Involvement of apamin-sensitive SK channels in spike frequency adaptation of neurons in nucleus tractus solitarii of the rat

We delineated the role of Ca²⁺-activated K⁺ channels in the phenomenon of spike frequency adaptation (SFA) exhibited by neurons in the caudal region of nucleus tractus solitarius (cNTS) using intracellular recording coupled with the current-clamp technique in rat brain slices. Intracellular injection of a constant depolarizing current evoked a train of action potentials whose discharge frequency declined rapidly to a lower steady-state level of irregular discharges. This manifested phenomenon of SFA was found to be related to extracellular Ca²⁺. Low Ca²⁺ (0.25 mM) or Cd²⁺ (0.5 mM) in the perfusing medium resulted in a significant increase in the adaptation time constant (_adap) and an appreciable reduction in the percentage adaptation of spike frequency (F_adap). In addition, the evoked discharges were converted from an irregular to a regular pattern, accompanied by a profound increase in mean firing rate. Intriguingly, similar alterations in _adap, F_adap, discharge pattern and discharge rate were elicited by apamin (1 µM), a selective blocker for small-conductance Ca²⁺-activated K⁺ (SK) channels. On the other hand, charybdotoxin (0.1 µM), a selective blocker for large-conductance Ca²⁺-activated K⁺ channels, was ineffective. Our results suggest that SK channels of cNTS neurons may subserve the generation of both SFA and irregular discharge patterns displayed by action potentials evoked with a prolonged depolarizing current.     

Small conductance potassium channels drive ATP-activated chloride secretion in the oviduct

The molecular mechanisms controlling fluid secretion within the oviduct have yet to be determined. As in other epithelia, both secretory and absorptive pathways are likely to work in tandem to drive appropriate ionic movement to support fluid movement across the oviduct epithelium. This study explored the role of potassium channels in basolateral extracellular ATP (ATP(e))-stimulated ion transport in bovine oviduct epithelium using the Ussing chamber short-circuit current (I(SC)) technique. Basal I(SC) in bovine oviduct epithelium comprises both chloride secretion and sodium absorption and was inhibited by treatment with basolateral K(+) channel inhibitors tetrapentlyammonium chloride (TPeA) or BaCl(2). Similarly, ATP-stimulated chloride secretion was significantly attenuated by pretreatment with BaCl(2,) tetraethylammonium (TEA), tolbutamide, and TPeA. Basolateral K(+) current, isolated using nystatin-perforation technique, was rapidly activated by ATP(e), and pretreatment of monolayers with thapsigargin or TPeA abolished this ATP-stimulated K(+) current. To further investigate the type of K(+) channel involved in the ATP response in the bovine oviduct, a number of specific Ca(2+)-activated K(+) channel inhibitors were tested on the ATP-induced ΔI(SC) in intact monolayers. Charbydotoxin, (high conductance and intermediate conductance inhibitor), or paxilline, (high conductance inhibitor) did not significantly alter the ATP(e) response. However, pretreatment with the small conductance inhibitor apamin resulted in a 60% reduction in the response to ATP(e). The presence of small conductance family member KCNN3 was confirmed by RT-PCR and immunohistochemistry. Measurements of intracellular calcium using Fura-2 spectrofluorescence imaging revealed the ability of ATP(e) to increase intracellular calcium in a phospholipase C-inositol 1,4,5-trisphosphate pathway-sensitive manner. In conclusion, these results provide strong evidence that purinergic activation of a calcium-dependent, apamin-sensitive potassium conductance is essential to promote chloride secretion and thus fluid formation in the oviduct.     

培养的大鼠三叉神经节细胞钙激活钾通道的氧化性调节

目的：研究氧化还原药物对三叉神经节细胞离子通道的调节作用。方法：采用全细胞膜片钳电生理方法,记录氧化还原药物对三叉神经节细胞大电导钙激活钾通道（BKca）的影响。结果：蛋氨酸特异性氧化剂chloramine-T（Ch—T）1mmol／L可轻微增加通道电流幅值,但该作用不能被半胱氨酸还原剂1,4-dithio-DL-threitol（DTT）所逆转。相反,半胱氨酸特异性氧化剂5,5’-dithio-bis（2-nitribenzoic acid）（DTNB）500μmol／L降低BKCa的电流幅值,此作用可被DTT 2mmol／L所逆转。结论：ROS通过氧化调节BKCa通道而参与三叉神经节细胞的功能调节,BKCa通道在氧化应激相关性生理、病理状态下起重要的调节作用。     

Ion conductance and ion selectivity of potassium channels in snail neurones

Summary Delayed potassium channels were studied in internally perfused neurone somata from land snails. Relaxation and fluctuation analysis of this class of ion channels revealed Hodgkin-Huxley type K channels with an average single channel conductance ( _K) of 2.40±0.15 pS. The conductance of open channels is independent of voltage and virtually all K channels seem to be open at maximum K conductance (g _K) of the membrane. Voltage dependent time constants of activation ofg _K, calculated from K current relaxation and from cut-off frequencies of power spectra, are very similar indicating dominant first-order kinetics. Ion selectivity of K channels was studied by ion substitution in the external medium and exhibited the following sequence: T1⁺>K⁺>Rb⁺>Cs⁺>NH ₄ ⁺ >Li⁺>Na⁺. The sequence of the alkali cations does not conform to any of the sequences predicted by Eisenman's theory. However, the data are well accommodated by a new theory assuming a single rate-limiting barrier that governs ion movement through the channel.This paper is dedicated to the memory of Walther Wilbrandt.     

Metabolic regulation and dysregulation of endothelial small conductance calcium activated potassium channels

The vascular endothelium is an important regulator of vascular reactivity and preserves the balance between vasoconstrictor and vasodilator tone during normal physiologic conditions. Example endothelial-derived vasoconstrictors include endothelin-1 and thromboxane A2; example vasodilators include nitric oxide and prostacyclin. A growing body of evidence points to the existence of a non-nitric oxide, non-prostacyclin endothelium-derived vasodilatory factor of currently unclear identity, often referred to as endothelium-derived hyperpolarizing factor (EDHF). Recent research testifies to the significance of EDHF in endothelium-dependent vascular smooth muscle relaxation. Special emphasis has been placed on the role of small conductance calcium-activated potassium channels (SK) in facilitating the endothelial and vascular responses to EDHF across the microcirculation, including coronary, mesenteric, and pulmonary vascular beds. Meanwhile, decreased activity of endothelial SK channel activity has been implicated in the pathology of a variety of disease states that alter the balance between vasodilator and vasoconstrictor tone. Hence the primary goal of this review is to characterize the physiology of endothelial SK channels in the microvasculature under normal and pathological conditions. Themes of regulation and dysregulation of SK channel activity through the action of protein kinases, reactive oxygen species, and byproducts of intermediary metabolism provide unifying principles to tie together vascular pathology in altered metabolic states ranging from hypertension to diabetes, to ischemia-reperfusion. A comprehensive understanding of SK channel pathophysiology may provide a foundation for development of new therapeutics targeting SK channels, particularly SK channel potentiators, that may have widespread application for many chronic disease states.     

Contribution of cyclic-nucleotide-gated channels to the resting conductance of olfactory receptor neurons

The basal conductance of unstimulated frog olfactory receptor neurons was investigated using whole-cell and perforated-patch recording. The input conductance, measured between -80 mV and -60 mV, averaged 0.25 nS in physiological saline. Studies were conducted to determine whether part of the input conductance is due to gating of neuronal cyclic-nucleotide-gated (CNG) channels. In support of this idea, the neuronal resting conductance was reduced by each of five treatments that reduce current through CNG channels: external application of divalent cations or amiloride; treatment with either of two adenylate cyclase inhibitors; and application of AMP-PNP, a competitive substrate for adenylate cyclase. The current blocked by divalent cations or by a cyclase inhibitor reversed near 0 mV, as expected for a CNG current. Under physiological conditions, gating of CNG channels contributes approximately 0.06 nS to the resting neuronal conductance. This implies a resting cAMP concentration of 0.1-0.3 micro M. A theoretical model suggests that a neuron containing 0.1-0.3 micro M cAMP is poised to give the largest possible depolarization in response to a very small olfactory stimulus. Although having CNG channels open at rest decreases the voltage change resulting from a given receptor current, it more substantially increases the receptor current resulting from a given increase in [cAMP].     

Small conductance chloride channels in the apical membrane of thyroid cells.

A small conductance chloride channel has been identified on the apical membrane of porcine thyroid cells using the patch-clamp technique. In cell attached membrane patches with NaCl in the pipette, the single channel conductance is 5.5 pS. The channel is highly selective for chloride over gluconate and iodide, and is impermeable to Na+, K+ and tetraethylammonium ions. The open state probability of the channel is not affected by voltage. The channel activity disappears after excision of the patch. The Cl- channel blocker 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) did not affect the activity of the thyroid Cl- channels. Treatment of thyroid cells with 8-(4-chlorophenylthio)adenosine-3',5'-cyclic monophosphate (8-chloro-cAMP) (0.5 mM) prior to giga-seal formation increased Cl- channel activity in the apical membrane of thyroid cells.     

Regulation of intrinsic excitability in hippocampal neurons by activity-dependent modulation of the KV2.1 potassium channel

K_V2.1 is the prominent somatodendritic sustained or delayed rectifier voltage-gated potassium (Kv) channel in mammalian central neurons, and is a target for activity-dependent modulation via calcineurin-dependent dephosphorylation. Using hanatoxin-mediated block of K_V2.1 we show that, in cultured rat hippocampal neurons, glutamate stimulation leads to significant hyperpolarizing shifts in the voltage-dependent activation and inactivation gating properties of the K_V2.1-component of delayed rectifier K⁺ (IK) currents. In computer models of hippocampal neurons, these glutamate-stimulated shifts in the gating of the K_V2.1-component of IK lead to a dramatic suppression of action potential firing frequency. Current-clamp experiments in cultured rat hippocampal neurons showed glutamate-stimulation induced a similar suppression of neuronal firing frequency. Membrane depolarization also resulted in similar hyperpolarizing shifts in the voltage-dependent gating properties of neuronal IK currents, and suppression of neuronal firing. The glutamate-induced effects on neuronal firing were eliminated by hanatoxin, but not by dendrotoxin-K, a blocker of K_V1.1-containing channels. These studies together demonstrate a specific contribution of modulation of K_V2.1 channels in the activity-dependent regulation of intrinsic neuronal excitability.     

Activation-inactivation of potassium channels and development of the potassium-channel spike in internally perfused squid giant axons

A spike that is the result of calcium permeability through potassium channels was separated from the action potential is squid giant axons internally perfused with a 30 mM NaF solution and bathed in a 100 mM CaCl2 solution by blocking sodium channels with tetrodotoxin. Currents through potassium channels were studied under voltage clamp. The records showed a clear voltage-dependent inactivation of the currents. The inactivation was composed of at least two components; one relatively fast, having a time constant of 20--30 ms, and the other very slow, having a time constant of 5--10 s. Voltage clamp was carried out with a variety of salt compositions in both the internal and external solutions. A similar voltage-dependent inactivation, also composed of the two components, was recognized in all the current through potassium channels. Although the direction and intensity of current strongly depended on the salt composition of the solutions, the time-courses of these currents at corresponding voltages were very similar. These results strongly suggest that the inactivation of the currents in attributable to an essential, dynamic property of potassium channels themselves. Thus, the generation of a potassium-channel spike can be understood as an event that occurs when the equilibrium potential across the potassium channel becomes positive.     

Ion conductance and selectivity of single calcium-activated potassium channels in cultured rat muscle

The conductance and selectivity of the Ca-activated K channel in cultured rat muscle was studied. Shifts in the reversal potential of single channel currents when various cations were substituted for Ki+ were used with the Goldman-Hodgkin-Katz equation to calculate relative permeabilities. The selectivity was Tl+ greater than K+ greater than Rb+ greater than NH4+, with permeability ratios of 1.2, 1.0, 0.67, and 0.11. Na+, Li+, and Cs+ were not measurably permeant, with permeabilities less than 0.05 that of K+. Currents with the various ions were typically less than expected on the basis of the permeability ratios, which suggests that the movement of an ion through the channel was not independent of the other ions present. For a fixed activity of Ko+ (77 mM), plots of single channel conductance vs. activity of Ki+ were described by a two-barrier model with a single saturable site. This observation, plus the finding that the permeability ratios of Rb+ and NH+4 to K+ did not change with ion concentration, is consistent with a channel that can contain a maximum of one ion at any time. The empirically determined dissociation constant for the single saturable site was 100 mM, and the maximum calculated conductance for symmetrical solutions of K+ was 640 pS. TEAi+ (tetraethylammonium ion) reduced single channel current amplitude in a voltage-dependent manner. This effect was accounted for by assuming voltage-dependent block by TEA+ (apparent dissociation constant of 60 mM at 0 mV) at a site located 26% of the distance across the membrane potential, starting at the inner side. TEAo+ was much more effective in reducing single channel currents, with an apparent dissociation constant of approximately 0.3 mM.     

神经元钙激活钾电流的特征和功能

钙激活钾电流是由动作电位激活的一类外向钾电流,它是由不同类型的钙激活钾通道所介导。钙激活钾电流参与动作电位复极化和后超极化的电位的形成,并通过调节神经元的放电频率和影响神经元的放电类型参与神经元的多种上生理功能。     

昆虫内向整流钾离子通道的结构与功能

内向整流钾离子通道(inwardly-rectifying potassium channels, Kir)在动物体内承担着重要的生理功能。有关昆虫Kir的研究虽然不多,但近5年来却取得许多重要进展,本文就昆虫Kir近年来的研究进展进行评述。目前对昆虫Kir的研究主要集中在双翅目与半翅目,对基因组的分析以及基因克隆研究表明,昆虫Kir基因数量较少,远低于哺乳动物。双翅目的冈比亚按蚊Anopheles gambiae与埃及伊蚊Aedes aegypti有5~6个Kir基因,黑腹果蝇Drosophila melanogaster仅3个Kir基因,半翅目的褐飞虱Nilaparvata lugens与热带臭虫Cimex lectularius也只有3个Kir基因,而大豆蚜Aphis glycines的Kir基因数则减少到2个,第3个Kir基因的丢失可能与其马氏管的退化有关。系统进化分析表明昆虫Kir具有3个亚家族,但与哺乳动物的7个Kir亚家族没有直系同源关系。尽管如此,昆虫Kir具有与哺乳动物Kir类似的基本结构特征:由4个亚基组成四聚体通道,每个亚基具有2个跨膜区(TM1与TM2),TM1与TM2之间具K^+选择过滤序列。昆虫的Kir基因主要在唾液腺与马氏管中高水平表达,Kir抑制剂可阻断唾液腺与马氏管的分泌活性,从而影响昆虫的取食与排泄活动并使昆虫致死,说明这2类组织器官的分泌活性与Kir有关,Kir介导的K^+跨膜转运驱动了这类组织中上皮细胞的分泌活动。更为重要的发现是氟啶虫酰胺对褐飞虱Kir具有很高的阻断活性,并影响其唾液分泌与排泄功能,证明了Kir就是该杀虫剂的分子靶标。最后本文还对昆虫Kir研究中存在的科学问题进行了分析,展望了开发靶向Kir的新型杀虫剂的研究前景。     

Expression and function of potassium channels in the human placental vasculature

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.     

New insights into the structure and function of potassium channels.

Potassium channels are surprisingly modular proteins. Well-defined regions that determine functional properties such as ion conduction and gating have recently been identified.     

Dual effects of nitric oxide on the large conductance calcium-activated potassium channels of rat brain

Previously, we have shown that nitric oxide (NO) directly activates the Maxi-K channels. In the present study, we have investigated whether NO has prolonged effects on the Maxi-K channels reconstituted in lipid bilayer. Application of S-nitroso-N-acetyl-D, L-penicillamine (SNAP), a NO donor, induced an immediate increase of open probability (Po) of Maxi-K channel in a dose-dependent manner.When SNAP was removed from the cytosolic solution, the Po did not simply returned to, but irreversibly decreased to a level lower than that of the control Po. At 0.2 mM, (Z)-[N-(3-Ammoniopropyl)-N-(n-propyl)amino] diazen-1-ium-1,2-diolate (PAPA-NO), another NO donor, produced a similar increase of Po and decrease of Po upon washout.The increasing effects of SNAP on Po were not blocked by either 50 U/ml superoxide dismutase (SOD) or 2 mM Nethylmaleimide (NEM) pre-treatments. However, NEM appears to be ineffective when applied after SNAP. These results suggest that NO can modulate Maxi-K channel via direct interaction and chemical modification, such as Snitrosylation in the brain.