Somatostatin receptor subtype 4 couples to the M-current to regulate seizures.

The K(+) M-current (I(M), Kv7) is an important regulator of cortical excitability, and mutations in these channels cause a seizure disorder in humans. The neuropeptide somatostatin (SST), which has antiepileptic properties, augments I(M) in hippocampal CA1 pyramidal neurons. We used SST receptor knock-out mice and subtype-selective ligands to investigate the receptor subtype that couples to I(M) and mediates the antiepileptic effects of SST. Using pentylenetetrazole as a chemoconvulsant, SST(2), SST(3), and SST(4) receptor knock-out mice all had shorter latencies to different seizure stages and increased seizure severity when compared with wild-type mice. However, the most robust differences were observed in the SST(4) knock-outs. When seizures were induced by systemic injection of kainate, only SST(4) knock-outs showed an increase in seizure sensitivity. We next examined the action of SST and subtype-selective SST agonists on electrophysiological parameters in hippocampal slices of wild-type and receptor knock-out mice. SST(2) and SST(4) appear to mediate the majority of SST inhibition of epileptiform activity in CA1. SST lacked presynaptic effects in mouse CA1, in contrast to our previous findings in rat. SST increased I(M) in CA1 pyramidal neurons of wild-type and SST(2) knock-out mice, but not SST(4) knock-out mice. Using M-channel blockers, we found that SST(4) coupling to M-channels is critical to its inhibition of epileptiform activity. This is the first demonstration of an endogenous enhancer of I(M) that is important in controlling seizure activity. SST(4) receptors could therefore be an important novel target for developing new antiepileptic and antiepileptogenic drugs.

Elevated extracellular K+ inhibits apoptosis of corneal epithelial cells exposed to UV-B radiation.

The goal of this study was to determine if the high [K(+)] in tears, 20-25 mM, serves to protect corneal epithelial cells from going into apoptosis after exposure to ambient UV-B radiation. Human corneal-limbal epithelial (HCLE) cells in culture were exposed to UV-B at doses of 50-200 mJ/cm(2) followed by measurement of K(+) channel activation and activity of apoptotic pathways. Patch-clamp recording showed activation of K(+) channels after UV-B exposure at 80 mJ/cm(2) or 150 mJ/cm(2) and a decrease in UV-induced K(+) efflux with increasing [K(+)](o). The UV-activated current was partially blocked by the specific K(+) channel blocker, BDS-1. DNA fragmentation, as measured by the TUNEL assay, was induced after exposure to UV-B at 100-200 mJ/cm(2). DNA fragmentation was significantly decreased when cells were incubated in 25, 50 or 100mM K(o)(+) after exposure to UV-B. The effector caspase, caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), but there was a significant decrease in activation when the cells were incubated in 25, 50 or 100mM K(o)(+) following exposure to UV-B. A decrease in mitochondrial potential, a possible activator of caspase-3, occurred after exposure to UV-B at 100-200 mJ/cm(2). This decrease in mitochondrial potential was prevented by 100mM K(o)(+); however, 25 or 50mM K(o)(+) provided minimal protection. Caspase-9, which is in the pathway from mitochondrial potential change to caspase-3 activation, showed little activation by UV-B radiation. Caspase-8, an initiator caspase that activates caspase-3, was activated by exposure to UV-B at 50-200 mJ/cm(2), and this UV-activation was significantly reduced by 25-100mM K(o)(+). The data show that the physiologically relevant [K(+)](o) of 25 mM can inhibit UV-B induced activation of apoptotic pathways. This suggests that the relatively high [K(+)] in tears reduces loss of K(+) from corneal epithelial cells in response to UV exposure, thereby contributing to the protection of the ocular surface from ambient UV radiation.

Potassium channel-based optogenetic silencing.

Optogenetics enables manipulation of biological processes with light at high spatio-temporal resolution to control the behavior of cells, networks, or even whole animals. In contrast to the performance of excitatory rhodopsins, the effectiveness of inhibitory optogenetic tools is still insufficient. Here we report a two-component optical silencer system comprising photoactivated adenylyl cyclases (PACs) and the small cyclic nucleotide-gated potassium channel SthK. Activation of this 'PAC-K' silencer by brief pulses of low-intensity blue light causes robust and reversible silencing of cardiomyocyte excitation and neuronal firing. In vivo expression of PAC-K in mouse and zebrafish neurons is well tolerated, where blue light inhibits neuronal activity and blocks motor responses. In combination with red-light absorbing channelrhodopsins, the distinct action spectra of PACs allow independent bimodal control of neuronal activity. PAC-K represents a reliable optogenetic silencer with intrinsic amplification for sustained potassium-mediated hyperpolarization, conferring high operational light sensitivity to the cells of interest.

Ultraviolet irradiation-induced K(+) channel activity involving p53 activation in corneal epithelial cells.

Recent studies from our lab found that ultraviolet (UV) irradiation induces a voltage-gated potassium (Kv) channel activation and subsequently activates JNK signaling pathway resulting in apoptosis. The present study in rabbit corneal epithelial (RCE) cells is to investigate mechanisms of UV irradiation-induced Kv channel activity involving p53 activation in parallel to DNA damage-induced signaling pathway. UV irradiation-induced signaling events were characterized by measurements of JNK activation and further downstream p53 phosphorylation. UV irradiation elicited an early response in the cell membrane through activation of Kv channels to activate the JNK signaling pathway and p53 phosphorylation. Exposure of RCE cells to UV irradiation within a few min resulted in JNK and p53 activations that were markedly inhibited by suppression of Kv channel activity. However, suppression of Kv channel activity failed to prevent p53 activation induced by extended DNA damages through prolonging UV exposure time (more than 15 min). In addition, caffeine inhibited UV-induced activation of SEK, an upstream MAPK kinase of JNK, resulting in suppression of both Kv channel-involved and DNA damage-induced p53 activation. Our results indicate in these cells that UV irradiation induces earlier and later intracellular events that link to activation of JNK and p53. The early event in response to UV irradiation is initiated by activating Kv channels in the cell membrane, and the later event is predominated by UV irradiation-caused DNA damage.

Diclofenac, a nonsteroidal anti-inflammatory drug, activates the transient outward K+ current in rat cerebellar granule cells.

Diclofenac, a nonsteroidal anti-inflammatory drug (NSAID), has been widely investigated in terms of its pharmacological action, but less is known about its direct effect on ion channels. Here, the effect of diclofenac on voltage-dependent transient outward K+ currents (I(A)) in cultured rat cerebellar granule cells was investigated using the whole-cell voltage-clamp technique. At concentrations of 10(-5)-10(-3) M, diclofenac reversibly increased the I(A) amplitude in a dose-dependent manner and significantly modulated the steady-state inactivation properties of the I(A) channels, but did not alter the steady-state activation properties. Furthermore, diclofenac treatment resulted in a slightly accelerated recovery from I(A) channel inactivation. Intracellular application of diclofenac could mimic the effects induced by extracellular application, although once the intracellular response reached a plateau, extracellular application of diclofenac could induce further increases in the current. These observations indicate that diclofenac might exert its effects on the channel protein at both the inner and outer sides of the cell membrane. Our data provide the first evidence that diclofenac is able to activate transient outward potassium channels in neurons. Although further work will be necessary to define the exact mechanism of diclofenac-induced I(A) channel activation, this study provides evidence that the nonsteroidal anti-inflammatory drug, diclofenac, may play a novel neuronal role that is worthy of future study.

Effects of helium-neon laser irradiation and local anesthetics on potassium channels in pond snail neurons.

Intracellular dialysis and membrane voltage clamping were used to show that He-Ne laser irradiation of a pond snail neuron at a dose of 0.7 x 10(-4) J (power density 1.5 x 10(2) W/m2) increases the amplitude of the potential-dependent slow potassium current, while a dose of 0.7 x 10(-3) J decreases this current. Bupivacaine suppresses the potassium current. Combined application of laser irradiation at a dose of 0.7 x 10(-3) J increased the blocking effect of 10 microM bupivacaine on the slow potassium current, while an irradiation dose of 0.7 x 10(-4) J weakened the effect of bupivacaine.

Preliminary microwave irradiation of water solutions changes their channel-modifying activity.

Earlier we have shown that millimetre microwaves (42.25 GHz) of non-thermal power, upon direct admittance into an experiment bath, greatly influence activation characteristics of single Ca(2+)-dependent K+ channels (in particular, the channel open state probability, Po). Here we present new data showing that similar changes in Po arise due to the substitution of a control bath solution for a preliminary microwave irradiated one of the same composition (100 mmol/l KCl with Ca2+ added), with irradiation time being 20-30 min. Therefore, due to the exposure to the field the solution acquires some new properties that are important for the channel activity. The irradiation terminated, the solution retains a new state for at least 10-20 min (solution memory). The data suggest that the effects of the field on the channels are mediated, at least partially, by changes in the solution properties.

Determination of the molecular mass of the native beta-cell sulfonylurea receptor.

In the present study we have determined the molecular mass of the beta-cell sulfonylurea receptor in its native form by two different experimental approaches; gel filtration chromatography and radiation inactivation analysis. We first confirmed that the denatured photolabelled MIN6 beta-cell receptor had a molecular size of 141 +/- 2 kDa (mean +/- S.E., n = 8). Under non-denaturing conditions, using gel filtration chromatography, apparent molecular masses of 166 +/- 1 kDa (mean +/- S.E., n = 3) and 182 +/- 5 kDa (mean +/- S.E., n = 4) were determined for the photoaffinity-labelled and unlabelled sulfonylurea receptor, respectively. We conclude that in the solubilized state the receptor exists as a monomer. Radiation inactivation analysis indicated that the receptor has a target size of 250 +/- 30 kDa (mean +/- S.E., n = 7). This value for the molecular mass is larger than that obtained from SDS-PAGE following photolabelling of the receptor (141 kDa) suggesting that the beta-cell sulfonylurea receptor is composed of more than one subunit in the native membrane.

Gamma irradiation induces acetylcholine-evoked, endothelium-independent relaxation and activates K-channels of isolated pulmonary artery of rats.

PURPOSE: To test the effects of irradiation (R*) on the pulmonary artery (PA). METHODS AND MATERIALS: Isolated PA rings were submitted to gamma irradiation (cesium, 8 Gy/min(-1)) at doses of 20 Gy-140 Gy. Rings were placed in an organ chamber, contracted with serotonin (10(-4) M 5-hydroxytryptamine [5-HT]), then exposed to acetylcholine (ACh) in incremental concentrations. Smooth muscle cell (SMC) membrane potential was measured with microelectrodes. RESULTS: A high dose of irradiation (60 Gy) increased 5HT contraction by 20%, whereas lower (20 Gy) doses slightly decreased it compared with control. In the absence of the endothelium, 5-HT precontracted rings exposed to 20 Gy irradiation developed a dose-dependent relaxation induced by acetylcholine (EI-ACh) with maximal relaxation of 60 +/- 17% (n = 13). This was totally blocked by L-NAME (10(-4) M), partly by 7-nitro indazole; it was abolished by hypoxia and iberiotoxin, decreased by tetra-ethyl-ammonium, and not affected by free radical scavengers. In irradiated rings, hypoxia induced a slight contraction which was never observed in control rings. No differences in SMC membrane potential were observed between irradiated and nonirradiated PA rings. CONCLUSION: Irradiation mediates endothelium independent relaxation by a mechanism involving the nitric oxide pathway and K-channels.

Single K+ channels closed by light and opened by cyclic GMP in molluscan extra-ocular photoreceptor cells.

We report the first recordings of the light-sensitive channel which is active during dark and is closed by light in the Onchidium extra-ocular photoreceptor cells. This light-sensitive channel was K-selective and was not blocked by extracellular Ca2+ and Mg2+. Application of cyclic GMP to excised inside-out patches activated (opened) a channel that appeared to be the same as the light-sensitive channel recorded from the same membrane in the intact cell.

Membrane potential can influence the rate of membrane photomodification.

Though cellular photomodification has been shown to change cellular resting membrane potential, an effect of membrane potential on the rate of photomodification has never been reported. Here we demonstrate that the rate of photomodification of potassium channels in frog atrial cells is voltage dependent. The rate of potassium channel photomodification using negatively charged Rose Bengal as the photosensitizer is about 2.5 times greater at the resting membrane potential of -70 mV compared to +40 mV. Similar results are obtained using the positively charged photosensitizer methylene blue. On the other hand, the rate of photomodified increase of leak current in the same cells does not significantly change in this voltage range with Rose Bengal as photosensitizer, but demonstrates a voltage dependence like that of potassium current when methylene blue is the photosensitizer. These observations cannot be explained based on voltage-dependent partitioning of the sensitizer, as similar effects on potassium current were obtained using either a positively charged or negatively charged sensitizer.

Membrane photomodification of cardiac myocytes: potassium and leakage currents.

Cardiac myocytes were isolated from the atria of frogs (Rana pipiens) and whole cell potassium (IK) and "leakage" (Ileak) currents were monitored using the patch clamp technique. Cells were photosensitized by exposure to Rose Bengal (0.125-0.5 microM). Illumination produced an exponential decrease in IK, and an increase in Ileak. Current modifications varied with light intensity and sensitizer concentration. IK stabilized when illumination ceased, while Ileak continued to increase at a slower rate after illumination ended. The exponential nature of IK modification suggests that potassium channels are photomodified with single hit kinetics. The stabilization of IK following illumination suggests (1) that the photomodification of the potassium channel does not involve long lasting (minutes) radical chain reactions and (2) that this photomodification is not repaired in the course of a few minutes.

GH3 cells, ionic currents and cell killing: photomodification sensitized by Rose Bengal.

Photosensitization using Rose Bengal (RB) modifies membrane ionic currents and kills cultured mouse pituitary, GH3, cells. Here we investigate the dose-response relationship for ionic current modification and for cell killing to assess a possible causal link. When exposed to 0.5 microM RB and 6.5 mW/cm2 of visible light, calcium current was blocked in 1.9 +/- 0.2 min (mean +/- SEM; 0.74 +/- 0.08 J/cm2; n = 18), a transient component of potassium current, tentatively identified as a delayed-rectifier potassium current, disappeared in 52 +/- 8 s (0.34 +/- 0.05 J/cm2; n = 10) and a steady-state component of potassium current, largely a calcium-activated potassium current, disappeared in 3.5 +/- 0.4 min (1.37 +/- 0.16 J/cm2; n = 11). Conversely, the background leak current increased in magnitude. At 5 min of illumination, the longest time studied here, it continued to increase nearly linearly, making it the only current component studied that is still changing after 5 min of light. Under the conditions used, cell killing increased to 100% in the exposure range of 4-10 min of illumination (1.6 J/cm2 to 3.9 J/cm2) when assessed using fluorescent markers, ethidium homodimer and calcein and required slightly longer exposure times when assessed using trypan blue. Thus, it is difficult to ascribe a causal role in cell killing by photosensitization to alterations of standard ion channels and known ionic currents. However, the increase in leak current has the correct dose-response characteristics to be involved.

Mutagenesis in mammalian cells can be modulated by radiation-induced voltage-dependent potassium channels.

In mammalian cells, little is known about the initial events whose ultimate consequence is mutagenesis or DNA repair. The role the plasma membrane may play as an initiator of such a pathway is not understood. We show, for the first time, that membrane voltage-dependent potassium (K+) currents, activated by ionizing radiation (Kuo et al., 1993), play a significant role in radiation mutagenesis. Specifically, we show that the frequency of mutation at the HGPRT locus is increased as expected to 37.6 +/- 4.0 mutations per 100,000 survivors by 800 cGy of ionizing radiation from a spontaneous frequency of 1.5 +/- 1.5. This increase, however, is abolished if either K+ channel blocker, CsCl or BaCl2, is present for 2 h following irradiation of the cells. RbCl, chemically similar to CsCl but known not to block K+ channels, is ineffective in reducing the mutation frequency. Treatment of cells with CsCl or BaCl2 had no effect on radiation-induced cell killing.

Neuronal ion channels and their sensitivity to extremely low frequency weak electric field effects.

Neuronal ion channels are gated pores whose opening and closing is usually regulated by factors such as voltage or ligands. They are often selectively permeable to ions such as sodium, potassium or calcium. Rapid signalling in neurons requires fast voltage sensitive mechanisms for closing and opening the pore. Anything that interferes with the membrane voltage can alter channel gating and comparatively small changes in the gating properties of a channel can have profound effects. Extremely low frequency electrical or magnetic fields are thought to produce, at most, microvolt changes in neuronal membrane potential. At first sight, such changes in membrane potential seem orders of magnitude too small to significantly influence neuronal signalling. However, in the central nervous system, a number of mechanisms exist which amplify signals. This may allow such small changes in membrane potential to induce significant physiological effects.

[Effects of microwave irradiation on ATPase activity and voltage dependent ion channel of rat hippocampus cell membrane].

OBJECTIVE: To study the effect of microwave irradiation on hippocampus cell. METHOD: Changes of ATPase activity and voltage dependent ion channel of hippocampus cell membrane were observed in mice exposed to 2 450 MHz microwave irradiation of 10 mW/cm2 from a physical therapy machine. Histochemical method and patch clamp method were used to determine the activity of Na+, K(+)-ATPase, Ca2+, Mg(2+)-ATPase and voltage dependent Na+, K+, Ca2+ channels respectively. RESULT: 1) Na+, K(+)-ATPase activity of microwave irradiated mice showed no significant change as compared with the control, but the activity of Ca2+, Mg(2+)-ATPase decreased significantly (P< 0.05); 2) In microwave irradiated mice, Na+, K+, Ca2+, current inducement rate in hippocampus neuron decreased significantly, the membrane voltage of Na+ current peak shifted to depolarization, and the attenuation rate of Na+ current and current A inducement rate decreased significantly as compared with control mice. CONCLUSION: Irradiation of 2 450 MHz microwave at a doze of 10 mW/cm2 was not fatal to mice hippocampus cell. But Ca2+, Mg(2+)-ATPase activity of hippocampal cell membrane and voltage dependent Na+, K+, Ca2+ ion channel of hippocampal nervous were affected which would affect study and memory.

Striatal potassium channel dysfunction in Huntington's disease transgenic mice.

Huntington's disease (HD) is a neurodegenerative disorder that mainly affects the projection neurons of the striatum and cerebral cortex. Genetic mouse models of HD have shown that neurons susceptible to the mutation exhibit morphological and electrophysiological dysfunctions before and during development of the behavioral phenotype. We used HD transgenic mouse models to examine inwardly and outwardly rectifying K+ conductances, as well as expression of some related K+ channel subunits. Experiments were conducted in slices and dissociated cells from two mouse models, the R6/2 and TgCAG100, at the beginning and after full development of overt behavioral phenotypes. Striatal medium-sized spiny neurons (MSNs) from symptomatic transgenic mice had increased input resistances, depolarized resting membrane potentials, and reductions in both inwardly and outwardly rectifying K+ currents. These changes were more dramatic in the R6/2 model than in the TgCAG100. Parallel immunofluorescence studies detected decreases in the expression of K+ channel subunit proteins, Kir2.1, Kir2.3, and Kv2.1 in MSNs, which contribute to the formation of the channel ionophores for these currents. Attenuation in K+ conductances and channel subunit expression contribute to altered electrophysiological properties of MSNs and may partially account for selective cellular vulnerability in the striatum.

Site-specific, photochemical proteolysis applied to ion channels in vivo.

A method for site-specific, nitrobenzyl-induced photochemical proteolysis of diverse proteins expressed in living cells has been developed based on the chemistry of the unnatural amino acid (2-nitrophenyl)glycine (Npg). Using the in vivo nonsense codon suppression method for incorporating unnatural amino acids into proteins expressed in Xenopus oocytes, Npg has been incorporated into two ion channels: the Drosophila Shaker B K+ channel and the nicotinic acetylcholine receptor. Functional studies in vivo show that irradiation of proteins containing an Npg residue does lead to peptide backbone cleavage at the site of the novel residue. Using this method, evidence is obtained for an essential functional role of the "signature" Cys128-Cys142 disulfide loop of the nAChR alpha subunit.

The sensitivity of cells and tissues to exogenous fields: effects of target system initial state.

The effect of the initial biochemical or metabolic state of a cell membrane target pathway on its sensitivity to exogenous electromagnetic (EMF) fields is considered. It is shown that the resting or initial transmembrane voltage can affect the frequency response of the membrane pathway and substantially alter the signal to thermal noise threshold (SNR) of the target. EMF sensitivity is examined using a model which describes the response to applied fields of both single cells and cells in gap junction contact via a distributed parameter electrical circuit analog, wherein a voltage-dependent membrane impedance, relating to the initial biochemical state of the target cell(s), is considered. Application of the Hodgkin-Huxley K(+)-conduction pathway membrane to this model results, at a given transmembrane voltage, in a preferential array response to applied field frequencies in the 1-100 Hz range, centered at approximately 16 Hz for 1-10 mm array lengths. Extension of the model to consider the voltage dependence of the Hodgkin-Huxley K+ pathway results in a significant modulation of array frequency response with changing membrane resting potential. The result is EMF sensitivity (SNR) depends upon the initial state of the target tissue, providing a possible explanation of why, e.g., repairing, rather than resting, bone exhibits a physiologically relevant response to certain weak EMF signals.