Channels In Inside-out Patches Research Articles

Binding and effect of K ATP channel openers in the absence of Mg2+.

1 Openers of ATP-sensitive K(+) channels (K(ATP) channels) are thought to act by enhancing the ATPase activity of sulphonylurea receptors (SURs), the regulatory channel subunits. At higher concentrations, some openers activate K(ATP) channels also in the absence of MgATP. Here, we describe binding and effect of structurally diverse openers in the absence of Mg(2+) and presence of EDTA. 2 Binding of openers to SUR2B was measured using a mutant with high affinity for [(3)H]glibenclamide ([(3)H]GBC). In the absence of Mg(2+), 'typical' openers (benzopyrans, cyanoguanidines and aprikalim) inhibited [(3)H]GBC binding with K(i) values approximately 200 x higher than in the presence of MgATP. Minoxidil sulphate and nicorandil were inactive, whereas binding of diazoxide was unaffected by MgATP. 3 In the absence/presence of MgATP, N-cyano-N'-(1,1-dimethylpropyl)-N"-3-pyridylguanidine (P1075) activated the Kir6.2/SUR2B channel in inside-out patches with EC(50)=2000/67nM and E(max)=32/134%. In the absence of Mg(2+), responses were variable with only a small part of the variability being explained by a decrease in channel responsiveness with time after patch excision and to differences in the ATP sensitivity between patches. 4 The rank order of efficacy of the openers was P1075>rilmakalim approximately nicorandil>diazoxide>minoxidil sulphate. 5 The data show that structurally diverse openers are able to bind to, and to activate the Kir6.2/SUR2B channel by a pathway independent of ATP hydrolysis. These effects are observed at concentrations used to define the biochemical mechanism of the openers in the presence of MgATP and allow the openers to be classified into 'typical' and 'atypical' KCOs with diazoxide standing apart.

Changes in cAMP Affinity Modulate HCN Channel Activity

Wang et al. describe a mechanism whereby activity-dependent changes in adenosine 3′,5′-monophosphate (cAMP) binding affinity, rather than changes in cAMP concentration, can modulate activity of the hyperpolarization-activated, cyclic nucleotide-gated, cation nonselective (HCN) channel and can help regulate rhythmic firing. HCN channels are involved in pace-making and are modulated by cAMP, which facilitates channel opening. This has been modeled as an allosteric interaction in which channel opening is coupled with a transition to a conformation that enhances cAMP binding. Wang et al. used inside-out patch-clamp and voltage-clamp analyses of oocytes expressing HCN2 channel mRNA to test this model. Concentrations of cAMP ≥10 μM accelerated current activation and shifted the voltage dependence to more positive potentials, whereas 10 nM cAMP produced a slow phase of activation, consistent with preferential binding of cAMP to the open channel, shifting the equilibrium between closed and open unbound channels and eliciting a delayed phase of channel opening. Activation kinetics of channels in inside-out patches without cAMP fit a single exponential time course; a second exponential was apparent when cAMP was present. Activation kinetics of channels in intact oocytes resembled those of cell-free patches in low concentrations of cAMP; mutant channels that were unable to bind cAMP, however, showed only a single exponential component. Normal--but not mutant--channels showed "memory" and responded to a train of hyperpolarizations with a prolonged tail of channel opening that lasted for tens of seconds. Modeling studies indicated that cAMP modulation of neuronal HCN channels could play an important role in regulating rhythmic activity. J. Wang, S. Chan, M. F. Nolan, S. A. Siegelbaum, Activity-dependent regulation of HCN pacemaker channels by cyclic AMP: Signaling through dynamic allosteric coupling. Neuron 36 , 451-461 (2002). [Online Journal]

BK channels in human glioma cells have enhanced calcium sensitivity

We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at −60 mV by 2.1 × 10−6 M cytosolic Ca2+, were highly K+-selective, and had a slope conductance of ≈210 pS. We characterized the Ca2+ sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca2+]i. The half-maximal voltage for channel activation, V0.5, of glioma BK currents in outside-out patches was +138 mV with 0 Ca2+/10 EGTA. V0.5 was shifted to +81 mV and −14 mV with free [Ca2+]i of 1.5 × 10−7 M and 2.1 × 10−6 M, respectively. These results suggest that glioma BK channels have a higher Ca2+ sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary β-subunits by glioma cells. Expression of the auxiliary β-subunit of BK channels by glioma cells may relate to the high Ca2+ sensitivity of glioma BK channels. GLIA 38:281–291, 2002. © 2002 Wiley-Liss, Inc.

BK channels in human glioma cells have enhanced calcium sensitivity

We have previously demonstrated the expression of large-conductance, calcium-activated potassium (BK) channels in human glioma cells. In the present study, we characterized the calcium sensitivity of glioma BK channels in excised membrane patches. Channels in inside-out patches were activated at -60 mV by 2.1 x 10(-6) M cytosolic Ca(2+), were highly K(+)-selective, and had a slope conductance of approximately iqual 210 pS. We characterized the Ca(2+) sensitivity of these channels in detail by isolating BK currents in outside-out patches with different free [Ca(2+)](i). The half-maximal voltage for channel activation, V(0.5), of glioma BK currents in outside-out patches was +138 mV with 0 Ca(2+)/10 EGTA. V(0.5) was shifted to +81 mV and -14 mV with free [Ca(2+)](i) of 1.5 x 10(-7) M and 2.1 x 10(-6) M, respectively. These results suggest that glioma BK channels have a higher Ca(2+) sensitivity than that described in many other human preparations. Data obtained from a cloned BK channel (hbr5) expressed in HEK cells support the conclusion that glioma BK channels have an unusually high sensitivity to calcium. In addition, the sensitivity of glioma BK channels to the BK inhibitor tetrandrine suggests the expression of BK channel auxiliary beta-subunits by glioma cells. Expression of the auxiliary beta-subunit of BK channels by glioma cells may relate to the high Ca(2+) sensitivity of glioma BK channels.

Cysteine Residues in the Nucleotide Binding Domains Regulate the Conductance State of CFTR Channels

Gating of cystic fibrosis transmembrane conductance regulator (CFTR) channels requires intermolecular or interdomain interactions, but the exact nature and physiological significance of those interactions remains uncertain. Subconductance states of the channel may result from alterations in interactions among domains, and studying mutant channels enriched for a single conductance type may elucidate those interactions. Analysis of CFTR channels in inside-out patches revealed that mutation of cysteine residues in NBD1 and NBD2 affects the frequency of channel opening to the full-size versus a 3-pS subconductance. Mutating cysteines in NBD1 resulted in channels that open almost exclusively to the 3-pS subconductance, while mutations of cysteines in NBD2 decreased the frequency of subconductance openings. Wild-type channels open to both size conductances and make fast transitions between them within a single open burst. Full-size and subconductance openings of both mutant and wild-type channels are similarly activated by ATP and phosphorylation. However, the different size conductances open very differently in the presence of a nonhydrolyzable ATP analog, with subconductance openings significantly shortened by ATP γS, while full-size channels are locked open. In wild-type channels, reducing conditions increase the frequency and decrease the open time of subconductance channels, while oxidizing conditions decrease the frequency of subconductance openings. In contrast, in the cysteine mutants studied, altering redox potential has little effect on gating of the subconductance.

PKC Helps BK Channels Choose

The activation of large conductance Ca 2+ -activated K + (BK) channels results in cellular hyperpolarization and decreased excitation. Protein kinase C (PKC), adenosine 3′,5′-monophosphate (cAMP)-dependent protein kinase (PKA), and guanosine 3′,5′-monophosphate (cGMP)-dependent protein kinase (PKG) regulate the activity of BK channels. However, the mechanism by which these kinases regulate BK channel activity remains largely unknown. Zhou et al. cloned three isoforms of the BK channel α subunit, which differ only in their COOH-termini: both the A (BK A ) and B (BK B ) isoforms have two serine residues that serve as sites for protein kinase C (PKC)-mediated phosphorylation, whereas the C (BK C ) isoform does not. Inside-out cell membrane patches from HEK293 cells that expressed BK A or BK B were activated by the application of PKG but not by PKA, whereas BK C -containing inside-out patches were only activated by the application of PKA. Upon mutation of the PKC-mediated phosphorylation sites (serine to alanine) on BK A , PKA (but not PKG) activated the mutated BK channel in inside-out patches, suggesting that PKC-dependent phosphorylation of BK A switches the sensitivity of the BK channel from PKA to PKG. Mutation of either of the two PKC-mediated phosphorylation sites on BKA reduced, but did not completely eliminate, the capacity of PKG to activate BK A , which suggests that both Ser residues in BK A are important for optimal PKG-dependent activation of the BK channel. Mutation of the known distinct PKA or PKG phosphorylation sites on BK A prevented the activation of BK channels by those kinases, indicating that PKA- or PKG-mediated phosphorylation is essential to BK channel activation. Thus, the data suggest that the activity of PKC determines the sensitivity of BK channel activation to PKA or PKG. X.-B. Zhou, C. Arntz, S. Kamm, K. Motejlek, U. Sausbier, G.-X. Wang, P. Ruth, M. Korth, A molecular switch for specific stimulation of the BK Ca channel by cGMP and cAMP kinase. J. Biol. Chem. 276 , 43239-43245 (2001). [Abstract] [Full Text]

Effects of protein tyrosine kinase and protein tyrosine phosphatase on apical K(+) channels in the TAL.

We have previously demonstrated that the protein level of c-Src, a nonreceptor type of protein tyrosine kinase (PTK), was higher in the renal medulla from rats on a K-deficient (KD) diet than that in rats on a high-K (HK) diet (Wang WH, Lerea KM, Chan M, and Giebisch G. Am J Physiol Renal Physiol 278: F165-F171, 2000). We have now used the patch-clamp technique to investigate the role of PTK in regulating the apical K channels in the medullary thick ascending limb (mTAL) of the rat kidney. Inhibition of PTK with herbimycin A increased NP(o), a product of channel number (N) and open probability (P(o)), of the 70-pS K channel from 0.12 to 0.42 in the mTAL only from rats on a KD diet but had no significant effect in tubules from animals on a HK diet. In contrast, herbimycin A did not affect the activity of the 30-pS K channel in the mTAL from rats on a KD diet. Moreover, addition of N-methylsulfonyl-12,12-dibromododec-11-enamide, an agent that inhibits the cytochrome P-450-dependent production of 20-hydroxyeicosatetraenoic acid, further increased NP(o) of the 70-pS K channel in the presence of herbimycin A. Furthermore, Western blot detected the presence of PTP-1D, a membrane-associated protein tyrosine phosphatase (PTP), in the renal outer medulla. Inhibition of PTP with phenylarsine oxide (PAO) decreased NP(o) of the 70-pS K channel in the mTAL from rats on a HK diet. However, PAO did not inhibit the activity of the 30-pS K channel in the mTAL. The effect of PAO on the 70-pS K channel was due to indirectly stimulating PTK because pretreatment of the mTAL with herbimycin A abolished the inhibitory effect of PAO. Finally, addition of exogenous c-Src reversibly blocked the activity of the 70-pS K channel in inside-out patches. We conclude that PTK and PTP have no effect on the low-conductance K channels in the mTAL and that PTK-induced tyrosine phosphorylation inhibits, whereas PTP-induced tyrosine dephosphorylation stimulates, the apical 70-pS K channel in the mTAL.

Lipopolysaccharide can activate BK channels of arterial smooth muscle in the absence of iNOS expression

The role of inducible nitric oxide synthase (iNOS) in the acute activation of large-conductance, Ca 2+-dependent K + channels (BK channels) by Escherichia coli endotoxin (lipopolysaccharide, LPS) was studied in murine vascular smooth muscle cells. Confocal laser scanning microscopy and patch clamp recordings were utilised. Within 2 h of donor rat sacrifice, iNOS-like immunoreactivity could be detected in cerebrovascular smooth muscle cells (CVSMCs) enzymatically dispersed from rat cerebral arteries. This staining was absent in cells fixed immediately after donor rat sacrifice. LPS was then applied to the cytoplasmic face of inside-out membrane patches excised from rat CVSMCs within 2–4 h of donor rat sacrifice. It was found that LPS (10–100 μg/ml) rapidly and reversibly increased the open probability of BK channels in these patches. This LPS response was not altered in the presence of the non-isoform specific NOS inhibitor N ω-nitro- l-arginine. LPS responses were then compared in aortic smooth muscle (ASMC) BK channels derived from wild-type and iNOS-knockout (iNOS-KO) mice. LPS activated BK channels in inside-out patches of ASMC membrane derived from both wild-type and iNOS-knockout mice. These studies establish that LPS can activate BK channels by a mechanism quite independent of the well-established pathway mediated by iNOS in vascular smooth muscle cells.

Volume-dependent ATP-conductive large-conductance anion channel as a pathway for swelling-induced ATP release.

In mouse mammary C127i cells, during whole-cell clamp, osmotic cell swelling activated an anion channel current, when the phloretin-sensitive, volume-activated outwardly rectifying Cl(-) channel was eliminated. This current exhibited time-dependent inactivation at positive and negative voltages greater than around +/-25 mV. The whole-cell current was selective for anions and sensitive to Gd(3)+. In on-cell patches, single-channel events appeared with a lag period of approximately 15 min after a hypotonic challenge. Under isotonic conditions, cell-attached patches were silent, but patch excision led to activation of currents that consisted of multiple large-conductance unitary steps. The current displayed voltage- and time-dependent inactivation similar to that of whole-cell current. Voltage-dependent activation profile was bell-shaped with the maximum open probability at -20 to 0 mV. The channel in inside-out patches had the unitary conductance of approximately 400 pS, a linear current-voltage relationship, and anion selectivity. The outward (but not inward) single-channel conductance was suppressed by extracellular ATP with an IC(50) of 12.3 mM and an electric distance (delta) of 0.47, whereas the inward (but not outward) conductance was inhibited by intracellular ATP with an IC(50) of 12.9 mM and delta of 0.40. Despite the open channel block by ATP, the channel was ATP-conductive with P(ATP)/P(Cl) of 0.09. The single-channel activity was sensitive to Gd(3)+, SITS, and NPPB, but insensitive to phloretin, niflumic acid, and glibenclamide. The same pharmacological pattern was found in swelling-induced ATP release. Thus, it is concluded that the volume- and voltage-dependent ATP-conductive large-conductance anion channel serves as a conductive pathway for the swelling-induced ATP release in C127i cells.

Regulation of ROMK1 Channels by Protein-tyrosine Kinase and -tyrosine Phosphatase

We have used the two-electrode voltage clamp technique and the patch clamp technique to investigate the regulation of ROMK1 channels by protein-tyrosine phosphatase (PTP) and protein-tyrosine kinase (PTK) in oocytes coexpressing ROMK1 and cSrc. Western blot analysis detected the presence of the endogenous PTP-1D isoform in the oocytes. Addition of phenylarsine oxide (PAO), an inhibitor of PTP, reversibly reduced K(+) current by 55% in oocytes coinjected with ROMK1 and cSrc. In contrast, PAO had no significant effect on K(+) current in oocytes injected with ROMK1 alone. Moreover, application of herbimycin A, an inhibitor of PTK, increased K(+) current by 120% and completely abolished the effect of PAO in oocytes coexpressing ROMK1 and cSrc. The effects of herbimycin A and PAO were absent in oocytes expressing the ROMK1 mutant R1Y337A in which the tyrosine residue at position 337 was mutated to alanine. However, addition of exogenous cSrc had no significant effect on the activity of ROMK1 channels in inside-out patches. Moreover, the effect of PAO was completely abolished by treatment of oocytes with 20% sucrose and 250 microg/ml concanavalin A, agents that inhibit the endocytosis of ROMK1 channels. Furthermore, the effect of herbimycin A is absent in the oocytes pretreated with either colchicine, an inhibitor of microtubules, or taxol, an agent that freezes microtubules. We conclude that PTP and PTK play an important role in regulating ROMK1 channels. Inhibiting PTP increases the internalization of ROMK1 channels, whereas blocking PTK stimulates the insertion of ROMK1 channels.

Properties and pharmacological modification of ATP-sensitive K(+) channels in cat tracheal myocytes.

The effects of levcromakalim and nucleoside diphosphates (NDPs) on both membrane currents and unitary currents in cat trachea myocytes were investigated by use of patch-clamp techniques. In conventional whole-cell configuration, levcromakalim produced a concentration-dependent K(+) current which was suppressed by additional application of 5 microM glibenclamide at -70 mV. When 3 mM ATP was added in the pipette solution, the peak amplitude of the levcromakalim-induced current was much smaller than that in the absence of ATP. When 3 mM uridine 5'-diphosphate (UDP) was included in the pipette solution, much higher concentrations of glibenclamide (>/=50 microM) were required to suppress the 100 microM levcromakalim-induced membrane current in comparison with those in the absence of UDP. In the cell-attached patches, levcromakalim activated a 40 pS K(+) channel which was inhibited by additional application of glibenclamide in symmetrical 140 mM K(+) conditions. UDP (>/=0.1 mM) was capable of reactivating the channel in inside-out patches in which the glibenclamide-sensitive K(+) channel had run down, in the presence of levcromakalim. The K(+) channel reactivated by UDP was suppressed by additional application of either intracellular 3 mM ATP or 100 microM glibenclamide. These results demonstrate that smooth muscle cells in the cat trachea possess ATP-sensitive 40 pS K(+) channels which are blocked by glibenclamide (i.e. K(ATP)) and can be activated by levcromakalim and that intracellular UDP causes a significant shift of the glibenclamide-sensitivity of these channels.

Protein tyrosine kinase regulates the number of renal secretory K channels.

The apical small conductance (SK) channel plays a key role in K secretion in the cortical collecting duct (CCD). A high-K intake stimulates renal K secretion and involves a significant increase in the number of SK channels in the apical membrane of the CCD. We used the patch-clamp technique to examine the role of protein tyrosine kinase (PTK) in regulating the activity of SK channels in the CCD. The application of 100 microM genistein stimulated SK channels in 11 of 12 patches in CCDs from rats on a K-deficient diet, and the mean increase in NP(o), a product of channel number (N) and open probability (P(o)), was 2.5. In contrast, inhibition of PTK had no effect in tubules from animals on a high-K diet in all 10 experiments. Western blot analysis further shows that the level of cSrc, a nonreceptor type of PTK, is 261% higher in the kidneys from rats on a K-deficient diet than those on a high-K diet. However, the effect of cSrc was not the result of direct inhibition of channel itself, because addition of exogenous cSrc had no effect on SK channels in inside-out patches. In cell-attached patches, application of herbimycin A increased channel activity in 14 of 16 patches, and the mean increase in NP(o) was 2.4 in tubules from rats on a K-deficient diet. In contrast, herbimycin A had no effect on channel activity in any of 15 tubules from rats on a high-K diet. Furthermore, herbimycin A pretreatment increased NP(o) per patch from the control value (0.4) to 2.25 in CCDs from rats on a K-deficient diet, whereas herbimycin failed to increase channel activity (NP(o): control, 3.10; herbimycin A, 3.25) in the CCDs from animals on a high-K diet. We conclude that PTK is involved in regulating the number of apical SK channels in the kidney.

TGFbeta1 regulates the gating properties of intermediate-conductance KCa channels in developing parasympathetic neurons.

The developmental expression of Ca2+-activated K+ channels (KCa) in chick ciliary ganglion (CG) neurons is regulated by a target-derived avian isoform of TGFbeta1, which evokes a robust increase in the number of functional large-conductance (BK) KCa channels but which produces no change in their kinetics. However, CG neurons express multiple KCa channel subtypes. Here we show that TGFbeta1 regulates the gating properties of intermediate-conductance (IK) KCa channels in developing CG neurons. IK channels in inside-out patches excised from control E9 CG neurons became active on exposure to 1-5 microM free Ca2+ but then remained active on return to Ca2+-free salines. In contrast, IK channels in TGFbeta1-treated cells became active on exposure to 1-5 microM Ca2+, but became quiescent immediately on return to Ca2+-free salines. In contrast to its effects on BK channels, TGFbeta1 had no effect on the mean number of IK channels detected in excised patches. IK channels were not activated in cell-attached patches on E9 neurons depolarized by bath application of 145 mM KCl in the presence of 5 mM external Ca2+. However, BK channels were activated immediately by this procedure and were detected at a higher density in TGFbeta1-treated cells. In addition, analyses of macroscopic KCa fluctuations, and the voltage-dependence of KCa tail currents, suggest that IK channels do not contribute to voltage-evoked whole cell KCa. IK channels therefore may have some other function. These results indicate that the effects of TGFbeta1 on CG neurons entail distinct actions on multiple KCa channel subtypes.

Imidazolines and the pancreatic B-cell. Actions and binding sites.

Stimulation of insulin secretion by imidazoline compounds displays variable characteristics. Phentolamine (10-100 microM) increased secretion of perifused mouse islets at nonstimulatory glucose concentrations (5 mM) and even in the absence of glucose. Idazoxan (20-100 microM) elicited a moderate increase in insulin secretion, which required the presence of a stimulatory glucose concentration (10 mM). Phentolamine is therefore a stimulator of secretion in its own right, whereas idazoxan may be termed an enhancer of secretion. Both compounds inhibited the activity of ATP-dependent K+ channels in inside-out patches from B-cells; however, idazoxan achieved only an incomplete block. Both compounds depolarized the B-cell plasma membrane to an extent that permitted the opening of voltage-dependent Ca2+ channels (-40 to -30 mV). An increase in cytoplasmic Ca2+ concentration was induced by phentolamine and much less so by idazoxan. Activation of protein kinase C, a possible mechanism to amplify Ca(2+)-induced secretion, could not be verified for phentolamine. It thus appears that stimulation of insulin secretion by phentolamine is due to its blocking effect on KATP channels, which may be the correlate of non-adrenergic imidazoline binding sites which were characterized in insulin-secreting HIT cells. Whether incomplete closure of KATP channels by idazoxan or additional effects are responsible for the requirement of high glucose to stimulate secretion remains to be clarified.

Mitochondria present in excised patches from pancreatic B-cells may form microcompartments with ATP-dependent potassium channels.

Experiments with inside-out patches excised from pancreatic B-cells have yielded evidence that mitochondria are often contained in the cytoplasmic plug protruding into the tip of patch pipette. When intact B-cells were loaded with the fluorescent mitochondrial stain, rhodamine 123, and membrane patches excised from these cells, a green fluorescence could be observed in the lumen at the tip of the patch pipette. The same result was obtained with the mitochondrial stain, MitoTracker Green FM, which is only fluorescent in a membrane-bound state. Furthermore, the open probability of ATP-dependent potassium (K(ATP)) channels in inside-out patches was influenced by mitochondrial fuels and inhibitors. Respiratory substrates like tetramethyl phenylene diamine (2 mM) plus ascorbate (5 mM) or alpha-ketoisocaproic acid (10 mM) reduced the open probability of K(ATP) channels in inside-out patches significantly (down to 57% or 65% of control, respectively). This effect was antagonized by the inhibitor of cytochrome oxidase, sodium azide (5 mM). Likewise, the inhibitor of succinate dehydrogenase, malonate (5 mM), increased the open probability of K(ATP) channels in the presence of succinate (1 mM). However, oligomycin in combination with antimycin and rotenone did not increase open probability. Although it cannot be excluded that these effects result from a direct interaction with the K(ATP) channels, the presence of mitochondria in the close vicinity permits the hypothesis that changes in mitochondrial metabolism are involved, mitochondria and K(ATP) channels thus forming functional microcompartments.

The A kinase anchoring protein is required for mediating the effect of protein kinase A on ROMK1 channels.

In the present study, we have used the two-electrode voltage-clamp and patch-clamp techniques to study the effects of forskolin and cAMP on the ROMK1 channels, which are believed to be the native K+ secretory channels in the kidney. Addition of 1 microM forskolin or 100 microM 8-bromo-cAMP, within 10 min, has no significant effect on the current of ROMK1 channels expressed in Xenopus oocytes. In contrast, application of 1 microM forskolin, within 3 min, significantly increased whole-cell K+ current by 35%, when ROMK1 channels were coexpressed with the A kinase anchoring protein AKAP79, which was cloned from neuronal tissue. Two lines of evidence indicate that the effect of forskolin is mediated by a cAMP-dependent pathway: (i) Addition of 100 microM 8-bromo-cAMP mimics the effect of forskolin and (ii) the effect of forskolin and cAMP is not additive. That AKAP is required for the effect of cAMP is further supported by experiments in which addition of ATP (100 microM) and cAMP (100 microM) restored the activity of run-down ROMK1 channels in inside-out patches in oocytes that coexpressed ROMK1 and AKAP79 but not in those that expressed ROMK1 alone. Moreover, when we used RII, the regulatory subunit of type II protein kinase A, in an overlay assay, we identified a RII-binding protein in membranes obtained from the kidney cortex but not in membranes from oocytes. This suggests that the insensitivity of ROMK1 channels to forskolin and cAMP is due to the absence of AKAPs. We conclude that AKAP may be a critical component that mediates the effect of protein kinase A on the ROMK channels in the kidney.

Effects of cytosolic ATP and other nucleotides on Ca 2+-activated K + channels in cultured bovine adrenal chromaffin cells

The effects of cytosolic ATP on Ca 2+-dependent K + (K Ca) channel activation in cultured bovine adrenal chromaffin cells were investigated by using single-channel recording patch-clamp techniques. Application of ATP to the intracellular surface of excised inside-out patches activated K Ca channels in a dose-dependent manner at 30 μM to 10 mM. The K Ca channels also were activated by 3 mM of adenosine 5′- O-(3′-thiotriphosphate) (ATP γS), a non-hydrolyzable analogue of ATP, but not by 5′-adenylylimidodiphosphate (AMP-PNP) (from 300 μM to 3 mM). Furthermore, other nucleotides also activated K Ca channels in inside-out patches. This modulation took place without addition of exogenous protein kinase and was dependent on the presence of Mg 2+ in the bathing solution. Staurosporine, a non-specific kinase inhibitor, or H-89 ( N-[2-( p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), a cAMP-dependent protein kinase inhibitor, was unable to alter ATP-mediated K Ca channel activation. Following complete removal of Mg 2+, a higher concentration of ATP (10 mM) and other nucleotides was required to activate K Ca channels; however, Mg 2+ was ineffective in altering the activation of K Ca channels by itself. It is concluded that intracellular ATP and other nucleotides activate K Ca channels directly. These nucleotides may regulate catecholamine release by changing the cell membrane potential in adrenal chromaffin cells.

Activation of Ca2+-activated K+ channels by an increase in intracellular Ca2+ induced by depolarization of mouse skeletal muscle fibres.

1. Ionic currents were simultaneously recorded at macroscopic and unitary level using the whole-cell and cell-attached patch-clamp procedures together on the same portion of isolated mouse skeletal muscle fibres. 2. In the presence of Tyrode solution in the patch pipette and Tyrode-TTX solution in the bath, macroscopic and unitary currents through delayed rectifier K+ channels were simultaneously recorded in response to depolarizing pulses of 1 s duration. 3. In five fibres, successive long-lasting incremental depolarizing levels induced, at -40 mV or -30 mV, the opening of a high conductance channel carrying an outward current superimposed on delayed rectifier K+ channel activity. Opening of this high conductance channel was not observed when the depolarization steps were applied in the patch pipette. 4. Using the same depolarizing protocol, activation of a high conductance channel was also observed in two fibres in the presence of a K+-rich solution in the pipette (145 mM K+) . 5. With either Tyrode or K+-rich solution in the pipette, unitary current amplitudes of the high conductance channel matched well with the values obtained for Ca2+-activated K+ (KCa) channels in inside-out patches under similar ionic conditions. 6. Indo-1 fluorescence measurements showed that the stimulation protocol that led to KCa channel opening induced stepwise increases in intracellular [Ca2+] in the submicromolar range. 7. Our results provide evidence that activation of sarcolemmal KCa channels can be induced by a rise in intracellular [Ca2+] following voltage-activated sarcoplasmic reticulum Ca2+ release.

Regulation of heterologously expressed transient receptor potential-like channels by calcium ions

The Drosophila melanogaster gene product TRPL (transient receptor potential-like) is a Ca 2+-permeable cation channel that contributes to the light-induced Ca 2+ entry in Drosophila photoreceptors and bears homology to several recently cloned mammalian channels. Intracellular Ca 2+ has been implicated to stimulate TRPL channels. This constitutes a potentially dangerous mechanism that may lead to Ca 2+ overload. Therefore, we studied whether TRPL channels, like other Ca 2+-permeable channels, are inhibited by intracellular Ca 2+ concentrations in the micromolar range and whether this effect is mediated by calmodulin. In Sf9 cells expressing the TRPL gene along with histamine H1 receptors after infection with baculoviruses containing the corresponding complementary DNA, histamine-induced TRPL currents were inhibited by intracellular Ca 2+ with an ic 50 of 2.3 μM. Moreover, TRPL currents were reversibly attenuated by a preceding hyperpolarization. This attenuation reflected the action of an increased Ca 2+ influx, since it was abolished in the absence of extracellular Ca 2+ and enhanced by raising extracellular Ca 2+ to 20 mM. Finally, the activity of TRPL channels in inside-out patches was reversibly inhibited by raising the Ca 2+ concentration on the cytosolic side of the patches to 10–50 μM. Addition of calmodulin or the calmodulin inhibitor calmidazolium did not modify the inhibition of the TRPL by Ca 2+. We conclude that high intracellular Ca 2+ concentrations inhibit the TRPL gene, but no evidence was found for the requirement of calmodulin. This mechanism makes Ca 2+ influx through the TRPL gene self-limiting. Furthermore, the TRPL may allow one to study the structural requirements for channel regulation by Ca 2+.

Characterization and function of Ca(2+)-activated K+ channels in arteriolar muscle cells.

We examined the functional role of large-conductance Ca(2+)-activated K+ (KCa) channels in the hamster cremasteric microcirculation by intravital videomicroscopy and characterized the single-channel properties of these channels in inside-out patches of membrane from enzymatically isolated cremasteric arteriolar muscle cells. In second-order (39 +/- 1 microns, n = 8) and third-order (19 +/- 2 microns, n = 8) cremasteric arterioles with substantial resting tone, superfusion with the KCa channel antagonists tetraethylammonium (TEA, 1 mM) or iberiotoxin (IBTX, 100 nM) had no significant effect on resting diameters (P > 0.05). However, TEA potentiated O2-induced arteriolar constriction in vivo, and IBTX enhanced norepinephrine-induced contraction of cremasteric arteriolar muscle cells in vitro. Patch-clamp studies revealed unitary K(+)-selective and IBTX-sensitive currents with a single-channel conductance of 240 +/- 2 pS between -60 and 60 mV (n = 7 patches) in a symmetrical 140 mM K+ gradient. The free Ca2+ concentration ([Ca2+]) for half-maximal channel activation was 44 +/- 3, 20 +/- 1, 6 +/- 0.4, and 3 +/- 0.5 microM at membrane potentials of -60, -30, +30, and +60 mV, respectively (n = 5), with a Hill coefficient of 1.9 +/- 0.2. Channel activity increased e-fold for a 16 +/- 1 mV (n = 6) depolarization. The plot of log[Ca2+] vs. voltage for half-maximal activation (V1/2) was linear (r2 = 0.9843, n = 6); the change in V1/2 for a 10-fold change in [Ca2+] was 84 +/- 5 mV, and the [Ca2+] for half-maximal activation at 0 mV (Ca0; the Ca2+ set point) was 9 microM. Thus, in vivo, KCa channels are silent in cremasteric arterioles at rest but can be recruited during vasoconstriction. We propose that the high Ca0 is responsible for the apparent lack of activity of these channels in resting cremasteric arterioles, and we suggest that this may result from expression of unique KCa channels in the microcirculation.