Sungkwon Chung

NITRIC OXIDE DIRECTLY ACTIVATES CALCIUM-ACTIVATED POTASSIUM CHANNELS FROM RAT BRAIN RECONSTITUTED INTO PLANAR LIPID BILAYER

Using the planar lipid bilayer technique, we tested whether NO directly activates calcium‐activated potassium (Maxi‐K) channels isolated from rat brain. We used streptozotocin (STZ) as NO donor, and the NO release was controlled with light. In the presence of 100–800 μM STZ, the Maxi‐K channel activity increased up to 3‐fold within several tens of seconds after the light was on, and reversed to the control level several minutes after shutting off the light. Similar activation was observed with other NO donors such as S‐nitroso‐N‐acetylpenicillamine and sodium nitroprusside. The degree of activity increase was dependent upon the initial open probability (P init). When the P init was lower, the activity increase was greater. These results demonstrate that NO can directly affect the Maxi‐K channel activity, and suggest that the Maxi‐K channel might be one of the physiological targets of NO in brain.

Expression of Kv1.5 K+ channels in activated microglia in vivo

We examined the expression of outward rectifier K+ channels in activated microglia in vivo. For this purpose, lipopolysaccharide (LPS, 2 μg) was injected into the cortex near the hippocampal region of rat brains, and K+ channel expression was examined using antibodies against shaker‐type K+ channels, Kv1.5 and Kv1.3. OX‐42‐positive microglia were found around the injection sites from 8 h after the LPS injection and remained there for 3 days. The OX‐42‐positive microglia expressed Kv1.5 immunoreactivity, and the time course of Kv1.5 expression was closely correlated with that of OX‐42. In saline‐injected brains, OX‐42‐positive cells also expressed Kv1.5 immunoreactivity even though far fewer OX‐42‐positive cells were found. Increase of Kv1.5 expression after LPS injection was also demonstrated by immunoblot analysis. On the other hand, Kv1.3 immunoreactivity was barely detected in OX‐42‐positive cells over the entire experimental period. The expression of Kv1.5 preceded that of inducible nitric oxide synthase (iNOS), which is a prominent indication of microglial activation. iNOS was not detectable until 12 h, and thereafter it was maintained for 3 days together with Kv1.5 and OX‐42. These results suggest that in vivo as well as in vitro activated microglia expressed outward K+ channels and that some of the channels at least are Kv1.5. GLIA 24:408–414, 1998.

Inward and outward rectifying potassium currents set membrane potentials in activated rat microglia.

Activation of cultured rat microglial cells with lipopolysaccharide (LPS), induced outward rectifying K+ (K(V)) current in addition to already existing inward rectifying K+ current (K(IR)). By measuring zero-current membrane-potentials using whole-cell patch-clamp method, we showed that K(V) current plays a direct role in setting membrane potential to near -45 mV. Since the membrane potentials of microglia show two prominent peaks at -45 and -70 mV, we hypothesize that K(IR) current might set the membrane potential to near -70 mV. We observed that cells with larger K(IR) current had a zero-current membrane-potential at around -70 mV, and that blocking of K(IR) current with Ba2+ depolarized membrane potentials to near -45 mV. These results indicate that the amounts of K(IR), and K(V) current determine the zero-current membrane-potentials in LPS-activated microglia.

Modulation of large conductance Ca2+-activated K+ channel by Gαh (transglutaminase II) in the vascular smooth muscle cell

Abstract Among G-proteins, Gh is unique in structural differences in the GTP-binding domain and possessing transglutaminase activity. We have studied the role of G protein in modulation of large conductance Ca2+-activated K+ (Maxi-K+) channel by the inside-out mode of patch clamp in smooth muscle cells from superior mesenteric artery of the rabbit. When the non-hydrolyzable GTP analogue, GTPγS, was applied, the channel activity was increased about 2.5-fold. Addition of GDPβS resulted in reversal of the GTPγS effect. When the Gαh7 antibody was applied, the GTPγS-stimulated channel activity was significantly inhibited to control level, suggesting that Gαh is involved in activation of the Maxi-K+ channel in smooth muscle cells.

beta-adrenergic modulation of maxi-K channels in vascular smooth muscle via Gi through a membrane-delimited pathway.

Abstract Direct modulation of large-conductance Ca2+-activated K+ (maxi-K) channel by receptor-associated G protein in rabbit mesenteric arterial smooth muscle cells was studied using the outside-out patch clamp technique. Applying a β-adrenoceptor agonist (isoproterenol) increased maxi-K channel activity by 75%, and the effect was almost completely abolished by pretreating the cells with pertussis toxin but not with cholera toxin. When the antibody against Gi protein was present in the pipette solution the stimulatory effect of isoproterenol disap-peared. These results suggest that β-adrenoceptor stimulation increases maxi-K channel activity via a membrane-delimited pathway, probably through pertussis toxin-sensitive G protein (Gi).

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.