ATP4− and ATP·Mg inhibit the ATP-sensitive K+ channel of rat ventricular myocytes

K⁺ currents were recorded from ATP-sensitive channels in inside-out membrane patches excised from isolated rat ventricular myocytes. ATP-sensitive K⁺ channel inhibition could be evoked by ATP in the absence of magnesium where most ATP would be present as the free acid ATP^4–. Channel inhibition was enhanced when the same total concentration of ATP was applied in the presence of magnesium, where most ATP would be bound as ATP·Mg. Dose-response relationships for ATP-sensitive K⁺ channel inhibition evoked by ATP had a Hill coefficient of 2 andK _i of 17 and 30 M for ATP in the presence and absence of magnesium respectively. This was the obverse of the expected results if ATP^4– were to be the sole form of ATP to effect channel closure. ATP-sensitive K⁺ channel inhibition evoked by ATPS, AMP-PNP and AMP-PCP was also enhanced in the presence of magnesium. It is concluded that the ATP-sensitive K⁺ channel of rat ventricular myocytes binds and is closed by both the free-acid and divalent-cationbound forms of ATP.     

A potassium current activated by lemakalim and metabolic inhibition in rabbit mesenteric artery

K⁺ channels which are inhibited by intracellular ATP (ATP_i) (K_ATP channels) are thought to be the physiological target site of the K⁺ channel opening drugs (2) and to underlie a variety of physiological phenomena including hypoxia induced vasodilation (3). However, electrophysiological evidence for ATP_i-regulated K⁺ currents in smooth muscle is scarce. We, therefore, investigated the effects of one K⁺ channel opener, lemakalim, and metabolic inhibition on the membrane conductance of freshly dissociated rabbit mesenteric artery smooth muscle cells, using the perforated-patch whole cell recording technique (6). The cells were metabolically inhibited with 1 mM iodoacetic acid and 50 M dinitrophenol. Both lemakalim (0.1–3 M) and metabolic inhibition activated a time-independent and glyburide sensitive K⁺ current at physiological membrane potentials. The similarities between the lemakalim and metabolic inhibition activated currents suggest that a single class of channels underlies both currents. These results are the first whole-cell current recordings to demonstrate the activation of a smooth muscle membrane conductance by metabolic inhibition, lending support to the view that hypoxia induced vasodilation arises from the activation of K_ATP channels.     

Adaptative modifications of right coronary myocytes voltage-gated K+ currents in rat with hypoxic pulmonary hypertension

Chronic hypoxia (CH)-induced pulmonary hypertension (PHT) is well known to alter K+ channels in pulmonary myocytes. PHT induces right ventricle hypertrophy that increases oxygen demand; however, coronary blood flow and K+ channel adaptations of coronary myocytes during PHT remain unknown. We determined whether CH and PHT altered K+ currents and coronary reactivity and what impact they might have on right myocardial perfusion. Right ventricle perfusion, as attested by microspheres, was redistributed toward hypertrophied right ventricle [RV/LV (%)=0.59+/-0.07% in CH rats vs. 0.29+/-0.03 in control rats, P<0.05]. Whole-cell patch clamping showed a reduction of global outward current in hypoxic right coronary artery myocytes (H-RCA), whereas hypoxic left coronary artery myocytes exhibited an increase. K+ channel blockers revealed that a 4-aminopyridine (4AP)-sensitive current (Kv current) was decreased in H-RCA (14.3+/-1.1 vs. 23.4+/-2.5 pA/pF at 60 mV in control RCA, P<0.05) and increased in hypoxic left coronary artery myocytes (H-LCA; 26.4+/-3.8 vs. 11.8+/-1.6 pA/pF at 60 mV in control LCA, P<0.05). Constriction to 4AP was decreased in H-RCA when compared to normoxic control and increased in H-LCA when compared to LCA. Finally, we observed that the expression of Kv1.2 and Kv1.5 were lower in H-RCA than that in H-LCA. This study reveals that CH differentially regulates Kv channels in coronary myocytes. Hypoxia decreases Kv currents and therefore reduces vasoreactivity that contributes to an adaptative response leading to right hypertrophied ventricle perfusion enhancement at rest.     

Actions of pinacidil on membrane currents in canine ventricular myocytes and their modulation by intracellular ATP and cAMP

We studied the effects of pinacidil (3–50 M) on the membrane currents of canine ventricular myocytes, using the whole-cell variant of the patch-clamp technique, and the modulation of these effects by intracellular environment, using the pipette perfusion technique. The following observations were obtained: (1) pinacidil induced a dosedependent outward shift in current at voltages positive to ±70 mV; (2) the pinacidil-induced current was largely timeindependent at voltages positive to ±50 mV and displayed an increase in current fluctuations at more positive voltages, resembling the kinetic properties of current through the ATP-regulated K⁺ channels; (3) elevating the extracellular potassium concentration ([K⁺]_o) caused a positive shift in the voltage where the pinacidil-induced current crossed the voltage axis and increased the slope conductance of this current; (4) the pinacidil-induced current was reduced by Ba²⁺ (0.5–1.5 mM) and abolished by intracellular Cs⁺ (125 mM); (5) glibenclamide reversibly reduced or abolished the pinacidil-induced current; (6) the action of pinacidil was decreased by elevating [ATP] in the pipette solution (from 1 to 10 mM); (7) the action of pinacidil was augmented by adding isoproterenol (1 M) to the superfusate or adding cAMP (0.1 mM) to the pipette solution; (8) elevating temperature augmented, and accelerated the onset of, pinacidil's action; (9) pinacidil reversibly decreased the Ca²⁺ -independent transient outward current (I_to1) but augmented the Ca²⁺ -dependent transient outward current (I_to2). Based on these observations, we reached the following conclusions: (1) the main effect of pinacidil is to increase an outward current through the ATP-regulated K⁺ channels; (2) pinacidil's action is modulated by an enzymatic reaction.     

Fast and slow blockades of the inward-rectifier K+ channel by external divalent cations in guinea-pig cardiac myocytes

The present patch-clamp study shows that external Mg²⁺, Ca²⁺ and Sr²⁺ decrease the unit amplitude of inward current through the inward-rectifier K⁺ channel in a concentration-dependent manner. Sr²⁺ produces a voltage-dependent flickering block as well, and the fractional electrical distance between the external orifice and the Sr²⁺ binding site () is 0.73. The decrease of unit amplitude is reversible and voltage independent while it does not increase the noise level on the open-channel current. Unit current decreased by Mg²⁺ or Ca²⁺ has a longer mean open time, which is inversely proportional to the unit amplitude. External Mg²⁺ does not decrease the amplitude of unit outward current. A surface potential shift, measured using voltage-dependent Cs⁺ block (=1.60), failed to explain the current decrease. Therefore, we conclude that (1) the external divalent cations cause an extremely fast channel block, which appears as a decreased amplitude of the unit current on the recording system; (2) the blocking site (fast site) is present near the external orifice of the channel, and it is separate from the blocking site (slow site) to which Cs⁺ and Sr²⁺ bind.     

ATP-sensitive K+ channels in rat ventricular myocytes are blocked and inactivated by internal divalent cations

K⁺ currents were recorded from ATP-sensitive channels in inside-out patches from isolated rat ventricular myocytes. In the absence of internal divalent cations the current voltage relationship could be described by constant-field assumptions with a permeability of 1.25×10^–13 cm²/s; outward currents saturated under a high driving force for K⁺ movement. Internal 0.1–5.0 mM Mg²⁺, 0.1 M Ca²⁺ and 10 mM Na⁺ each depressed the flux of K⁺ ions moving outwards through open channels. Internal 0.1–5.0 mM Mg²⁺, 0.1–1.0 M Ca²⁺ and 1–10 M Ba²⁺ and Sr²⁺ blocked K⁺ channel activity in a dose-and voltage-dependent manner. Run-down channels could be reactivated by Mg-ATP, but not by AMP-PNP, ATPS or Mg-free ATP which suggested that phosphorylation of the channels was involved in their activity. Ca²⁺ (>=1 M) and Sr²⁺ (1 mM) markedly inactivated K⁺ ATP channels, millimolar Ba²⁺ or Mg²⁺ were less effective. This suggested that the run down of the channels was a Ca²⁺-dependent dephosphorylation of the K⁺ channel protein.     

Dual regulation of the ATP-sensitive potassium channel by activation of cGMP-dependent protein kinase

Adenosine triphosphate (ATP)-sensitive potassium (K(ATP)) channels couple cellular metabolic status to membrane electrical activity. In this study, we performed patch-clamp recordings to investigate how cyclic guanosine monophosphate (cGMP)-dependent protein kinase (PKG) regulates the function of K(ATP) channels, using both transfected human SH-SY5Y neuroblastoma cells and embryonic kidney (HEK) 293 cells. In intact SH-SY5Y cells, the single-channel currents of Kir6.2/sulfonylurea receptor (SUR) 1 channels, a neuronal-type K(ATP) isoform, were enhanced by zaprinast, a cGMP-specific phosphodiesterase inhibitor; this enhancement was abolished by inhibition of PKG, suggesting a stimulatory role of cGMP/PKG signaling in regulating the function of neuronal K(ATP) channels. Similar effects of cGMP accumulation were confirmed in intact HEK293 cells expressing Kir6.2/SUR1 channels. In contrast, direct application of purified PKG suppressed rather than activated Kir6.2/SUR1 channels in excised, inside-out patches, while tetrameric Kir6.2LRKR368/369/370/371AAAA channels expressed without the SUR subunit were not modulated by zaprinast or purified PKG. Lastly, reconstitution of the soluble guanylyl cyclase/cGMP/PKG signaling pathway by generation of nitric oxide led to Kir6.2/SUR1 channel activation in both cell types. Taken together, here, we report novel findings that PKG exerts dual functional regulation of neuronal K(ATP) channels in a SUR subunit-dependent manner, which may provide new means of therapeutic intervention for manipulating neuronal excitability and/or survival.     

Biophysical,pharmacological and developmental properties of ATP-sensitive K+ channels in cultured myotomal muscle cells from Xenopus embryos

Unlike mammalian muscle cells in culture, cultured myotomal muscle cells of Xenopus embryos express ATP-sensitive K⁺ (K_ATP) channels. The K_ATP channels are blocked by internal ATP (half-maximal inhibition K _0.5 = 16 M) and to a lesser extent by internal ADP, are voltage independent, have an inward rectification at positive potentials and are inhibited by glibenclamide (K _0.5 = 2 M). Surprisingly, these K_ATP channels are not sensitive to K⁺ channel openers such as cromakalim. Opening of these K_ATP channels does not occur under normal physiological conditions. It is elicited by metabolic exhaustion of the muscle cell and it precedes the development of an irreversible rigor state. Neither intracellular acidosis nor an increase of intracellular Ca²⁺ are involved in K_ATP channel opening. Different types of K⁺ channels are successively expressed after plating of myotomal muscle cells: (1) sustained delayed-rectifier K⁺ channels; (2) K_ATP channels; (3) inward-rectifier K⁺ channels; (4) transient delayed-rectifier K⁺ channels. The current density associated with K_ATP channels far exceeds that of voltage-dependent K⁺ channels. Innervation controls the expression of these K_ATP channels. Co-culture of muscle cells with neurons from the neural tube decreases the number of active K_ATP channels per patch. Similarly, in situ innervated submaxillaris muscle of tadpoles at stage 50–55 has a very low density of K_ATP channels.     

Pinacidil activates the ATP-sensitive K+ channel in inside-out and cell-attached patch membranes of guinea-pig ventricular myocytes

Patch-clamp techniques were used to study the effects of pinacidil on the adenosine-5-triphosphate (ATP)-sensitive K⁺ channel current in guinea-pig ventricular myocytes. In inside-out patches, the ATP-sensitive K⁺ channel current could be recorded at an internal ATP concentration of 0.5 mM or less and almost complete inhibition was achieved by raising the concentration to 2 mM. Application of pinacidil (10–30 M) in the presence of 2 mM ATP restored the current, whereas 5 mM ATP antagonized the effect of pinacidil. The conductance of the channel at symmetrical K⁺ concentrations of 140 mM was 75 pS with a slight inward rectification at voltages positive to +40 mV. There was no significant change in the conductance after application of pinacidil. In 0.5 mM ATP, at –80 mV, both the distributions of the open time and the life-time of bursts could be fitted by a single exponential. An increase in ATP concentration decreased the mean life-time of bursts, whereas pinacidil increased it with little increase in the mean open time. Closed time distributions of the channel were fitted by at least two exponentials, with a fast and a slow time constant. An increase in ATP concentration markedly increased the slow time constant associated with a decrease in the number of bursts, whereas the effect of pinacidil was opposite to that of increased ATP. These results indicate that pinacidil increases the open-state probability of the ATP-sensitive K⁺ channel. In cell-attached patches, application of pinacidil (100–200 M) to the extracellular solution reversibly induced the channel activity, which showed similar properties to those of the ATP-sensitive K⁺ channel recorded in cell-free patches.     

The effects of over-expression of the FK506-binding protein FKBP12.6 on K+ currents in adult rabbit ventricular myocytes

This study examines the effects of the intracellular protein FKBP12.6 on action potential and associated K⁺ currents in isolated adult rabbit ventricular cardiomyocytes. FKBP12.6 was over-expressed by ~6 times using a recombinant adenovirus coding for human FKBP12.6. This over-expression caused prolongation of action potential duration (APD) by ~30%. The amplitude of the transient outward current (I _to) was unchanged, but rate of inactivation at potentials positive to +40 mV was increased. FKBP12.6 over-expression decreased the amplitude of the inward rectifier current (I _K1) by ~25% in the voltage range −70 to −30 mV, an effect prevented by FK506 or lowering intracellular [Ca²⁺] below 1 nM. Over-expression of an FKBP12.6 mutant, which cannot bind calcineurin, prolonged APD and affected I _to and I _K1 in a similar manner to wild-type protein. These data suggest that FKBP12.6 can modulate APD via changes in I _K1 independently of calcineurin binding, suggesting that FKBP12.6 may affect APD by direct interaction with I _K1.     

Positive cooperativity in activation of the cardiac muscarinic K+ channel by intracellular GTP

Concentration-dependent effects of intracellular GTP on activation of the muscarinic K⁺ channel were examined in inside-out patches of cardiac atrial myocytes. The pipette solution contained 0.1 M ACh. GTP (0.01–30 M) and 0.5 mM MgCl₂ were applied to the inside side of the patch membrane. K⁺ channels were activated with GTP concentration above 0.1 M. Channel activation reached a maximal value with 1–3 M GTP. It decreased at GTP concentrations larger than 3 M, probably due to desensitization. The dependence of the open probability of the channel on intracellular GTP showed a sigmoidal relationship with a Hill coefficient of around 3. A positive cooperative effect of intracellular GTP on the K⁺ channel may play an important role in amplifying the signal from the membrane receptor to the K⁺ channel.     

Activation of the Na+/K+-ATPase by insulin and glucose as a putative negative feedback mechanism in pancreatic beta-cells

Pancreatic beta-cells of sulfonylurea receptor type 1 knock-out (SUR1(-/-)) mice exhibit an oscillating membrane potential (V (m)) demonstrating that hyper-polarisation occurs despite the lack of K(ATP) channels. We hypothesize that glucose activates the Na(+)/K(+)-ATPase thus increasing a hyper-polarising current. Elevating glucose in SUR1(-/-) beta-cells resulted in a transient fall in V (m) and [Ca(2+)](c) independent of sarcoplasmic and endoplasmic reticulum Ca(2+)-activated ATPase (SERCA) activation. This was not affected by K(+) channel blockade but inhibited by ATP depletion and by ouabain. Increasing glucose also reduced [Na(+)](c), an effect reversed by ouabain. Exogenously applied insulin decreased [Na(+)](c) and hyper-polarised V (m). Inhibiting insulin signalling in SUR1(-/-) beta-cells blunted the glucose-induced decrease of [Ca(2+)](c). Tolbutamide (1 mmol/l) disclosed the SERCA-independent effect of glucose on [Ca(2+)](c) in wild-type beta-cells. The data show that in SUR1(-/-) beta-cells, glucose activates the Na(+)/K(+)-ATPase presumably by increasing [ATP](c). Insulin can also stimulate the pump and potentiate the effect of glucose. Pathways involving the pump may thus serve as potential drug targets in certain metabolic disorders.     

Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca-activated K channel current in cultured rat hippocampal astrocytes

Brief hypoxia differentially regulates the activities of Ca²⁺-activated K⁺ channels (K_Ca) in a variety of cell types. We investigated the effects of hypoxia (<2% O₂) on K_Ca channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K_Ca current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K_Ca currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K_Ca channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K_Ca current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca²⁺ external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K_Ca channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K_Ca currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K_Ca single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K_Ca currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.     

Age-dependent differences in the inhibition of HCN2 current in rat ventricular myocytes by the tyrosine kinase inhibitor erbstatin

Previously, we have shown that murine HCN2 channels over-expressed in newborn and adult cardiac myocytes produce currents with different biophysical characteristics. To investigate the role of tyrosine kinase modulation in these age-dependent differences, we employed the broad spectrum tyrosine kinase inhibitor erbstatin. Our results demonstrated distinct and separable effects of erbstatin on channel gating and current amplitude and a marked age dependence to these effects. In newborn myocytes, erbstatin decreased current amplitude, shifted the activation relation negative, and slowed activation kinetics. The effect on activation voltage but not that on amplitude was absent when expressing a cAMP-insensitive mutant (HCN2R/E), while a C-terminal truncated form of HCN2 (HCN2ΔCx) exhibited only the voltage dependent but not the amplitude effect of erbstatin. Thus, the action of erbstatin on the activation relation and current amplitude are distinct and separable in newborn myocytes, and the effect on activation voltage depends on the cAMP status of HCN2 channels. In contrast to newborn myocytes, erbstatin had no effect on HCN2 under control conditions in adult myocytes but induced a negative shift with no change in amplitude when saturated cAMP was added to the pipette solution. We conclude that erbstatin’s effects on HCN2 current magnitude and voltage dependence are distinct and separable, and there are fundamental developmental differences in the heart that affect channel function and its modulation by the tyrosine kinase inhibitor erbstatin. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Regional expression of the anesthetic-activated potassium channel TRESK in the rat nervous system

The two-pore-domain potassium (K_2P) channels contribute to background (leak) potassium currents maintaining the resting membrane potential to play an important role in regulating neuronal excitability. As such they may contribute to nociception and the mechanism of action of volatile anesthetics. In the present study, we examined the protein expression pattern of the K_2P channel TRESK in the rat central nervous system (CNS) and peripheral nervous system (PNS) by immunohistochemistry. The regional distribution expression pattern of TRESK has both similarities and significant differences from that of other K_2P channels expressed in the CNS. TRESK expression is broadly found in the brain, spinal cord and dorsal root ganglia (DRG). TRESK expression is highest in important CNS structures, such as specific cortical layers, periaqueductal gray (PAG), granule cell layer of the cerebellum, and dorsal horn of the spinal cord. TRESK expression is also high in small and medium sized DRG neurons. These results provide an anatomic basis for identifying functional roles of TRESK in the rat nervous system.     

Increased Ca2+ channel currents in cerebellar Purkinje cells of the ataxic groggy rat

The ataxic groggy rat (strain name; GRY) is an autosomal recessive neurological mutant found in a closed colony of Slc:Wistar rats. Recent genetic analysis has identified the missense (M251K) mutation in the alpha(1) subunit of the Ca(V)2.1 (P/Q-type) voltage-dependent Ca(2+) channel gene (Cacna1a) of GRY rat. In this study, we found that high-voltage-activated (HVA) Ca(2+) channel currents in acutely dissociated Purkinje cells of GRY rats showed increased (not decreased) current density and depolarizing shift of the activation and inactivation curves compared with those of normal Wistar rats. In contrast low-voltage-activated (LVA) Ca(2+) channel currents of GRY rats showed no significant changes. These results suggest that functional alteration of Ca(2+) channel currents in cerebellar Purkinje cells of GRY rats is attributed to the change of HVA Ca(2+) channel currents, and that increased HVA Ca(2+) channel function underlies the cerebellar dysfunction and ataxic phenotype of GRY rats.     

Tetraethylammonium ion sensitivity of a 35-pS Ca2+-activated K+ channel in GH3 cells that is activated by thyrotropin-releasing hormone

Single Ca²⁺-activated K⁺ channels were studied in membrane patches from the GH₃ anterior pituitary cell line. We have previously demonstrated the coexistence of large-conductance and small-conductance (280 pS and 11 pS in symmetrical 150 mM K⁺, respectively) Ca²⁺-activated K⁺ channels in this cell line (Lang and Ritchie 1987). Here we report the existence of a third type of Ca²⁺-activated K⁺ channel that has a conductance of about 35 pS under similar conditions. In excised inside-out patches, this channel can be activated by elevations of the internal free Ca²⁺ concentration, and the open probability increases as the membrane potential is made more positive. In excised patches, the sensitivity of this 35-pS channel to internal Ca²⁺ is low; at positive membrane potentials, this channel requires Ca²⁺ concentrations greater than 10 M for activation. However, 35-pS channels have a much higher sensitivity to Ca²⁺ in the first minute after excision (activated by 1 M Ca²⁺ at –50 mV). Therefore, it is possible that the Ca²⁺ sensitivity of this channel is stabilized by intracellular factors. In cell-attached patches, this intermediate conductance channel can be activated (at negative membrane potentials) by thyrotropin-releasing hormone-induced elevations of the intracellular Ca²⁺ concentration and by Ca²⁺ influx during action potentials. The intermediate conductance channel is inhibited by high concentrations of external tetraethylammonium ions (K _d=17 mM) and is relatively resistant to inhibition by apamin.     

5-HT inhibition of rat insulin 2 promoter Cre recombinase transgene and proopiomelanocortin neuron excitability in the mouse arcuate nucleus

A number of anti-obesity agents have been developed that enhance hypothalamic 5-HT transmission. Various studies have demonstrated that arcuate neurons, which express proopiomelanocortin peptides (POMC neurons), and neuropeptide Y with agouti-related protein (NPY/AgRP) neurons, are components of the hypothalamic circuits responsible for energy homeostasis. An additional arcuate neuron population, rat insulin 2 promoter Cre recombinase transgene (RIPCre) neurons, has recently been implicated in hypothalamic melanocortin circuits involved in energy balance. It is currently unclear how 5-HT modifies neuron excitability in these local arcuate neuronal circuits. We show that 5-HT alters the excitability of the majority of mouse arcuate RIPCre neurons, by either hyperpolarization and inhibition or depolarization and excitation. RIPCre neurons sensitive to 5-HT, predominantly exhibit hyperpolarization and pharmacological studies indicate that inhibition of neuronal firing is likely to be through 5-HT_1F receptors increasing current through a voltage-dependent potassium conductance. Indeed, 5-HT_1F receptor immunoreactivity co-localizes with RIPCre green fluorescent protein expression. A minority population of POMC neurons also respond to 5-HT by hyperpolarization, and this appears to be mediated by the same receptor-channel mechanism. As neither POMC nor RIPCre neuronal populations display a common electrical response to 5-HT, this may indicate that sub-divisions of POMC and RIPCre neurons exist, perhaps serving different outputs.     

A newly identified Ca2+ dependent K+ channel in the smooth muscle membrane of single cells dispersed from the rabbit portal vein

We found a new type of Ca²⁺-dependent K⁺ channel in smooth muscle cell membranes of single cells of the rabbit portal vein. A slope conductance of the current was 180 pS when 142 mM K⁺ solution was exposed to both sides of the membrane (this channel was named the K_M channel, in comparison to the known K_L and K_S channels from the same membrane patch; Inoue et al. 1985). This K_M channel was less sensitive to the cytoplasmic Ca²⁺ concentration, [Ca²⁺]_i, but was sensitive to the extracellular Ca²⁺, [Ca²⁺]_o, e.g. in the outside-out membrane patch, lowering the [Ca²⁺]_o in the bath markedly reduced the open probability of this channel, and also in cell-attached configuration, lowering of the [Ca²⁺]_o using the internally perfused patch clamp electrode device reduced the opening of K_M channel. TEA⁺ (1–10 mM) reduced the amplitude of the elementary current through the K_M channel applied from each side of the membrane, but this agent inhibited the K_M channel to a greater extent when applied to the inner than to the outer surface of the membrane. Furthermore, this K_M channel had a weak voltage dependency, and the open probability of the channel remained much the same within a wide range of potential (from –60 mV to +60 mV). Whereas most Ca²⁺-dependent K⁺ channels are regulated mainly by [Ca²⁺]_i and possess a voltage dependency, these properties of the K_M channel differed from other Ca²⁺-dependent K⁺ channels. The elucidation of this K_M channel should facilitate explanations of the actions of external Ca²⁺ or TEA⁺ on the membrane potential, in the smooth muscles of the rabbit portal vein.     

Neuropeptide Y inhibits Ca2+-activated K+ channels in vascular smooth muscle cells from the rat tail artery

The effects of neuropeptide Y (NPY) on the Ca²⁺-activated K⁺ channel in smooth muscle cells from the rat tail artery were studied by whole-cell and single-channel patch-clamp recording techniques. In the presence of nifedipine (1 M), whole-cell outward currents through Ca²⁺-activated K⁺ channels were inhibited by NPY in a dose-dependent manner from 20 to 200 nM. A maximum inhibition to about 48% of the control current could be achieved. Recordings from outside-out patches showed that the open probability of Ca²⁺-activated K⁺ channels were similarly inhibited by NPY. At 200 nM NPY, the open probability was reduced to about 36% of the control value. NPY did not affect the open times or current amplitude, but increased significantly the short (from 0.49 to 0.58 ms) and long (from 441 to 728 ms) closed times. Inhibition of Ca²⁺-activated K⁺ channels by NPY may contribute to its excitatory action on vascular smooth muscle cells.