deformation of the membrane, and temperature changes. Physiologically, these channels appear to control the noxious input threshold for temperature and pressure sensitivity in dorsal root ganglia neurons. We present the crystal structure of human TRAAK at a resolution of 3.8 angstroms. The channel comprises two protomers, each containing two distinct pore domains, which create a two-fold symmetric K(+) channel. The extracellular surface features a helical cap, 35 angstroms tall, that creates a bifurcated pore entryway and accounts for the insensitivity of two-pore domain K(+) channels to inhibitory toxins. Two diagonally opposed gate-forming inner helices form membrane-interacting structures that may underlie this channel's sensitivity to chemical and mechanical properties of the cell membrane.     

Ion channels in yeast

Voltage-dependent ion channels have been found in the plasma membrane of the yeast Saccharomyces cerevisiae. Ion channel activities were recorded from spheroplasts or patches of plasma membrane with the patch-clamp technique. The most prominent activities came from a set of potassium channels with the properties of activation by positive but not negative voltages, high selectivity for potassium over sodium ion, unit conductance of 20 picosiemens, inhibition by tetraethylammonium or barium ions, and bursting kinetics.     

胞外ATP、H2O2、Ca2+与NO调控木榄根系离子平衡机理研究

运用非损伤微测技术(NMT),研究了短期盐胁迫下胞外ATP(eATP)、H2 O2 、Ca2 + 与NO 对非泌盐红树木榄根 系K+/Na+ 平衡的调控作用。NaCl(100 mmol/L,24 h)与等渗甘露醇处理的实验表明,木榄根尖对盐胁迫的响应具 有高度的离子特异性。盐胁迫增强了木榄根尖的Na+ 外流,但Na+ 外流被Na+ /H+ 逆向转运蛋白抑制剂Amiloride 和质膜H+ -ATPase 抑制剂Vanadate 抑制,表明Na+ 外流源于根尖表皮细胞质膜Na+ /H+ 逆向转运系统驱动的Na+ 外排。短期盐胁迫处理能诱导木榄根尖K+ 外流,但被氯化四乙胺(TEA,外向K+ 通道抑制剂)明显抑制,证明K+ 外流是由激活的去极化外向型离子通道KORCs 介导。胞外ATP(300 mol/L)、H2 O2 (10 mmol/L)、Ca2 + (10 mmol/ L)与SNP(NO 供体,100 mol/L)均能增加短期盐胁迫下的Na+ 外流,同时抑制K+ 外流。其中,促进Na+ 外流效果 较强的是H2 O2 和Ca2 + ,而Ca2 + 和NO 抑制K+ 外流的效果突出。这些实验结果表明,胞外ATP、H2 O2 、Ca2 + 与NO 这4 种盐胁迫信使是通过上调木榄根系细胞质膜Na+ /H+ 逆向转运体系(Na+ /H+ 逆向转运体和H+ 泵)活性,在促 进Na+ 和H+ 逆向跨膜转运的同时,抑制去极化激活的K+ 离子通道来减少盐诱导的K+ 外流。      

Atrial natriuretic peptide inhibits a cation channel in renal inner medullary collecting duct cells

The patch-clamp technique was used to examine the effects of atrial natriuretic peptide (ANP) and its second messenger guanosine 3',5'-monophosphate (cGMP) on an amiloride-sensitive cation channel in the apical membrane of renal inner medullary collecting duct cells. Both ANP (10(-11) M) and dibutyryl guanosine 3',5'-monophosphate (10(-4) M) inhibited the channel in cell-attached patches, and cGMP (10(-5) M) inhibited the channel in inside-out patches. The inner medullary collecting duct is the first tissue in which ANP, via its second messenger cGMP, has been shown to regulate single ion channels. The results suggest that the natriuretic action of ANP is related in part to cGMP-mediated inhibition of electrogenic Na+ absorption by the inner medullary collecting duct.     

Functional conversion between A-type and delayed rectifier K+ channels by membrane lipids

Voltage-gated potassium (Kv) channels control action potential repolarization, interspike membrane potential, and action potential frequency in excitable cells. It is thought that the combinatorial association between distinct alpha and beta subunits determines whether Kv channels function as non-inactivating delayed rectifiers or as rapidly inactivating A-type channels. We show that membrane lipids can convert A-type channels into delayed rectifiers and vice versa. Phosphoinositides remove N-type inactivation from A-type channels by immobilizing the inactivation domains. Conversely, arachidonic acid and its amide anandamide endow delayed rectifiers with rapid voltage-dependent inactivation. The bidirectional control of Kv channel gating by lipids may provide a mechanism for the dynamic regulation of electrical signaling in the nervous system.     

Exchange of conduction pathways between two related K+ channels

The structure of the ion conduction pathway or pore of voltage-gated ion channels is unknown, although the linker between the membrane spanning segments S5 and S6 has been suggested to form part of the pore in potassium channels. To test whether this region controls potassium channel conduction, a 21-amino acid segment of the S5-S6 linker was transplanted from the voltage-activated potassium channel NGK2 to another potassium channel DRK1, which has very different pore properties. In the resulting chimeric channel, the single channel conductance and blockade by external and internal tetraethylammonium (TEA) ion were characteristic of the donor NGK2 channel. Thus, this 21-amino acid segment controls the essential biophysical properties of the pore and may form the conduction pathway of these potassium channels.     

Voltage sensor of Kv1.2: structural basis of electromechanical coupling

Voltage-dependent ion channels contain voltage sensors that allow them to switch between nonconductive and conductive states over the narrow range of a few hundredths of a volt. We investigated the mechanism by which these channels sense cell membrane voltage by determining the x-ray crystal structure of a mammalian Shaker family potassium ion (K+) channel. The voltage-dependent K+ channel Kv1.2 grew three-dimensional crystals, with an internal arrangement that left the voltage sensors in an apparently native conformation, allowing us to reach three important conclusions. First, the voltage sensors are essentially independent domains inside the membrane. Second, they perform mechanical work on the pore through the S4-S5 linker helices, which are positioned to constrict or dilate the S6 inner helices of the pore. Third, in the open conformation, two of the four conserved Arg residues on S4 are on a lipid-facing surface and two are buried in the voltage sensor. The structure offers a simple picture of how membrane voltage influences the open probability of the channel.     

Sequence of a probable potassium channel component encoded at Shaker locus of Drosophila

Potassium currents are crucial for the repolarization of electrically excitable membranes, a role that makes potassium channels a target for physiological modifications that alter synaptic efficacy. The Shaker locus of Drosophila is thought to encode a K+ channel. The sequence of two complementary DNA clones from the Shaker locus is reported here. The sequence predicts an integral membrane protein of 70,200 daltons containing seven potential membrane-spanning sequences. In addition, the predicted protein is homologous to the vertebrate sodium channel in a region previously proposed to be involved in the voltage-dependent activation of the Na+ channel. These results support the hypothesis that Shaker encodes a structural component of a voltage-dependent K+ channel and suggest a conserved mechanism for voltage activation.     

Mutations affecting TEA blockade and ion permeation in voltage-activated K+ channels

Voltage-dependent ion channels are responsible for electrical signaling in neurons and other cells. The main classes of voltage-dependent channels (sodium-, calcium-, and potassium-selective channels) have closely related molecular structures. For one member of this superfamily, the transiently voltage-activated Shaker H4 potassium channel, specific amino acid residues have now been identified that affect channel blockade by the small ion tetraethylammonium, as well as the conduction of ions through the pore. Furthermore, variation at one of these amino acid positions among naturally occurring potassium channels may account for most of their differences in sensitivity to tetraethylammonium.     

Mitogens and oncogenes can block the induction of specific voltage-gated ion channels

The mechanisms underlying the ontogeny of voltage-gated ion channels in muscle are unknown. Whether expression of voltage-gated channels is dependent on mitogen withdrawal and growth arrest, as is generally true for the induction of muscle-specific gene products, was investigated in the BC3H1 muscle cell line by patch-clamp techniques. Differentiated BC3H1 myocytes expressed functional Ca2+ and Na+ channels that correspond to those found in T tubules of skeletal muscle. However, Ca2+ and Na+ channels were first detected after about 5 days of mitogen withdrawal. In order to test whether cellular oncogenes, as surrogates for exogenous growth factors, could prevent the expression of ion channels whose induction was contingent on mitogen withdrawal, BC3H1 cells were modified by stable transfection with oncogene expression vectors. Expression vectors containing v-erbB, or c-myc under the control of the SV40 promoter, delayed but did not prevent the appearance of functional Ca2+ and Na+ channels. In contrast, transfection with a Val12 c-H-ras vector, or cotransfection of c-myc together with v-erbB, suppressed the formation of functional Ca2+ and Na+ channels for greater than or equal to 4 weeks. Potassium channels were affected neither by mitogenic medium nor by transfected oncogenes. Thus, the selective effects of certain oncogenes on ion channel induction corresponded to the suppressive effects of mitogenic medium.     

Functional differences between two classes of sodium channels in developing rat skeletal muscle

Excitability is generated in developing skeletal muscle by the incorporation of sodium-selective ion channels into the surface membrane. Whole-cell and patch voltage-clamp recording from myotubes and their embryologic precursors, myoblasts, indicated that voltage-activated sodium current in myoblasts was more resistant to block by tetrodotoxin (TTX) than that in myotubes. Single-channel recording from both cell types showed two classes of sodium channels. One class had a lower single-channel conductance, activated at more hyperpolarized voltages, and was more resistant to TTX than the other. The proportion of TTX-resistant to TTX-sensitive sodium channels was higher in myoblasts than in myotubes. Thus, the difference in TTX sensitivity between myoblasts and myotubes can be explained by a difference in the proportion of the two classes of sodium channels. In addition, the lower conductance of TTX-resistant channels provides insight into the relationship between the TTX binding site and the external mouth of the sodium channel.     

Distal initiation and active propagation of action potentials in interneuron dendrites

Fast and reliable activation of inhibitory interneurons is critical for the stability of cortical neuronal networks. Active conductances in dendrites may facilitate interneuron activation, but direct experimental evidence was unavailable. Patch-clamp recordings from dendrites of hippocampal oriens-alveus interneurons revealed high densities of voltage-gated sodium and potassium ion channels. Simultaneous recordings from dendrites and somata suggested that action potential initiation occurs preferentially in the axon with long threshold stimuli, but can be shifted to somatodendritic sites when brief stimuli are applied. After initiation, action potentials propagate over the somatodendritic domain with constant amplitude, high velocity, and reliability, even during high-frequency trains.     

Changes in sodium channel gating produced by point mutations in a cytoplasmic linker

Voltage-gated sodium channels are transmembrane proteins of approximately 2000 amino acids and consist of four homologous domains (I through IV). In current topographical models, domains III and IV are linked by a highly conserved cytoplasmic sequence of amino acids. Disruptions of the III-IV linker by cleavage or antibody binding slow inactivation, the depolarization-induced closed state characteristic of sodium channels. This linker might be the positively charged "ball" that is thought to cause inactivation by occluding the open channel. Therefore, groups of two or three contiguous lysines were neutralized or a glutamate was substituted for an arginine in the III-IV linker of type III rat brain sodium channels. In all cases, inactivation occurred more rapidly rather than more slowly, contrary to predictions. Furthermore, activation was delayed in the arginine to glutamate mutation. Hence, the III-IV linker does not simply act as a charged blocker of the channel but instead influences all aspects of sodium channel gating.     

昆虫离子通道及抗性的研究进展

对钠、氯、钾和钙离子通道的结构、药理、分子抗性及基因调控机理的研究进展进行了综述，认为研究昆虫离子通道对于害虫抗性治理和开发新农药具有重要意义。     

Glucose, sulfonylureas, and neurotransmitter release: role of ATP-sensitive K+ channels

Sulfonylurea-sensitive adenosine triphosphate (ATP)-regulated potassium (KATP) channels are present in brain cells and play a role in neurosecretion at nerve terminals. KATP channels in substantia nigra, a brain region that shows high sulfonylurea binding, are inactivated by high glucose concentrations and by antidiabetic sulfonylureas and are activated by ATP depletion and anoxia. KATP channel inhibition leads to activation of gamma-aminobutyric acid (GABA) release, whereas KATP channel activation leads to inhibition of GABA release. These channels may be involved in the response of the brain to hyper- and hypoglycemia (in diabetes) and ischemia or anoxia.     

Calmodulin activation of calcium-dependent sodium channels in excised membrane patches of Paramecium

Calmodulin is a calcium-binding protein that participates in the transduction of calcium signals. The electric phenotypes of calmodulin mutants of Paramecium have suggested that the protein may regulate some calcium-dependent ion channels. Calcium-dependent sodium single channels in excised patches of the plasma membrane from Paramecium were identified, and their activity was shown to decrease after brief exposure to submicromolar concentrations of calcium. Channel activity was restored to these inactivated patches by adding calmodulin that was isolated from Paramecium to the cytoplasmic surface. This restoration of channel activity did not require adenosine triphosphate and therefore, probably resulted from direct binding of calmodulin, either to the sodium channel itself or to a channel regulator that was associated with the patch membrane.