慢性心衰大鼠下丘脑室旁核瞬时外向钾通道蛋白Kv4.2和Kv4.3低表达促进肾交感神经兴奋性

 目的: 观察慢性心衰大鼠下丘脑室旁核内瞬时外向钾通道蛋白Kv4.2和Kv4.3的变化及其对交感神经活性的影响。方法: 采用冠状动脉左前降支结扎术建立大鼠心衰模型或假手术模型,造模4周后超声心动图测定心功能;酶联免疫吸附法测定血浆去甲肾上腺素(NE)及血清N端前脑钠肽(NT-proBNP)含量;Western blot和real-time PCR法测定室旁核内Kv4.2和Kv4.3的表达情况;室旁核部位注射钾通道阻滞剂4-氨基吡啶(4-AP),电生理记录仪记录血压、心率和肾交感神经放电的变化。结果: 与假手术组比,心衰组大鼠心功能明显降低,血浆NE及血清NT-proBNP明显升高,室旁核内Kv4.2和Kv4.3表达明显下调;注射4-AP后导致血压、心率和交感神经放电升高,但心衰组的升高幅度小于假手术组。结论: 心力衰竭时室旁核内Kv4.2和Kv4.3表达下调并伴有交感神经放电增加,促进心衰进展。     

Restoration of cardiac transient outward potassium current by norepinephrine in diabetic rats

In cardiac ventricle, the density of the transient outward potassium current, Ito, is clearly related to sympathetic nervous system integrity. This sympathetic regulation of Ito expression may be greatly significant to the genesis of cardiac complications of several diseases such us diabetes mellitus. Autonomic neuropathy, including cardiac neuropathy, is a complication of chronic diabetes. The objective of the present study was to identify the possible role of cardiac sympathetic neuropathy in the reduction of Ito current density in diabetic ventricular myocardium. Thus, we employed the patch-clamp technique to test whether Ito can be restored in diabetic myocytes incubated with norepinephrine. We also measured, using HPLC, the catecholamine content of the stellate ganglion, which is responsible for cardiac sympathetic innervation, in normal and diabetic animals. The main result of the present study was to show that a 24-h incubation of diabetic cells with norepinephrine restores Ito density to control values. The restoration of Ito current density by norepinephrine suggests that the diabetes-induced reduction of Ito is at least partially attributable to a reduced trophic effect of norepinephrine on the expression of Ito.     

Inhibition of transient outward current by intracellular ion substitution unmasks slow inward calcium current in cardiac Purkinje fibers

The ionophore nystatin was used to replace intracellular K⁺ with Cs⁺ in calf Purkinje fibers. Such replacement inhibited the transient outward current, and unmasked a large slow inward current (I_si). These results support the involvement of K⁺ as a charge carrier of the transient outward current, and suggest a method for better analysis of I_si.     

Potentiation of a transient outward current by Na+ influx in crayfish neurones

1. In voltage-clamped motor-giant neurones of the crayfishOrconectes limosus a depolarizing voltage step clicits a transient inward current carried by Na⁺ which is followed by an early and a delayed outward current. 2. The early outward current is reduced if the Na⁺ current is suppressed by tetrodotoxin or the removal of external Na⁺. It is also abolished if the K⁺ channel blocking agents tetraethylammonium and 3,4-diaminopyridine are applied to the neurone. 3. The outward current was not depressed if Li⁺ was substituted for Na⁺ in the external solution or if the Na–K pump was inhibited by ouabain or the removal of external K⁺. 4. Ionophoretic injections of EGTA did not depress the early outward current. 5. Short ionophoretic injections of Na⁺ into the neurone increased the outward current elicited by a depolarization but did not affect the leakage current. 6. It is suggested that the influx of Na⁺ leads to a transient increment of the Na⁺ concentration near K⁺ channels and that internal Na⁺ ions exert an activating or modulating effect on K⁺ channels.     

Biophysical and pharmacological properties of the voltage-gated potassium current of human pancreatic β-cells

Voltage-gated potassium (Kv) currents of human pancreatic islet cells were studied by whole-cell patch clamp recording. On average, 75% of the cells tested were identified as β-cells by single cell, post-recording RT-PCR for insulin mRNA. In most cells, the dominant Kv current was a delayed rectifier. The delayed rectifier activated at potentials above −20 mV and had a V _½ for activation of −5.3 mV. Onset of inactivation was slow for a major component (τ= 3.2 s at +20 mV) observed in all cells; a smaller component (τ= 0.30 s) with an amplitude of ∼25% was seen in some cells. Recovery from inactivation had a τ of 2.5 s at −80 mV and steady-state inactivation had a V _½ of −39 mV. In 12% of cells (21/182) a low-threshold, transient Kv current (A-current) was present. The A-current activated at membrane potentials above −40 mV, inactivated with a time constant of 18.5 ms at −20 mV, and had a V _½ for steady-state inactivation of −52 mV. TEA inhibited total Kv current with an  IC₅₀= 0.54 m m   and PAC, a disubstituted cyclohexyl Kv channel inhibitor, inhibited with an  IC₅₀= 0.57 μ m   . The total Kv current was insensitive to margatoxin (100 n m ), agitoxin-2 (50 n m ), kaliotoxin (50 n m ) and ShK (50 n m ). Hanatoxin (100 n m ) inhibited total Kv current by 65% at +20 mV. Taken together, these data provide evidence of at least two distinct types of Kv channels in human pancreatic β-cells and suggest that more than one type of Kv channel may be involved in the regulation of glucose-dependent insulin secretion.     

Roles of nicotinic acetylcholine receptor β subunits in function of human α4-containing nicotinic receptors

Naturally expressed nicotinic acetylcholine receptors (nAChR) containing α4 subunits (α4*-nAChR) in combination with β2 subunits (α4β2-nAChR) are among the most abundant, high-affinity nicotine binding sites in the mammalian brain. β4 subunits are also richly expressed and colocalize with α4 subunits in several brain regions implicated in behavioural responses to nicotine and nicotine dependence. Thus, α4β4-nAChR also may exist and play important functional roles. In this study, properties were determined of human α4β2- and α4β4-nAChR heterologously expressed de novo in human SH-EP1 epithelial cells. Whole-cell currents mediated via human α4β4-nAChR have ∼4-fold higher amplitude than those mediated via human α4β2-nAChR and exhibit much slower acute desensitization and functional rundown. Nicotinic agonists induce peak whole-cell current responses typically with higher functional potency at α4β4-nAChR than at α4β2-nAChR. Cytisine and lobeline serve as full agonists at α4β4-nAChR but are only partial agonists at α4β2-nAChR. However, nicotinic antagonists, except hexamethonium, have comparable affinities for functional α4β2- and α4β4-nAChR. Whole-cell current responses show stronger inward rectification for α4β2-nAChR than for α4β4-nAChR at a positive holding potential. Collectively, these findings demonstrate that human nAChR β2 or β4 subunits can combine with α4 subunits to generate two forms of α4*-nAChR with distinctive physiological and pharmacological features. Diversity in α4*-nAChR is of potential relevance to nervous system function, disease, and nicotine dependence.     

Regulation of cardiac shal-related potassium channel Kv 4.3 by serum- and glucocorticoid-inducible kinase isoforms in Xenopus oocytes

The human cardiac transient outward potassium current I_to is formed by co-assembly of voltage-dependent K⁺ channel (Kv 4.3) pore-forming -subunits with differently spliced K channel interacting protein (KChIP) accessory proteins. I_to is of considerable importance for the normal course of the cardiac ventricular action potential. The present study was performed to determine whether isoforms of the serum- and glucocorticoid-inducible kinase (SGK) family influence Kv 4.3/KChIP2b channel activity in the Xenopus laevis heterologous expression system. Co-expression of SGK1, but not of SGK2 or SGK3, increased Kv 4.3/KChIP2b channel currents. The up-regulation of the current was not due to changes in the activation curve or changes of channel inactivation. The currents in oocytes expressing Kv 4.3 alone were smaller than those in Kv 4.3/KChIP2b expressing oocytes, but were still stimulated by SGK1. The effect of wild-type SGK1 was mimicked by constitutively active SGK1 (SGK1 S422D) but not by an inactive mutant (SGK1 K127N). The current amplitude increase mediated by SGK1 was not dependent on NEDD4.2 or RAB5, nor did it reflect increased cell surface expression. In conclusion, SGK1 stimulates Kv 4.3 potassium channels expressed in Xenopus oocytes by a novel mechanism distinct from the known NEDD4.2-dependent pathway.     

Temperature and redox state dependence of native Kv2.1 currents in rat pancreatic β-cells

In pancreatic β-cells, voltage-dependent K⁺ (Kv) channels repolarise glucose-stimulated action potentials. Kv channels are therefore negative regulators of Ca²⁺ entry and insulin secretion. We have recently demonstrated that Kv2.1 mediates the majority of β-cell voltage-dependent outward K⁺ current and now investigate the function of native β-cell Kv2.1 channels at near-physiological temperatures (32-35 °C). While β-cell voltage-dependent outward K⁺ currents inactivated little at room temperature, both fast-inactivation (111.5 ± 14.3 ms) and slow-inactivation (1.21 ± 0.12 s) was observed at 32-35 °C. Kv2.1 mediates the fast-inactivating current observed at 32-35 °C, since it could be selectively ablated by expression of a dominant-negative Kv2.1 construct (Kv2.1N). The surprising ability of Kv2.1N to selectively remove the fast-inactivating component, together with its sensitivity to tetraethylammonium (TEA), demonstrate that this component is not mediated by the classically fast-inactivating and TEA-resistant channels such as Kv1.4 and 4.2. Increasing the intracellular redox state by elevating the cytosolic NADPH/NADP⁺ ratio from 1/10 to 10/1 increased the rates of both fast- and slow-inactivation. In addition, increasing the intracellular redox state also increased the relative contribution of the fast-inactivation component from 38.8 ± 2.1 % to 55.9 ± 1.8 %. The present study suggests that, in β-cells, Kv2.1 channels mediate a fast-inactivating K⁺ current at physiological temperatures and may be regulated by the metabolic generation of NADPH.     

Crucial function of the pre-T-cell receptor (TCR) in TCRP selection, TCRβ allelic exclusion and αβ versus γδ lineage commitment

Summary: The analysis of T-cell receptor (TCR) βselection, TCRβ allelic exclusion and TCRβ rearrangement in γδ T cells from normal and pre-TCR-deficient mice has shown that the pre-TCR has a crucial role in T-lyinpbocyte development:

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  The pre-TCR is by far the most effective receptor that generates large numbers of CD4⁺8⁺ T cells with productive TCRβ rearrangements.
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  In the absence of the pre-TCR, TCRβ rearrangement proceeds in developing cells irrespective of whether they already contain a productive TCRβ gene.
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  The pre-TCR directs developing T cells to the αβ lineage because y5 T cells from pTα^-/- mice proceed much further in TCRβ rearrangement than γδ T cells from wild-type mice. It is argued that the pre-TCR commits developing T cells to the αβ lineage by an instructive mechanism, which has largely replaced an evolutionarily more ancient mechanism that involves stochastic αβ lineage commitment.

     

An early outward transient K+ current that depends on a preceding Na+ current and is enhanced by insulin

A whole-cell early transient outward current occurs in rat myoballs if and only if there is an immediatly preceding current of large amplitude through the voltage-gated, tetrodotoxin-inhibitable Na⁺ channel. This early outward transient is a K⁺ current, designated I _K(Na⁺). Under the conditions in which I _K(Na⁺) appears, simultaneous measurement of voltage and current, under voltage clamp, demonstrates that there is transient voltage escape to depolarized levels, peaking at about the time of peak inward Na⁺ current arid resembling an action potential. I _K(Na⁺) was never seen in the absence of this breach of the voltage clamp, suggesting that I _K(Na⁺) might be an artefact due to transient depolarization from the clamp. However, when the voltage escape was mimicked by voltage commands under conditions in which the Na⁺ channel was not activated, there was no I _K(Na). Insulin increased or produced I _K(Na⁺) even though insulin had no effect on I _Na or on the delayed rectifier K⁺ current or on the escape from voltage clamp. It is concluded that there is a population of rat myoballs in which there is an early outward K⁺ current that requires an immediately preceding current through the voltagegated tetrodotoxin-inhibitable Na⁺ channel and is enhanced by insulin.