Ca(2+)-dependent inhibition of inwardly rectifying K(+) channel in opossum kidney cells

The effect of intracellular Ca(2+) on the activity of the inwardly rectifying ATP-regulated K(+) channel with an inward conductance of about 90 pS was examined by using the patch-clamp technique in opossum kidney proximal tubule (OKP) cells. The activity of the inwardly rectifying K(+) channel rapidly declined with an application of ionomycin (1 microM) in the presence of 10(-6) M Ca(2+) in cell-attached patches. The application of 10 microM phorbor-12-myristate-acetate (PMA) with 10(-6) M Ca(2+) reduced the K(+) channel activity. Although the channel activity was not influenced by an increase of bath Ca(2+) from 10(-7.5) to 10(-6) M, the activity was inhibited by protein kinase C (PKC, 1 U/ml) with 10(-6) M Ca(2+) in inside-out patches. The inhibitory effect of Ca(2+) with ionomycin on the channel activity was diminished by the pretreatment with a specific PKC inhibitor, GF 109203X (5 microM), in cell-attached patches. By contrast, the application of Ca(2+)/calmodulin kinase II (CaMK II, 300 pM) dramatically increased this channel activity in inside-out patches. In cell-attached patches, the addition of both GF 109203X and cyclospolin A (5 microM), a potent inhibitor of protein phosphatase 2B (calcineurin), instead stimulated the K(+) channel activity with ionomycin and 10(-6) M Ca(2+). The addition of protein phosphatase 2B (calcineurin) (2 U/ml) to the bath with calmodulin (1 microM) and Ni(2+) (10 microM) to stimulate calcineurin inhibited the channel activity in inside-out patches. Furthermore, the inhibitory effect of PKC or calcineurin on this channel activity was abolished by a removal of Ca(2+) from bath solution. These results suggest that Ca(2+)-dependent inhibitory effect on the inwardly rectifying K(+) channel in OKP cells was mainly mediated by Ca(2+)-PKC-mediated phosphorylation, and that the Ca(2+)-calmodulin-dependent phosphorylation process may be counterbalanced by the Ca(2+)-calmodulin-dependent dephosphorylation process.     

An ATP-regulated and pH-sensitive inwardly rectifying K(+) channel in cultured human proximal tubule cells

Although renal K(+) channels along the nephron have been explored in various animal species, little is known about the K(+) channels in human proximal tubule cells. Using the patch-clamp technique, we investigated the properties of an inwardly rectifying K(+) channel present in the surface membrane of cultured human proximal tubule cells of normal kidney origin. This channel was the most frequently observed K(+) channel in cell-attached patches, and cytoplasmic ATP was required to maintain channel activity in inside-out patches. Its single channel conductance was about 42 pS for inward currents and 7 pS for outward currents under the symmetrical K(+) condition. The ATP effect on channel activity was dose-dependently stimulatory within a range of 0.1 to 10 mM, and a nonhydrolyzable ATP analog, AMP-PNP (3 mM), had no effect on channel activity in either the presence or absence of ATP (1 mM). The channel activity observed in cell-attached patches was reduced to 30 to 50% of controls by a membrane-permeable nonspecific protein kinase inhibitor, K252a (1 microM), or a potent protein kinase A inhibitor, KT5720 (500 nM). In contrast, a membrane-permeable cAMP analog, 8Br-cAMP (100 microM), induced a twofold increase in channel activity. The addition of a catalytic subunit of protein kinase A (PKA-CS, 100 U/ml) to the bath in inside-out patches stimulated channel activity in the presence of 1 mM ATP. Furthermore, the channel activity maintained with 1 mM ATP in inside-out patches was suppressed by internal acidification and enhanced by alkalization. These results suggest that the activity of the inwardly rectifying K(+) channel in cultured human proximal tubule cells was ATP-dependent and regulated at least in part by cAMP/PKA-mediated phosphorylation processes and intracellular pH.     

Activation of Ca(2+)-dependent K+ channel and Cl- conductance in canine pancreatic acinar cells through a cyclic AMP pathway.

Effects of adenosine 3',5'-cyclic monophosphate (cAMP) on Ca(2+)-dependent K+ channel and Cl- conductance in the plasma membrane of isolated canine pancreatic acinar cells were studied by patch-clamp methods. In whole-cell current recordings on isolated cells dialyzed with K(+)-rich solution containing 0.5 mM EGTA, addition of 0.5 mM dibutyryl cAMP (dbcAMP), or 50 microM forskolin to the bath increased outward K+ and inward Cl- currents associated with depolarizing and hyperpolarizing voltage jumps, respectively. In intact cells (cell-attached configurations), addition of 0.5 mM dbcAMP or 50 microM forskolin to the bath increased the opening of single K+ channel. In Ca(2+)-free external solution (bath and pipette) 50 microM forskolin or 0.5 mM dbcAMP application evoked an increase in the opening of single K+ channel in intact cells. Addition of 0.5 mM dbcAMP to the bath solution containing 10 mM EGTA without Ca2+ increased the currents of whole-cell dialyzed with K(+)-rich solution containing 10 mM EGTA. When cell was dialyzed with 20 mM EGTA, dbcAMP, or forskolin application did not increase the whole-cell currents. In excised inside-out patches, addition of the catalytic subunit of cAMP-dependent protein kinase (16 U/ml) in the presence of 0.3 mM ATP to the cytoplasmic face of membrane activated the K+ channel, but 0.1 mM cAMP did not. These results suggest that cAMP-dependent phosphorylation can activate Ca(2+)-dependent K+ channels without increase in intracellular free Ca2+ and cAMP-dependent mechanism can activate Ca(2+)-dependent Cl- conductances without the increase in Ca2+ in canine pancreatic acinar cells.     

Constitutive activity of inwardly rectifying K+ channel at physiological [Ca]i is mediated by Ca2+/CaMK II pathway in opossum kidney proximal tubule cells

Using patch-clamp technique, we studied the role of the Ca(2+)/calmodulin kinase II (CaMK II)-mediated phosphorylation process on the K(+) channel with an inward conductance of 90 pS in opossum kidney proximal tubule cells (OKPCs). The intracellular Ca(2+) concentration ([Ca](i)) was measured by use of the fluorescent dye fura 2. The following results were obtained: (i) In cell-attached patches, the channel activity was inhibited by a decrease in [Ca](i) induced by perfusion with low Ca(2+) (10(-8) M), La(3+) (100 microM), or EGTA/AM (100 microM) contained in the bath solution. The application of KN-62 (10 microM) or KN-93 (5 microM), inhibitors of CaMK II, also inhibited the channel activity. (ii) The membrane potential measured with nystatin-perforated patches was significantly decreased by the fall in [Ca](i) induced by the perfusion with EGTA- or La(3+)-containing solution. Also, the application of KN-62 (10 microM) or KN-93 (5 microM) to the bath significantly decreased the membrane potential. (iii) In inside-out patches, the channel activity was significantly stimulated by the application of CaMK II (300 pM) at 10(-7) M Ca(2+) in the bath. Furthermore, the application of KN-62 (10 microM) to the bath significantly decreased the channel activity. Our findings show that the constitutive activity of inwardly rectifying K(+) channel at physiological [Ca](i) is mediated by the Ca(2+)/CaMK II pathway in OKPCs.     

Number of K(Ca) channels underlying spontaneous miniature outward currents (SMOCs) in mudpuppy cardiac neurons

Spontaneous miniature outward currents (SMOCs) in parasympathetic neurons from mudpuppy cardiac ganglia are caused by activation of TEA- and iberiotoxin-sensitive, Ca(2+)-dependent K(+) (BK) channels. Previously we reported that SMOCs are activated by Ca(2+)-induced Ca(2+) release (CICR) from caffeine- and ryanodine-sensitive intracellular Ca(2+) stores. In the present study, we analyzed the single channel currents that contribute to SMOC generation in mudpuppy cardiac neurons. The slope conductance of BK channels, determined from the I-V relationship of single-channel currents recorded with cell-attached patches in physiological K(+) concentrations, was 84 pS. The evidence supporting the identity of this channel as the channel involved in SMOC generation was its sensitivity to internal Ca(2+), external TEA, and caffeine. In cell-attached patch recordings, 166 microM TEA applied in the pipette reduced single-channel current amplitude by 32%, and bath-applied caffeine increased BK channel activity. The ratio between the averaged SMOC amplitude and the single-channel current amplitude was used to estimate the average number of channels involved in SMOC generation. The estimated number of channels involved in generation of an averaged SMOC ranged from 18 to 23 channels. We also determined that the Po of the BK channels at the peak of a SMOC remains constant at voltages more positive than -20 mV, suggesting that the transient rise in intracellular Ca(2+) from ryanodine-sensitive intracellular stores in the vicinity of the BK channel reached concentrations most likely exceeding 40 microM.     

Properties of a Ca(2+)-activated large conductance K(+) channel with ATP sensitivity in human renal proximal tubule cells

The properties of a native Ca(2+)-activated large conductance K(+) channel (BK channel) present in the surface membrane of cultured human renal proximal tubule epithelial cells (RPTECs) were investigated by using the patch-clamp technique. The slope conductance of the BK channel was about 295 pS, and the channel was selective to K(+) over Na(+), with a selectivity ratio of about 12.2. The activity of the channel was almost maximally enhanced by 10(-4 )M or more Ca(2+) in the cytoplasmic surface of the patch membrane and was markedly diminished by reducing the cytoplasmic Ca(2+) to 10(-6) M at the membrane potential of about 0 mV. The depolarization of the patch membrane also enhanced the channel activity, and hyperpolarization lowered it. K(+) channel blockers, Ba(2+) (0.1-1 mM), tetraethylammonium (1 mM), and charybdotoxin (100 nM), were effective for the suppression of channel activity. A significant feature of the K(+) channel was that channel activity maintained by 10(-5)-10(-4 )M Ca(2+) in inside-out patches was inhibited by the addition of ATP (1-10 mM) to the bath solution. ATPgammaS, and a nonhydrolyzable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP), also had inhibitory effects on channel activity. However, an inhibitor of ATP-sensitive K(+) channels, glibenclamide (0.1 mM), induced no appreciable change in channel activity from both intra- and extracellular sides. These results suggest that besides the common natures of the BK channel family such as regulation by cytoplasmic Ca(2+) and membrane potential, the BK channel in RPTECs is directly inhibited by intracellular ATP independent of phosphorylation processes and sulfonylurea receptor.     

Isoprenaline-stimulated differential adrenergic response of K+ channels in skeletal muscle under hypokalaemic conditions

The mechanism underlying the hyperpolarization induced by isoprenaline in mouse lumbrical muscle fibres was studied using cell-attached patch and intracellular membrane potential ( V(m)) recordings. Sarcolemmal inwardly rectifying K(+) channels (K(IR): 45 pS) and Ca(2+)-activated K(+) channels (BK: 181 pS) were identified. Exposure to isoprenaline closed K(IR) channels and increased BK channel activity. This increase was observed as a shift from 50 to -40 mV in the voltage dependence of channel activation. Isoprenaline prevented hysteresis of V(m) when the extracellular [K(+)] fell below 3.8 mM. This hysteresis was due to the properties of the K(IR). The effects of chloride transport and isoprenaline on V(m) did not interact purely competitively, but isoprenaline could prevent the depolarization induced by hyperosmotic media equally as well as bumetanide, which inhibits the Na(+)/K(+)/2Cl(-) cotransporter. In lumbrical muscle this leads to hyperpolarization, but this might vary among muscles. The switch from K(IR) to BK as the component of total K(+) conductance was due to isoprenaline.     

Single-channel L-type Ca2+ currents in chicken embryo semicircular canal type I and type II hair cells

Few data are available concerning single Ca channel properties in inner ear hair cells and particularly none in vestibular type I hair cells. By using the cell-attached configuration of the patch-clamp technique in combination with the semicircular canal crista slice preparation, we determined the elementary properties of voltage-dependent Ca channels in chicken embryo type I and type II hair cells. The pipette solutions included Bay K 8644. With 70 mM Ba(2+) in the patch pipette, Ca channel activity appeared as very brief openings at -60 mV. Ca channel properties were found to be similar in type I and type II hair cells; therefore data were pooled. The mean inward current amplitude was -1.3 +/- 0.1 (SD) pA at - 30 mV (n = 16). The average slope conductance was 21 pS (n = 20). With 5 mM Ba(2+) in the patch pipette, very brief openings were already detectable at -80 mV. The mean inward current amplitude was -0.7 +/- 0.2 pA at -40 mV (n = 9). The average slope conductance was 11 pS (n = 9). The mean open time and the open probability increased significantly with depolarization. Ca channel activity was still present and unaffected when omega-agatoxin IVA (2 microM) and omega-conotoxin GVIA (3.2 microM) were added to the pipette solution. Our results show that types I and II hair cells express L-type Ca channels with similar properties. Moreover, they suggest that in vivo Ca(2+) influx might occur at membrane voltages more negative than -60 mV.     

Inhibition of large-conductance Ca(2+)-activated K(+) channels in hippocampal neurons by toosendanin

The effect of toosendanin, a selective presynaptic blocker and effective antibotulismic agent, on large-conductance Ca(2+)-activated K(+) channels was studied in inside-out patches of pyramidal neurons freshly isolated from the hippocampal CA1 region of the rat. Toosendanin (1 x 10(-6)g/ml approximately 1 x 10(-4)g/ml) was found to inhibit large-conductance Ca(2+)-activated K(+) channels by reducing its open probability significantly in a concentration-dependent manner, although the effective concentration of toosendanin was lower in a symmetrical K(+) (150 mM) solution than under asymmetrical conditions (changing K(+) concentration in pipette solution to 5mM). The action was partially reversible by washing. By decreasing the slow open time constant, toosendanin shortened the open dwell time of large-conductance Ca(2+)-activated K(+) channels in a dose-dependent manner. A dose-dependent reduction of unitary current amplitude of the channel was detected after toosendanin perfusion. On elevating the intracellular free calcium concentration from 1 to 10 microM, a similar effect on large-conductance Ca(2+)-activated K(+) channels by toosendanin was also observed, but its efficacy was diminished.These results show that toosendanin inhibits large-conductance Ca(2+)-activated K(+) channels in hippocampal neurons by reducing the open probability and unitary current amplitude of the channel, and that Ca(2+) interferes with the effect. These data provide an explanation for toosendanin-induced facilitation of neurotransmitter release and the antibotulismic effect of the drug.     

A Ba(2+)-sensitive K(+) current contributes to the resting membrane potential of neurons in rat suprachiasmatic nucleus

A Ba(2+)-sensitive K(+) current was studied in neurons of the suprachiasmatic nucleus (SCN) using the whole cell patch-clamp technique in acutely prepared brain slices. This Ba(2+)-sensitive K(+) current was found in approximately 90% of the SCN neurons and was uniformly distributed across the SCN. Current-clamp studies revealed that Ba(2+) (500 microM) reversibly depolarized the membrane potential by 6.7 +/- 1.3 mV (n = 22) and concomitantly Ba(2+) induced an increase in the spontaneous firing rate of 0.8 +/- 0.2 Hz (n = 12). The Ba(2+)-evoked depolarizations did not depend on firing activity or spike dependent synaptic transmission. No significant day/night difference in the hyperpolarizing contribution to the resting membrane potential of the present Ba(2+)-sensitive current was observed. Voltage-clamp experiments showed that Ba(2+) (500 microM) reduced a fast-activating, voltage-dependent K(+) current. This current was activated at levels below firing threshold and exhibited outward rectification. The Ba(2+)-sensitive K(+) current was strongly reduced by tetraethylammonium (TEA; 20 and 60 mM) but was insensitive to 4-aminopyridine (4-AP; 5 mM) and quinine (100 microM). A component of Ba(2+)-sensitive K(+) current remaining in the presence of TEA exhibited no clear voltage dependence and is less likely to contribute to the resting membrane potential. The voltage dependence, kinetics and pharmacological properties of the Ba(2+)- and TEA-sensitive K(+) current make it unlikely that this current is a delayed rectifier, Ca(2+)-activated K(+) current, ATP-sensitive K(+) current, M-current or K(+) inward rectifier. Our data are consistent with the Ba(2+)- and TEA-sensitive K(+) current in SCN neurons being an outward rectifying K(+) current of a novel identity or belonging to a known family of K(+) channels with related properties. Regardless of its precise molecular identity, the current appears to exert a significant hyperpolarizing effect on the resting potential of SCN neurons.     

Potentiation of large conductance, Ca2+-activated K+ (BK) channels by alpha5beta1 integrin activation in arteriolar smooth muscle

Injury/degradation of the extracellular matrix (ECM) is associated with vascular wall remodelling and impaired reactivity, a process in which altered ECM-integrin interactions play key roles. Previously, we found that peptides containing the RGD integrin-binding sequence produce sustained vasodilatation of rat skeletal muscle arterioles. Here, we tested the hypothesis that RGD ligands work through alpha5beta1 integrin to modulate the activity of large conductance, Ca(2+)-activated K(+) (BK) channels in arteriolar smooth muscle. K(+) currents were recorded in single arteriolar myocytes using whole-cell and single-channel patch clamp methods. Activation of alpha5beta1 integrin by an appropriate, insoluble alpha5beta1 antibody resulted in a 30-50% increase in the amplitude of iberiotoxin (IBTX)-sensitive, whole-cell K(+) current. Current potentiation occurred 1-8 min after bead-antibody application to the cell surface. Similarly, the endogenous alpha5beta1 integrin ligand fibronectin (FN) potentiated IBTX-sensitive K(+) current by 26%. Current potentiation was blocked by the c-Src inhibitor PP2 but not by PP3 (0.1-1 mum). In cell-attached patches, number of open channels x open probability (NP(o)) of a 230-250 pS K(+) channel was significantly increased after FN application locally to the external surface of cell-attached patches through the recording pipette. In excised, inside-out patches, the same method of FN application led to large, significant increases in NP(o) and caused a leftward shift in the NP(o)-voltage relationship at constant [Ca(2+)]. PP2 (but not PP3) nearly abolished the effect of FN on channel activity, suggesting that signalling between the integrin and channel involved an increase in Ca(2+)sensitivity of the channel via a membrane-delimited pathway. The effects of alpha5beta1 integrin activation on both whole-cell and single-channel BK currents could be reproduced in HEK 293 cells expressing the BK channel alpha-subunit. This is the first demonstration at the single-channel level that integrin signalling can regulate an ion channel. Our results show that alpha5beta1 integrin activation potentiates BK channel activity in vascular smooth muscle through both Ca(2+)- and c-Src-dependent mechanisms. This mechanism is likely to play a role in the arteriolar dilatation and impaired vascular reactivity associated with ECM degradation.     

Single-channel currents through transient-receptor-potential-like (TRPL) channels

Single-channel current recordings were used to examine the properties and modulation of Drosophila transient-receptor-potential-like (TRPL) channels transiently expressed in HEK and COS cells. Recombinant TRPL channels were constitutively active and characterized by a conductance of 104 pS in on-cell membrane patches with 115 mM Na+ and 2 mM Mg2+ in the pipette solution. In inside-out membrane patches exposed to 115 mM Na+ plus 2 mM Mg2+, 115 mM Na+ plus 10 mM Mg2+, 90 mM Ca2+ and 90 mM Ba2+ on both sides, the single-channel conductances were 72 pS, 36 pS, 48 pS and 46 pS, respectively. The single TRPL channel currents reversed close to 0 mV and displayed a linear voltage dependence between -120 mV and +120 mV. Removal of cations from the pipette and bath solutions abolished inward and outward currents, respectively. Similar currents were not observed in mock-transfected and native cells. The opening probability of TRPL channels increased by depolarizing the membrane and accounted for the outward rectification of whole-cell TRPL currents. In on-cell membrane patches, the TRPL channel activity was enhanced by cell dialysis of 300 microM guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma-S]) and by a rise of intracellular Ca2+ (>2 microM). Constitutively active TRPL channels depolarized the host cells to -10 mV and the membrane potential was restored by cell dialysis with 10 mM BAPTA. The present results suggest that TRPL forms non-selective cationic channels modulated by intracellular Ca2+ in mammalian cells.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.     

Fast BK-type channel mediates the Ca(2+)-activated K(+) current in crayfish muscle.

The role of the Ca(2+)-activated K(+) current (I(K(Ca))) in crayfish opener muscle fibers is functionally important because it regulates the graded electrical activity that is characteristic of these fibers. Using the cell-attached and inside-out configurations of the patch-clamp technique, we found three different classes of channels with properties that matched those expected of the three different ionic channels mediating the depolarization-activated macroscopic currents previously described (Ca(2+), K(+), and Ca(2+)-dependent K(+) currents). We investigated the properties of the ionic channels mediating the extremely fast activating and persistent I(K(Ca)). These voltage- and Ca(2+)-activated channels had a mean single-channel conductance of approximately 70 pS and showed a very fast activation. Both the single-channel open probability and the speed of activation increased with depolarization. Both parameters also increased in inside-out patches, i.e., in high Ca(2+) concentration. Intracellular loading with the Ca(2+) chelator bis(2-aminophenoxy) ethane-N, N,N',N'-tetraacetic acid gradually reduced and eventually prevented channel openings. The channels opened at very brief delays after the pulse depolarization onset (<5 ms), and the time-dependent open probability was constant during sustained depolarization (< or =560 ms), matching both the extremely fast activation kinetics and the persistent nature of the macroscopic I(K(Ca)). However, the intrinsic properties of these single channels do not account for the partial apparent inactivation of the macroscopic I(K(Ca)), which probably reflects temporal Ca(2+) variations in the whole muscle fiber. We conclude that the channels mediating I(K(Ca)) in crayfish muscle are voltage- and Ca(2+)-gated BK channels with relatively small conductance. The intrinsic properties of these channels allow them to act as precise Ca(2+) sensors that supply the exact feedback current needed to control the graded electrical activity and therefore the contraction of opener muscle fibers.     

ATP-sensitive potassium channels in freshly dissociated adult rat striatal neurons: activation by metabolic inhibitors and the dopaminergic receptor agonist quinpirole

The characteristics of adenosine 5'-triphosphate (ATP)-sensitive K+ channels in acutely isolated striatal neurons from adult rats were examined. Neurons had a resting membrane potential of -53.9+/-1.2 mV (n=66), with evoked or spontaneous action potentials firing at 10+/-0.7 Hz, and large inwards and outwards whole-cell currents. In cell-attached patches with a high [K+] in the pipette, a voltage-independent, ATP-insensitive 16.5+/-1.5 pS channel was observed in 375 out of 452 cells. Bath application of Na+-azide (0.5-2 mM) to 108 neurons revealed another 145.7+/-3.5 pS (LKATP) channel in 65 neurons; this channel was blocked by tolbutamide. The LKATP channel exhibited a high open probability (Po, 0.8+/-0.05) at 0 mV pipette potential. Varying the pipette [K+] shifted the reversal potential of LKATP, showing the channel's K+ selectivity. Cytoplasmic ATP (ATPi) reversibly inhibited LKATP, with an inhibitory constant (Ki) of 0.12 mM. LKATP was sensitive to intracellular Ca2+ but insensitive to iberiotoxin. In 25% of cell-attached patches, the presence of quinpirole in the pipette opened a third type of channel (90.6+/-1.7 pS, termed D2KATP). Sulpiride, a dopamine D2-receptor antagonist, inhibited D2KATP. ATPi reversibly inhibited D2KATP, with a Ki of 0.212 mM. The Na+-azide- or quinpirole-induced current caused a tolbutamide-sensitive membrane hyperpolarization and a marked reduction in action potential frequency. We propose that ATP-sensitive K+ channels play a metabolism-dependent role in striatal neurons.     

Ca(2+)-induced Ca(2+) release activates spontaneous miniature outward currents (SMOCs) in parasympathetic cardiac neurons.

Mudpuppy parasympathetic cardiac neurons exhibit spontaneous miniature outward currents (SMOCs) that are thought to be due to the activation of clusters of large conductance Ca(2+)-activated K(+) channels (BK channels) by localized release of Ca(2+) from internal stores close to the plasma membrane. Perforated-patch whole cell recordings were used to determine whether Ca(2+)-induced Ca(2+) release (CICR) is involved in SMOC generation. We confirmed that BK channels are involved by showing that SMOCs are inhibited by 100 nM iberiotoxin or 500 microM tetraethylammonium (TEA), but not by 100 nM apamin. SMOC frequency is decreased in solutions that contain 0 Ca(2+)/3.6 mM Mg(2+), and also in the presence of 1 microM nifedipine and 3 microM omega-conotoxin GVIA, suggesting that SMOC activation is dependent on calcium influx. However, Ca(2+) influx alone is not sufficient; SMOC activation is also dependent on Ca(2+) release from the caffeine- and ryanodine-sensitive Ca(2+) store, because exposure to 2 mM caffeine consistently caused an increase in SMOC frequency, and 10-100 microM ryanodine altered the configuration of SMOCs and eventually inhibited SMOC activity. Depletion of intracellular Ca(2+) stores by the Ca-ATPase inhibitor cyclopiazonic acid (10 microM) inhibited SMOC activity, even when Ca(2+) influx was not compromised. We also tested the effects of the membrane-permeable Ca(2+) chelators, bis-(o-aminophenoxy)-N,N,N', N'-tetraacetic acid-AM (BAPTA-AM) and EGTA-AM. EGTA-AM (10 microM) caused no inhibition of SMOC activation, whereas 10 microM BAPTA-AM consistently inhibited SMOCs. After SMOCs were completely inhibited by BAPTA, 3 mM caffeine caused SMOC activity to resume. This effect was reversible on removal of caffeine and suggests that the source of Ca(2+) that triggers the internal Ca(2+) release channel is different from the source of Ca(2+) that activates clusters of BK channels. We propose that influx of Ca(2+) through voltage-dependent Ca(2+) channels is required for SMOC generation, but that the influx of Ca(2+) triggers CICR from intracellular stores, which then activates the BK channels responsible for SMOC generation.