Characterization of BK channel splice variants using membrane potential dyes.

BACKGROUND AND PURPOSE: Large conductance calcium- and voltage-activated potassium (BK) channels are encoded by a single gene that displays extensive pre-mRNA splicing. Here we exploited a membrane potential assay to investigate the sensitivity of different BK splice variants to elevations in intracellular free calcium and their inhibition by the BK channel blocker paxilline. EXPERIMENTAL APPROACH: Murine BK channel splice variants were expressed in human embryonic kidney 293 cells and their properties analysed in response to ionomycin-induced calcium influx in both fluorescent membrane potential (fluorescent-imaging plate reader) and patch clamp electrophysiological assays. The dose-dependent inhibition of distinct splice variants by the BK channel-specific blocker paxilline was also investigated. KEY RESULTS: Ionomycin-induced calcium influx induced a robust hyperpolarization of human embryonic kidney 293 cells expressing distinct BK channel splice variants: stress regulated exon (STREX), e22 and ZERO. Splice variant expression resulted in membrane hyperpolarization that displayed a rank order of potency in response to calcium influx of STREX > e22 > ZERO. The BK channel inhibitor paxilline exhibited very similar potency on all three splice variants with IC(50)s in membrane potential assays of 0.35 +/- 0.04, 0.37 +/- 0.03 and 0.70 +/- 0.02 micromol x L(-1) for STREX, ZERO and e22 respectively. CONCLUSIONS AND IMPLICATIONS: BK channel splice variants can be rapidly discriminated using membrane potential based assays, based on their sensitivity to calcium. BK channel splice variants are inhibited by the specific blocker paxilline with similar IC(50)s. Thus, paxilline may be used in functional assays to inhibit BK channel function, irrespective of the variant expressed.

The effects of neuroleptic and tricyclic compounds on BKCa channel activity in rat isolated cortical neurones.

1. The actions of several neuroleptic and tricyclic compounds were examined on the large conductance Ca(2+)-activated K+ (BKCa) channel present in neurones isolated from the rat motor cortex. 2. Classical neuroleptic compounds including chlorpromazine and haloperidol applied to the intracellular surface of inside-out patches produced a concentration-dependent reduction in BKCa channel activity. Similar effects were observed when these compounds were applied to the extracellular surface of outside-out patches. 3. In contrast, the atypical neuroleptic compounds clozapine and sulpiride did not affect BKCa channel activity (100 nM-1 mM) in either inside-out or outside-out patches, while 10 microM pimozide produced 73% of the inhibition produced by 10 microM chlorpromazine. 4. BKCa channel activity was also unaffected by application of structurally related tricyclic compounds including the anti-cholinesterase tacrine and the anti-epileptic carbamazepine. The tricyclic antidepressant drug amitriptyline was found to inhibit BKCa channel activity but was much less effective than the classical neuroleptic compounds. 5. It is concluded that compounds belonging to the classical neuroleptic group of drugs inhibit BKCa channel activity in the rat motor cortex in a structurally-specific manner. This observation may be of clinical significance as it may contribute to some of the side effects associated with classical neuroleptic drug therapy.

Effect of englitazone on KATP and calcium-activated non-selective cation channels in CRI-G1 insulin-secreting cells.

1. The effects of englitazone sodium, an antidiabetic agent, on ion channel activity in the CRI-G1 insulin secreting cell line was examined by use of the patch clamp technique. 2. Application of englitazone to the outside of CRI-G1 cells in the whole-cell recording configuration produced concentration-dependent inhibition of KATP currents with an IC50 value of 8 microM. The inhibition of the K+ current was not affected by the removal of Mg2+ ions from or the addition of trypsin to the solution bathing the intracellular surface of the cell membrane. 3. Englitazone also inhibited KATP channel activity in recordings from inside out excise membrane patches. The concentration-dependence of inhibition was identical to that observed in whole-cell recordings and was voltage-independent. Single channel recordings confirmed that neither the absence or presence of Mg2+ ions nor the addition of trypsin at the intracellular surface of the membrane influenced the inhibition of KATP channels by englitazone. 4. Englitazone also inhibited Ca(2+)-activated non-selective cation (NSCa) channels in inside-out patches in a concentration-dependent and voltage-independent manner with an IC50 value of 10 microM. In comparison, the non-sulphonylurea KATP channel blocker ciclazindol produced a slight voltage-dependent inhibition of the NSCa channel at a concentration of 20 microM. 5. In whole-cell recordings englitazone, at a relatively high concentration (50 microM) in comparison with that required to block KATP and NSCa channels, inhibited voltage-activated Ca2+ currents by 33% but did not inhibit voltage-activated K+ and Na+ currents. 6. It is concluded that englitazone is a novel blocker of NSCa and KATP channels. The inhibition of KATP channels occurs following procedures that dissociate sulphonylurea receptor coupling to the channel. The equipotent and voltage-independent inhibition of NSCa and KATP channels by englitazone may indicate a common mechanism of block.

Potassium channel dysfunction in hypothalamic glucose-receptive neurones of obese Zucker rats.

1. We have shown, using intracellular and cell-attached recordings, that glucose-receptive (GR) neurones of obese Zucker rats exhibit abnormal electrophysiological responses to changes in extracellular glucose concentration, whereas GR neurones of lean Zucker and control rats respond normally. 2. In inside-out recordings from obese rat GR neurones it was shown that the 150 pS ATP-sensitive K+ (KATP) and the 160 pS calcium-activated K+ (KCa) channels were absent, whereas both were present in GR neurones of lean Zucker and control rats. 3. The potassium channel most frequently observed in inside-out patches from obese GR neurones was characterized by a conductance of 213 pS, was activated by raising internal calcium and inhibited by application of internal ATP. This channel (which we have termed Kfa) was not observed in lean or control rat GR neurones. 4. Tolbutamide (100 microM) was found to induce no effect or to elicit a small depolarization of obese rat GR neurones in the absence of glucose, in contrast to its clear excitatory actions on control or lean Zucker GR neurones. 5. Intracellular, cell-attached and inside-out recordings from obese rat non-GR neurones showed that there was no alteration in their membrane properties or firing characteristics or in the characteristics of the large-conductance calcium-activated K+ channel (KCa) present in these neurones as compared with lean and control rats. 6. It is concluded that the Kfa channel is specific to GR neurones of obese Zucker rats and that the presence of this channel coupled with the absence of KATP and KCa channels results in the abnormal glucose-sensing response of these neurones.

The high-affinity sulphonylurea receptor regulates KATP channels in nerve terminals of the rat motor cortex.

The coexpression of sulphonylurea binding sites and ATP-sensitive K+(KATP) channels was examined in the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. These channels were not detected on neuronal cell bodies, but sulphonylurea-sensitive KATP channels and charybdotoxin-sensitive, large-conductance calcium-activated K+ BKCa channels were detected by patch clamping of fused nerve terminals from the motor cortex. Subcellular fractionation revealed that high-affinity sulphonylurea binding sites were enriched in the nerve terminal fraction, whereas glibenclamide increased calcium-independent glutamate efflux from isolated nerve terminals. It is concluded that neuronal sulphonylurea receptors and KATP channels are functionally linked in the motor cortex and that they are both selectively expressed in nerve terminals, where the KATP channel may serve to limit glutamate release under conditions of metabolic stress.

Ciclazindol inhibits ATP-sensitive K+ channels and stimulates insulin secretion in CR1-G1 insulin-secreting cells.

Ciclazindol, an anorectic drug, was shown to inhibit ATP-sensitive K+ (K(ATP)) channel currents and stimulate insulin secretion from CRI-G1 insulin-secreting cells. In contrast, the structurally related anorectic agent mazindol and the amphetamine-based anorectic compounds diethylpropion, fenfluramine, and phentermine had no effect on K(ATP) channel activity in this cell line. Similarly, cicliazindol elicited insulin secretion from CRI-G1 cells, whereas mazindol had no secretagogue action. The mechanism by which ciclazindol acts to inhibit K(ATP) channel activity is different than that of the sulfonylureas as ciclazindol is effective after procedures that decouple the sulfonylurea receptor from the K(ATP) channel. In agreement with this finding, ciclazindol failed to displace [3H]glibenclamide from CRI-G1 microsomal membranes. Further experiments demonstrated that ciclazindol has no significant effect on voltage-activated currents in this cell line.

Activation by intracellular ATP of a potassium channel in neurones from rat basomedial hypothalamus.

1. Cell-attached recordings from isolated glucose-sensitive hypothalamic neurones show that on removal of extracellular glucose there is an increased action current frequency concomitant with decreased single-channel activity. Conversely activation of single K+ channels was observed when extracellular glucose was increased. Isolation of membrane patches into the inside-out configuration following cell-attached recording demonstrated the presence of an ATP-activated K+ channel. 2. The ATP-activated K+ channel was characterized by a mean single-channel conductance of 132 pS in symmetrical 140 mM KCl solutions. Single-channel open-state probability (Po) was not calcium dependent, and the presence of calcium did not prevent activation of the channel by ATP. 3. Activation of the channel by ATP was concentration dependent and the Po of the ATP-activated channel was unaffected by membrane voltage, regardless of the degree of activation elicited by ATP. 4. Open and closed time histograms were constructed from inside-out and cell-attached recordings and were consistent with a single open and two closed states. Channel openings were grouped in bursts. Application of ATP, in isolated patches, and glucose, in cell-attached patches, increased the burst duration and number of bursts per second and decreased the slow closed-state time constant. In neither case was there a significant change in the fast closed-state time constant nor the open-state time constant. 5. The non-hydrolysable ATP analogue adenylylimidodiphosphate (AMP(PNP)) and 'Mg2(+)-free' ATP produced little change in the Po of the ATP-activated K+ channel when applied to the intracellular surface of excised patches. These results suggest that activation of this channel is via an enzymic mechanism. 6. ADP, GTP and GDP also activated the channel in a Mg(2+)-dependent manner. ADP and ATP activated the channel in an additive manner and neither GTP nor GDP inhibited channel activity induced by ATP. 7. It is concluded that the ATP-activated K+ channel observed in isolated inside-out patches from hypothalamic neurones is the same as the channel activated by an increase in the concentration of extracellular glucose in cell-attached recordings from glucose-sensitive neurones.

Characterization of an ATP-modulated large conductance Ca(2+)-activated K+ channel present in rat cortical neurones.

1. Single channel current recordings were used to study the characteristics of a large conductance Ca(2+)-activated K+ (BKCa) channel present in neurones acutely dissociated from the rat motor cortex. Application of ATP to the intracellular surface of excised inside-out patches produced a large, concentration-dependent increase in BKCa channel activity. 2. This ATP-mediated activation was dependent upon the presence of Mg2+ in the intracellular bathing solution and was diminished by the phosphatases 2,3-butanedione monoxime (BDM) or alkaline phosphatase and by the protein kinase inhibitors staurosporine, H-7 and PKI. 3. ADP stimulated BKCa channel activity in a Mg(2+)-dependent manner, an action also inhibited by the concomitant application of PKI or BDM. The effect of ADP was reduced by application of hexokinase and glucose or by application of the adenylate kinase inhibitor Ap5A. 4. Of other nucleotides tested, only CTP consistently activated BKCa channel activity. 5. Using the cell-attached configuration, bath application of forskolin or dibutyryl cAMP stimulated BKCa channel activity. 6. It is concluded that BKCa channel activity in the rat motor cortex is subject to modulation by the activity of a closely associated kinase. The ability of cAMP activators to stimulate BKCa channel activity in the intact cell suggests that this system may be of physiological importance.

NS 1619 activates BKCa channel activity in rat cortical neurones.

Single channel recordings of large conductance Ca2(+)-activated K+ (BKCa) channels were made from neurones isolated from rat motor cortex. Application of levcromakalim, pinacidil or diazoxide had no effect on BKCa channel activity in excised patches. In contrast, NS 1619 (1-(2'-hydroxy-5'-trifluoromethylphenyl)-5-trifluoromethyl- 2(3H)benzimidazalone) induced concentration-dependent activation of BKCa channels with a calculated EC50 of 32 microM. The NS 1619-induced activity was dependent on the presence of free Ca2+ at the intracellular surface, but was not associated with a change in channel voltage sensitivity. Niflumic acid had no effect on BKCa activity per se but prevented NS 1619-mediated activation.

Cloning and functional expression of the cDNA encoding an inwardly-rectifying potassium channel expressed in pancreatic beta-cells and in the brain.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1;[6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K(+)-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and beta-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

Direct demonstration of sulphonylurea-sensitive KATP channels on nerve terminals of the rat motor cortex.

We examined whether ATP-sensitive potassium (KATP) channels are present on presynaptic terminals of the rat motor cortex, an area of the CNS exhibiting a high density of sulphonylurea binding. A novel fused nerve terminal preparation was developed which produced structures amenable to patch clamp methods. In inside-out recordings a K+ channel was observed which possessed all the major features of the Type 1 KATP channel, including sensitivity to ATP and the antidiabetic sulphonylureas.

The effects of trypsin on ATP-sensitive potassium channel properties and sulfonylurea receptors in the CRI-G1 insulin-secreting cell line.

The effects of the proteolytic enzyme trypsin upon ATP-sensitive potassium (KATP) channel activity were examined in the CRI-G1 insulin-secreting cell line. Trypsin activated channels only when applied to the intracellular surface of the cell membrane. The activation could be prevented by the concomitant application of trypsin inhibitor or by heat inactivation of the enzyme. The trypsin-induced change in channel activity was accompanied by a reduction in the rate of channel rundown. However, trypsin did not affect the mean single channel conductance (55.2 pS), the ionic selectivity, or rectification of the KATP channel. Concentration response curves for various KATP channel inhibitors were constructed in the presence and absence of intracellular trypsin. The EC50 for tolbutamide was shifted from 30.0 +/- 4.5 microM, with 100 micrograms/ml heat-inactivated trypsin present to 9.7 +/- 1.0 mM with active trypsin in the intracellular solution. Treatment of the cells' external surface with 1 mg/ml trypsin did not alter the potency of tolbutamide. Intracellular trypsin also produced a significant fall in the potency of glibenclamide, meglitinide, and phentolamine but did not alter the effectiveness of thiopentone. Radioligand binding studies demonstrated a total loss of 3H-labeled glibenclamide binding when the intracellular surface of the cells was exposed to trypsin. In contrast, 3H-labeled glibenclamide binding was not affected when the enzyme was applied to the external surface. Trypsin treatment, therefore, alters a number of characteristics of KATP channel pharmacology, and we suggest that this is due to action at possibly more than one site but includes the functional cleavage of the sulfonylurea receptor from the KATP channel.

Potassium channel activity in sarcolemmal vesicles formed from skeletal muscle fibres of normal and dystrophic mice.

Sarcolemmal vesicles were produced from adult mouse extensor digitorum longus muscle (EDL) by treating swollen muscle fibres with collagenase. Vesicles formed from dystrophic (C57BL/6J dy/dy) and phenotypically normal animals were patch clamped and the single channel activity was recorded. Three types of K+ channel were observed in excised patches taken from normal and dystrophic muscle. A large conductance (300 pS) Ca2(+)-dependent K+ channel (KCa) was the most frequently observed of the K+ channels in both types of muscle preparation. In a number of patches taken from dystrophic muscle the open probability-voltage relationship for the KCa channel was markedly different from that in normal muscle, suggesting a possible reduction in Ca2+ sensitivity. An ATP-sensitive K+ channel (90 pS) was common to both normal and dystrophic muscle vesicles and was present in a large number of patches. An inwardly rectifying K+ channel (40 pS) was also observed in both types of sarcolemmal vesicles. The properties of all three K+ channels types were broadly consistent with other observations of skeletal muscle K+ channels, though all had higher conductances than had previously been noted in other species.

Sarcolemmal K+ channel activity in developing rat skeletal muscle membranes.

A method has been adapted to produce membrane vesicles suitable for routine membrane patch clamping from neonate rat skeletal muscle. Single K+ channel activity was recorded from cell-free inside-out patches. Most Ca2(+)-activated voltage sensitive channels had large conductances of up to 300 pS, as determined from their current/voltage relationship, and an open probability (Po) approaching unity at positive membrane potentials. A lower conductance K+ channel, probably responsible for inward rectification, had a lower conductance of about 100 pS. Outward rectifying K+ channels were also observed with the lowest conductance, about 40 pS. 0.1 mM ATP when applied to the inner membrane surface reduced or blocked activity, drastically reducing Po without altering single channel conductance. Such an effect has been reported in other preparations but was different in the neonate preparation in that it blocked channels with conductances as high as 300 pS. The simple preparation described, which we have also used successfully on mature rat and mouse skeletal muscle, has potential in the analysis of channel activities in various conditions and pathologies without the need for tissue culture to produce suitable membrane preparations.