Fañanas cells-the forgotten cerebellar glia cell type: Immunocytochemistry reveals two potassium channel-related polypeptides, Kv2.2 and Calsenilin (KChIP3) as potential marker proteins.

For long times astrocytes had been regarded as supporting cells, passively filling the spaces between neuronal cell bodies and their extensions. Now it is known that astrocytes are actively involved in a variety of important biological functions such as regulating cerebral blood flow, supporting neuronal metabolism, controlling the extracellular potassium concentration, and clearing neurotransmitters from the extracellular space. In line with this multitude of tasks astrocytes display conspicuous functional and regional heterogeneity. Using three complementary labeling methods nine classes of astrocytes have been differentiated, which were termed protoplasmic, fibrous, velate, radial, and perivascular astrocytes in addition to Bergmann, marginal, and ependymal glial cells. To complete this list retinal Müller cells and a largely forgotten astrocytic cell type, the "feathered cell" of Fañanas need to be added. So far, Fañanas cells could be only recognized with the tedious gold-sublimate procedure. Consequently, data indicating a potential biological function are completely missing. In a parallel investigation we used a battery of antibodies against potassium channels and related proteins to identify potential marker proteins for the immunocytochemical visualization of distinct cell types in the cerebellar cortex. Here we present novel marker proteins, the Kv2.2 potassium channel and calsenilin, to visualize Fañanas cells in the cerebellar Purkinje cell layer. Such markers will allow to identify Fañanas cell subsequent to patching and electrophysiological characterization. This may pave the path to obtain new functional data, which may be helpful to understand the role of these enigmatic cells in normal biological function and disease.

Voltage-sensitive potassium channels expressed after 20-Hydroxyecdysone treatment of a mosquito cell line.

The goal of this research was to express receptors and ion channels in hormone-treated insect cell lines. Treatment of Anopheles gambiae Sua1B cells with 20-hydroxyecdysone showed an inhibition of cell growth over a time course of three days, with no change in cellular morphology. The effect of 20-hydroxyecdysone was enhanced in the presence of the potassium channel blocker 4-aminopyridine, but not tetraethylammonium. Concentration-response curves of 4-aminopyridine in the presence of 42 µM (1 mg/ml) 20-hydroxyecdysone showed similar IC50 values (6-10 µM) across 3 day exposures. Whole cell patch clamp confirmed the expression of delayed-rectifier (Kv2) potassium channels in hormone-supplemented Sua1B cells, whereas untreated Sua1B cells showed no evidence of Kv2 expression. The hormone-induced expression of Kv2 channels occurred in as little as 4 h after treatment, but were not observed after 24 h of exposure to 20-hydroxyecdysone, suggesting they played a role in cell death. The expressed channels had current-voltage relationships diagnostic for the Kv2 subtype, and were inhibited with an IC50 = 13 mM of tetraethylammonium. Overall, these parameters were similar to Anopheles gambiae Kv2 potassium channels expressed in HEK-293 cells. The induced presence of ion channels (and possibly receptors) in these cells has potential utility for high throughput screening and basic neuroscience research.

Neuregulin 1 confers neuroprotection in SOD1-linked amyotrophic lateral sclerosis mice via restoration of C-boutons of spinal motor neurons.

INTRODUCTION: Increasing evidence implicates the role of the cell types surrounding motor neurons, such as interneurons and glial cells, in non-cell autonomous neurodegeneration of amyotrophic lateral sclerosis (ALS). C-boutons, the large cholinergic synapses that innervate spinal α-motor neurons to control their excitability, are progressively lost from motor neurons in both human ALS and mutant Cu/Zn superoxide dismutase 1 (SOD1)-ALS mice. Neuregulin-1 (NRG1), a trophic factor implicated in neural development, transmission, and synaptic plasticity, has been reported to localize in the synapse of C-boutons. However, the roles of NRG1 in maintenance of motor neuron health and activity, as well as the functional consequences of its alteration in motor neuron disease, are not fully understood. RESULTS: NRG1 was localized to the post-synaptic face of C-boutons and its expression was significantly lost in SOD1-ALS mice and human ALS patients. Losses of NRG1 expression and C-boutons occurred almost contemporaneously in SOD1-ALS mice. In addition, expressions of ErbB3 and ErbB4, receptors for NRG1, were reduced in the motor neurons of SOD1-ALS mice. Furthermore, viral-mediated delivery of type III-NRG1 to the spinal cord restored the number of C-boutons and extended the survival time of SOD1-ALS mice. CONCLUSIONS: These results suggest that maintenance of NRG1-ErbB4/3 axis by supplementation of NRG1 confers neuroprotection in motor neuron disease, partly through the maintenance of C-boutons of spinal motor neurons.

The inhibitory effect of shakuyakukanzoto on K+ current in H9c2 cells.

Shakuyakukanzoto (shao-yao-gan-cao-tang) is a commonly used Chinese traditional herbal medicine for the treatment of acute pain with muscle cramp. However, its mechanism of action is unclear. We previously reported that a low concentration of Kanzo (licorice) and isoliquiritigenin, a component of licorice, inhibited the potassium (K(+)) current in H9c2 cells. Therefore, in the present study, we examined the effects of Shakuyakukanzoto, Shakuyaku or Kanzo on the K(+) current (IKur) in H9c2 cells. Shakuyakukanzoto inhibited IKur in a concentration-dependent manner. The half-maximal concentration of Shakuyakukanzoto was approximately 1.3 mg/mL and the Hill coefficient was 1.2. The order of potency of inhibiting IKur was Kanzo>Shakuyakukanzoto>Shakuyaku. Glycyrrhizin, a major component of licorice, had no inhibitory effect on IKur. A small interfering RNA experiment indicated that IKur was most likely to be Kv2.1 in H9c2 cells. Our results suggest that Shakuyakukanzoto may normalize intracellular and extracellular K(+) balance by inhibiting IKur and reducing K(+) efflux, while the Na(+)-K(+) pump promotes K(+) influx into myofibers. Consequently, excess K(+) may be reduced from external space of myofibers. This may be a part of the Shakuyakukanzoto mechanism for improving muscle pain.

Thalamic neuropeptide mediating the effects of nursing on lactation and maternal motivation.

Nursing has important physiological and psychological consequences on mothers during the postpartum period. Tuberoinfundibular peptide of 39 residues (TIP39) may contribute to its effects on prolactin release and maternal motivation. Since TIP39-containing fibers and the receptor for TIP39, the parathyroid hormone 2 receptor (PTH2 receptor) are abundant in the arcuate nucleus and the medial preoptic area, we antagonized TIP39 action locally to reveal its actions. Mediobasal hypothalamic injection of a virus encoding an antagonist of the PTH2 receptor markedly decreased basal serum prolactin levels and the suckling-induced prolactin release. In contrast, injecting this virus into the preoptic area had no effect on prolactin levels, but did dampen maternal motivation, judged by reduced time in a pup-associated cage during a place preference test. In support of an effect of TIP39 on maternal motivation, we observed that TIP39 containing fibers and terminals had the same distribution within the preoptic area as neurons expressing Fos in response to suckling. Furthermore, TIP39 terminals closely apposed the plasma membrane of 82% of Fos-ir neurons. Retrograde tracer injected into the arcuate nucleus and the medial preoptic area labeled TIP39 neurons in the posterior intralaminar complex of the thalamus (PIL), indicating that these cells but not other groups of TIP39 neurons project to these hypothalamic regions. We also found that TIP39 mRNA levels in the PIL markedly increased around parturition and remained elevated throughout the lactation period, demonstrating the availability of the peptide in postpartum mothers. Furthermore, suckling, but not pup exposure without physical contact, increased Fos expression by PIL TIP39 neurons. These results indicate that suckling activates TIP39 neurons in the PIL that affect prolactin release and maternal motivation via projections to the arcuate nucleus and the preoptic area, respectively.

Kv2 channels regulate firing rate in pyramidal neurons from rat sensorimotor cortex.

The largest outward potassium current in the soma of neocortical pyramidal neurons is due to channels containing Kv2.1 α subunits. These channels have been implicated in cellular responses to seizures and ischaemia, mechanisms for intrinsic plasticity and cell death, and responsiveness to anaesthetic agents. Despite their abundance, knowledge of the function of these delayed rectifier channels has been limited by the lack of specific pharmacological agents. To test for functional roles of Kv2 channels in pyramidal cells from somatosensory or motor cortex of rats (layers 2/3 or 5), we transfected cortical neurons with DNA for a Kv2.1 pore mutant (Kv2.1W365C/Y380T: Kv2.1 DN) in an organotypic culture model to manipulate channel expression. Slices were obtained from rats at postnatal days (P7-P14) and maintained in organotypic culture. We used biolistic methods to transfect neurons with gold 'bullets' coated with DNA for the Kv2.1 DN and green fluorescent protein (GFP), GFP alone, or wild type (WT) Kv2.1 plus GFP. Cells that fluoresced green, contained a bullet and responded to positive or negative pressure from the recording pipette were considered to be transfected cells. In each slice, we recorded from a transfected cell and a control non-transfected cell from the same layer and area. Whole-cell voltage-clamp recordings obtained after 3-7 days in culture showed that cells transfected with the Kv2.1 DN had a significant reduction in outward current (∼45% decrease in the total current density measured 200 ms after onset of a voltage step from -78 to -2 mV). Transfection with GFP alone did not affect current amplitude and overexpression of the Kv2.1 WT resulted in greatly increased currents. Current-clamp experiments were used to assess the functional consequences of manipulation of Kv2.1 expression. The results suggest roles for Kv2 channels in controlling membrane potential during the interspike interval (ISI), firing rate, spike frequency adaptation (SFA) and the steady-state gain of firing. Specifically, firing rate and gain were reduced in the Kv2.1 DN cells. The most parsimonious explanation for the effects on firing is that in the absence of Kv2 channels, the membrane remains depolarized during the ISIs, preventing recovery of Na(+) channels from inactivation. Depolarization and the number of inactivated Na(+) channels would build with successive spikes, resulting in slower firing and enhanced spike frequency adaptation in the Kv2.1 DN cells.

Atomic-level simulation of current-voltage relationships in single-file ion channels.

The difficulty in characterizing ion conduction through membrane channels at the level of individual permeation events has made it challenging to elucidate the mechanistic principles underpinning this fundamental physiological process. Using long, all-atom simulations enabled by special-purpose hardware, we studied K(+) permeation across the KV1.2/2.1 voltage-gated potassium channel. At experimentally accessible voltages, which include the physiological range, the simulated permeation rate was substantially lower than the experimentally observed rate. The current-voltage relationship was also nonlinear but became linear at much higher voltages. We observed permeation consistent with a "knock-on" mechanism at all voltages. At high voltages, the permeation rate was in accordance with our previously reported KV1.2 pore-only simulations, after the simulated voltages from the previous study were recalculated using the correct method, new insight into which is provided here. Including the voltage-sensing domains in the simulated channel brought the linear current-voltage regime closer to the experimentally accessible voltages. The simulated permeation rate, however, still underestimated the experimental rate, because formation of the knock-on intermediate occurred too infrequently. Reducing the interaction strength between the ion and the selectivity filter did not increase conductance. In complementary simulations of gramicidin A, similar changes in interaction strength did increase the observed permeation rate. Permeation nevertheless remained substantially below the experimental value, largely because of infrequent ion recruitment into the pore lumen. Despite the need to apply large voltages to simulate the permeation process, the apparent voltage insensitivity of the permeation mechanism suggests that the direct simulation of permeation at the single-ion level can provide fundamental physiological insight into ion channel function. Notably, our simulations suggest that the knock-on permeation mechanisms in KV1.2 and KcsA may be different.

Molecular mechanisms underlying the apoptotic effect of KCNB1 K+ channel oxidation.

Potassium (K(+)) channels are targets of reactive oxygen species in the aging nervous system. KCNB1 (formerly Kv2.1), a voltage-gated K(+) channel abundantly expressed in the cortex and hippocampus, is oxidized in the brains of aging mice and of the triple transgenic 3xTg-AD mouse model of Alzheimer's disease. KCNB1 oxidation acts to enhance apoptosis in mammalian cell lines, whereas a KCNB1 variant resistant to oxidative modification, C73A-KCNB1, is cytoprotective. Here we investigated the molecular mechanisms through which oxidized KCNB1 channels promote apoptosis. Biochemical evidence showed that oxidized KCNB1 channels, which form oligomers held together by disulfide bridges involving Cys-73, accumulated in the plasma membrane as a result of defective endocytosis. In contrast, C73A-mutant channels, which do not oligomerize, were normally internalized. KCNB1 channels localize in lipid rafts, and their internalization was dynamin 2-dependent. Accordingly, cholesterol supplementation reduced apoptosis promoted by oxidation of KCNB1. In contrast, cholesterol depletion exacerbated apoptotic death in a KCNB1-independent fashion. Inhibition of raft-associating c-Src tyrosine kinase and downstream JNK kinase by pharmacological and molecular means suppressed the pro-apoptotic effect of KCNB1 oxidation. Together, these data suggest that the accumulation of KCNB1 oligomers in the membrane disrupts planar lipid raft integrity and causes apoptosis via activating the c-Src/JNK signaling pathway.

Effects of sodium tanshinone II A sulphonate on hypoxic pulmonary hypertension in rats in vivo and on Kv2.1 expression in pulmonary artery smooth muscle cells in vitro.

AIM OF THE STUDY: To investigate the effect of sodium tanshinone IIA sulphonate (STS), a water-soluble derivative of tanshinone II A, on hypoxic pulmonary hypertension (HPH) in rats and its underlying mechanisms. MATERIALS AND METHODS: Rats were exposed to hypoxia for two or three weeks, pretreated with or without STS. We detected mean pulmonary arterial pressure (mPAP), the ratio of right ventricle weight to left ventricle with septum weight [RV/(LV+S)], wall thickness and voltage-activated potassium channel (Kv) 2.1 mRNA level of pulmonary arteries (PAs), respectively, and the in vitro effects of STS on proliferation and Kv2.1 expression of cultured pulmonary smooth muscle cells (PASMCs) from normal rats. Cell proliferation was determined by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyltetrazoliumbromiede (MTT) assay and direct cell counting. Kv2.1 mRNA and protein level were evaluated by reverse transcription-polymerase chain reaction and Western blot, respectively. RESULTS: Chronic hypoxia increased values of mPAP and RV/(LV+S) and inhibited Kv2.1 mRNA level in PAs. Three weeks' daily STS pretreatment inhibited the hypoxia-induced increased mPAP and RV/(LV+S), pulmonary arterial thickening and up-regulated Kv2.1 mRNA level in PAs. Further study in vitro showed that STS suppressed significantly hypoxia-induced PASMCs proliferation and inhibition of Kv2.1 expression in PASMCs. CONCLUSIONS: STS might play protective effects on HPH through decreasing mPAP, V/(LV+S) and inhibiting structural remodeling in distal PAs. The mechanism of these effects may be attributed to inhibiting PASMCs proliferation and stimulating Kv2.1 expression.

Mutation of histidine 105 in the T1 domain of the potassium channel Kv2.1 disrupts heteromerization with Kv6.3 and Kv6.4.

The voltage-activated K(+) channel subunit Kv2.1 can form heterotetramers with members of the Kv6 subfamily, generating channels with biophysical properties different from homomeric Kv2.1 channels. The N-terminal tetramerization domain (T1) has been shown previously to play a role in Kv channel assembly, but the mechanisms controlling specific heteromeric assembly are still unclear. In Kv6.x channels the histidine residue of the zinc ion-coordinating C3H1 motif of Kv2.1 is replaced by arginine or valine. Using a yeast two-hybrid assay, we found that substitution of the corresponding histidine 105 in Kv2.1 by valine (H105V) or arginine (H105R) disrupted the interaction of the T1 domain of Kv2.1 with the T1 domains of both Kv6.3 and Kv6.4, whereas interaction of the T1 domain of Kv2.1 with itself was unaffected by this mutation. Using fluorescence resonance energy transfer (FRET), interaction could be detected between the subunits Kv2.1/Kv2.1, Kv2.1/Kv6.3, and Kv2.1/Kv6.4. Reduced FRET signals were obtained after co-expression of Kv2.1(H105V) or Kv2.1(H105R) with Kv6.3 or Kv6.4. Wild-type Kv2.1 but not Kv2.1(H105V) could be co-immunoprecipitated with Kv6.4. Co-expression of dominant-negative mutants of Kv6.3 reduced the current produced Kv2.1, but not of Kv2.1(H105R) mutants. Co-expression of Kv6.3 or Kv6.4 with wt Kv2.1 but not with Kv2.1(H105V) or Kv2.1(H105R) changed the voltage dependence of activation of the channels. Our results suggest that His-105 in the T1 domain of Kv2.1 is required for functional heteromerization with members of the Kv6 subfamily. We conclude from our findings that Kv2.1 and Kv6.x subunits have complementary T1 domains that control selective heteromerization.

Insensitivity of cardiac delayed-rectifier I(Kr) to tyrosine phosphorylation inhibitors and stimulators.

1. The rapidly activating delayed-rectifying K+ current (I(Kr)) in heart cells is an important determinant of repolarisation, and decreases in its density are implicated in acquired and inherited long QT syndromes. The objective of the present study on I(Kr) in guinea-pig ventricular myocytes was to evaluate whether the current is acutely regulated by tyrosine phosphorylation. 2. Myocytes configured for ruptured-patch or perforated-patch voltage-clamp were depolarised with 200-ms steps to 0 mV for measurement of I(Kr) tail amplitude on repolarisations to -40 mV. 3. I(Kr) in both ruptured-patch and perforated-patch myocytes was only moderately (14-20%) decreased by 100 microM concentrations of protein tyrosine kinase (PTK) inhibitors tyrphostin A23, tyrphostin A25, and genistein. However, similar-sized decreases were induced by PTK-inactive analogues tyrphostin A1 and daidzein, suggesting that they were unrelated to inhibition of PTK. 4. Ruptured-patch and perforated-patch myocytes were also treated with promoters of tyrosine phosphorylation, including phosphotyrosyl phosphatase (PTP) inhibitor orthovanadate, exogenous c-Src PTK, and four receptor PTK activators (insulin, insulin-like growth factor-1, epidermal growth factor, and basic fibroblast growth factor). None of these treatments had a significant effect on the amplitude of I(Kr). 5. We conclude that Kr channels in guinea-pig ventricular myocytes are unlikely to be regulated by PTK and PTP.