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P Proks

Publications and source records attributed to P Proks.

35 records · Page 2Linked to original sources
PubMed1998

Molecular analysis of ATP-sensitive K channel gating and implications for channel inhibition by ATP.

The beta cell KATP channel is an octameric complex of four pore-forming subunits (Kir6.2) and four regulatory subunits (SUR1). A truncated isoform of Kir6.2 (Kir6.2DeltaC26), which expresses independently of SUR1, shows intrinsic ATP sensitivity, suggesting that this subunit is primarily responsible for mediating ATP inhibition. We show here that mutation of C166, which lies at the cytosolic end of the second transmembrane domain, to serine (C166S) increases the open probability of Kir6.2DeltaC26 approximately sevenfold by reducing the time the channel spends in a long closed state. Rundown of channel activity is also decreased. Kir6.2DeltaC26 containing the C166S mutation shows a markedly reduced ATP sensitivity: the Ki is reduced from 175 microM to 2.8 mM. Substitution of threonine, alanine, methionine, or phenylalanine at position C166 also reduced the channel sensitivity to ATP and simultaneously increased the open probability. Thus, ATP does not act as an open channel blocker. The inhibitory effects of tolbutamide are reduced in channels composed of SUR1 and Kir6.2 carrying the C166S mutation. Our results are consistent with the idea that C166 plays a role in the intrinsic gating of the channel, possibly by influencing a gate located at the intracellular end of the pore. Kinetic analysis suggests that the apparent decrease in ATP sensitivity, and the changes in other properties, observed when C166 is mutated is largely a consequence of the impaired transition from the open to the long closed state.

Adenosine Triphosphate↗
PubMed1998

Electrophysiological and metabolic characterization of single beta-cells and islets from diabetic GK rats.

We have used the whole-cell recording technique to determine whether ATP-sensitive potassium (K[ATP]) currents, voltage-dependent Ca2+ currents, and exocytosis are different in single beta-cells from pancreatic islets of Goto-Kakizaki (GK) rats, a novel model of NIDDM, and normal rats. In addition, we have also measured the insulin secretory responses, ATP content, and the rate of glucose metabolism in intact islets. Although the glucose sensitivity of the K(ATP) current was similar between GK rats and controls, in the absence of glucose, K(ATP) current density was larger in GK rats, which resulted in a more hyperpolarized membrane potential. Whole-cell Ca2+ currents were similar. By monitoring the cell capacitance with a fixed intracellular solution, no difference was detected in the exocytotic responses of beta-cells from normal and GK rats. In islets from GK rats, the rates of glucose utilization ([3H]H2O production from 5-[3H]glucose) and oxidation ([14C]CO2 production from U-[14C]glucose) were not significantly different from controls. Insulin secretion, however, was impaired (by 50%), and this was paralleled by a smaller increase in ATP content in response to stimulation by 10 mmol/l glucose in islets from GK rats when compared with controls. Under conditions in which K(ATP) channels were held open and the effects of glucose were independent of membrane potential, insulin release was still significantly lower in GK rat islets than in controls. These findings suggest that the impaired insulin secretion in islets from GK rats does not simply result from a failure to close K(ATP) channels, nor does it result from an impairment in calcium secretion coupling.

Adenosine Triphosphate↗
PubMed1997

Phentolamine block of KATP channels is mediated by Kir6.2.

The ATP-sensitive K+-channel (KATP channel) plays a key role in insulin secretion from pancreatic beta cells. It is closed both by glucose metabolism and the sulfonylurea drugs that are used in the treatment of noninsulin-dependent diabetes mellitus, thereby initiating a membrane depolarization that activates voltage-dependent Ca2+ entry and insulin release. The beta cell KATP channel is a complex of two proteins: Kir6.2 and SUR1. The former is an ATP-sensitive K+-selective pore, whereas SUR1 is a channel regulator that endows Kir6.2 with sensitivity to sulfonylureas. A number of drugs containing an imidazoline moiety, such as phentolamine, also act as potent stimulators of insulin secretion, but their mechanism of action is unknown. We have used a truncated form of Kir6.2, which expresses independently of SUR1, to show that phentolamine does not inhibit KATP channels by interacting with SUR1. Instead, our results argue that phentolamine may interact directly with Kir6.2 to produce a voltage-independent reduction in channel activity. The single-channel conductance is unaffected. Although the ATP molecule also contains an imidazoline group, the site at which phentolamine blocks is not identical to the ATP-inhibitory site, because phentolamine block of an ATP-insensitive mutant (K185Q) is normal. KATP channels also are found in the heart where they are involved in the response to cardiac ischemia: they also are blocked by phentolamine. Our results suggest that this may be because Kir6.2, which is expressed in the heart, forms the pore of the cardiac KATP channel.

ATP-Binding Cassette Transporters↗
PubMed1997

Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells.

1. The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B-cells by monitoring changes in cell capacitance evoked by voltage-clamp depolarizations, infusion of high [Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, using the perforated patch whole-cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3. Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80% when ATP was replaced by its non-hydrolysable analogue adenosine 5'-[beta, gamma-methylene]triphosphate (AMP-PCP) in standard whole-cell experiments. Exocytosis elicited by GTP gamma S was similarly affected by replacement of ATP with the stable analogue. 4. Photoreleasing ATP in the presence of 170 nM [Ca2+]i, following the complete wash-out of endogenous ATP produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5. Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca(2+)-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting > or = 10 s) sustained component. Replacement of ATP with AMP-PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N-ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion. We propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell.

Adenosine Triphosphate↗
PubMed1997

Electrogenic arginine transport mediates stimulus-secretion coupling in mouse pancreatic beta-cells.

1. We have investigated the mechanism by which L-arginine stimulates membrane depolarization, an increase of intracellular calcium ([Ca2+]i) and insulin secretion in pancreatic beta-cells. 2. L-Arginine failed to affect beta-cell metabolism, as monitored by NAD(P)H autofluorescence. 3. L-Arginine produced a dose-dependent increase in [Ca2+]i, which was dependent on membrane depolarization and extracellular calcium. 4. The cationic amino acids L-ornithine, L-lysine, L-homoarginine (which is not metabolized) and NG-monomethyl-L-arginine (L-NMMA, a nitric oxide synthase inhibitor) produced [Ca2+]i responses similar to that produced by L-arginine. The neutral nitric oxide synthase inhibitors NG-nitro-L-arginine (L-NNA) and N omega-monomethyl-L-arginine (L-NAME) also increased [Ca2+]i. D-Arginine was ineffective. 5. L-Arginine did not affect whole-cell Ca2+ currents or ATP-sensitive K+ currents, but produced an inward current that was carried by the amino acid. 6. The reverse transcriptase-polymerase chain reaction demonstrated the presence of messenger RNA for the murine cationic amino acid transporters mCAT2A and mCAT2B within the beta-cell. 7. L-Arginine did not affect beta-cell exocytosis as assayed by changes in cell capacitance. 8. Our data suggest that L-arginine elevates [Ca2+]i and stimulates insulin secretion as a consequence of its electrogenic transport into the beta-cell. This uptake is mediated by the mCAT2A transporter.

Alternative Splicing↗
PubMed1996

Ca(2+)- and GTP-dependent exocytosis in mouse pancreatic beta-cells involves both common and distinct steps.

1. The effects of GTP and Ca2+ on secretion from single pancreatic beta-cells were studied using capacitance measurements as an indicator of exocytosis. 2. GTP or GTP gamma S produced a concentration-dependent increase in cell capacitance in the absence of intracellular calcium. There was no effect of cyclic AMP or BAPTA an GTP-induced secretion. 3. In the absence of GTP, the relationship between intracellular calcium concentration and the maximum rate of secretion was fitted by the Hill equation with a slope factor of 2.5 and half-maximal activation at 1.6 microM intracellular Ca2+. Similar values were obtained in the presence of GTP gamma S, suggesting GTP does not alter the sensitivity of the secretory machinery to Ca2+. 4. GDP beta S alone had no effect on cell capacitance but caused a dose-dependent inhibition of exocytosis induced by infusion of either GTP gamma S or Ca2+, suggesting both stimuli involve G-protein activation. GDP beta S was without effect on exocytosis evoked by depolarization-mediated Ca2+ entry. 5. The time course of exocytosis following rapid elevation of GTP gamma S by photolysis of a caged precursor was dependent on the intracellular Ca2+ and cyclic AMP concentrations. 6. Our results are interpreted in terms of a model in which the secretory pathways stimulated by Ca2+ and GTP contain both common and separate parts.

Animals↗
PubMed1996

Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium.

1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta-cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50-100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo- and endocytosis was studied by photorelease of Ca2+ from the 'caged' precursor Ca(2+)-nitrophenyl-EGTA (Ca(2+)-NP-EGTA). Both exo- and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg-ATP. 7. Our data indicate that in the pancreatic beta-cell, exocytosis and endocytosis are regulated by different mechanisms.

Adenosine Triphosphate↗
PubMed1996

Promiscuous coupling between the sulphonylurea receptor and inwardly rectifying potassium channels.

Sulphonylureas are a class of drugs widely used to treat non-insulin-dependent diabetes mellitus. These drugs act by binding to a sulphonylurea receptor (SUR) in the pancreatic beta-cell membrane which inhibits an ATP-sensitive potassium (K-ATP) channel and thereby stimulates insulin secretion. There has been much debate as to whether SUR and the K-ATP channel are the same or separate proteins, whether SUR confers ATP-sensitivity on an ATP-insensitive pore-forming subunit, and whether sulphonylureas can also modulate other types of K-channel. We show here that SUR itself does not possess intrinsic channel activity but that it endows sulphonylurea sensitivity on several types of inwardly-rectifying K-channels. It does not necessarily confer ATP-sensitivity on these channels.

ATP-Binding Cassette Transporters↗
PubMed1995

Effects of divalent cations on exocytosis and endocytosis from single mouse pancreatic beta-cells.

1. The effects of the divalent cations Ca2+, Ba2+ and Sr2+ on exocytosis and endocytosis from single isolated mouse pancreatic beta-cells were investigated by monitoring changes in cell capacitance. 2. The immediate increase in capacitance elicited by a single depolarization from -70 to +20 mV was dependent on the divalent cation species, with Ca2+ (8.2 +/- 1.1 fF pC-1) > Ba2+ (1.0 +/- 0.2 fF pC-1) > Sr2+ (0.7 +/- 0.2 fF pC-1) in perforated-patch recordings. 3. In Ba2+ solutions alone there was subsequently an additional slow increase in capacitance (to 4.3 +/- 1.1 fF pC-1). This second phase of exocytosis was unaffected by preincubation with colcemid (20 microM, 45 min) or cytochalasin D (10 microM, 15 min), suggesting that interaction of secretory granules with microtubules or microfilaments is not involved. 4. An increase in cell capacitance was elicited by depolarization in Ba2+ solutions when intracellular Ca2+ was buffered with 10 mM EGTA. Infusion of the beta-cell with Ba2+ also stimulated exocytosis although the rate was much slower (1.1 +/- 0.2 fF s-1; 8 microM free Ba2+) than for Ca2+ (39 +/- 5 fF s-1; 2 microM free Ca2+). These data indicate that Ba2+ does not evoke secretion by promoting Ca2+ release from internal stores. 5. The lower efficacy of Ba2+ in supporting exocytosis may be related to the fact that this cation does not activate calmodulin-dependent processes and the slow second phase of secretion may result from this ion being removed only slowly from the cytoplasm. 6. Endocytosis was faster in Sr2+ than in Ca2+ or Ba2+ solution, and the speed increased when the external concentration of all three divalent cation species was raised. The ability of Ba2+ to support endocytosis suggests calmodulin-dependent processes are not involved. These data suggest membrane retrieval is regulated differently from exocytosis in beta-cells.

Animals↗
PubMed1995

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.

Amino Acid Sequence↗
PubMed1994

Effects of intracellular pH on ATP-sensitive K+ channels in mouse pancreatic beta-cells.

1. The effects of intracellular pH (pHi) on the ATP-sensitive K+ channel (K+ATP channel) from mouse pancreatic beta-cells were examined in inside-out patches exposed to symmetrical 140 mM K+ solutions. 2. The relationship between channel activity and pHi was described by the Hill equation with half-maximal inhibition (Ki) at pHi 6.25 and a Hill coefficient of 3.7. 3. Following exposure to pHi < 6.8, channel activity did not recover to its original level. Subsequent application of trypsin to the intracellular membrane surface restored channel activity to its initial level or above. 4. At -60 mV the relationship between pHi and the single-channel current amplitude was described by a modified Hill equation with a Hill coefficient of 2.1, half-maximal inhibition at pHi 6.48 and a maximum inhibition of 18.5%. 5. A decrease in pHi reduced the extent of channel inhibition by ATP: Ki was 18 microM at pH 7.2 and 33 microM at pH 6.4. The Hill coefficient was also reduced, being 1.65 at pH 7.2 and 1.17 at pH 6.4. 6. When channel activity was plotted as a function of ATP4- (rather than total ATP) there was no effect of pHi on the relationship. This suggests that ATP4- is the inhibitory ion species and that the effects of reducing pHi are due to the lowered concentration of ATP4-. 7. Changes in external pH had little effect on either single-channel or whole-cell K+ATP currents. 8. The effects of pHi do not support a role for H+ in linking glucose metabolism to K+ATP channel inhibition in pancreatic beta-cells.

Adenosine Triphosphate↗
PubMed1994

Stimulus-secretion coupling in pancreatic beta cells.

Insulin secretion is triggered by a rise in the intracellular Ca2+ concentration that results from the activation of voltage-gated Ca2+ channels in the beta-cell plasma membrane. Multiple types of beta-cell Ca2+ channel have been identified in both electrophysiological and molecular biological studies, but it appears that the L-type Ca2+ channel plays a dominant role in regulating Ca2+ influx. Activity of this channel is potentiated by protein kinases A and C and is inhibited by GTP-binding proteins, which may mediate the effects of potentiators and inhibitors of insulin secretion on Ca2+ influx, respectively. The mechanisms by which elevation of intracellular Ca2+ leads to the release of insulin granules is not fully understood but appears to involve activation of Ca2+/calmodulin-dependent protein kinase. Phosphorylation by either protein kinase A or C, probably at different substrates, potentiates insulin secretion by acting at some late stage in the secretory process. There is also evidence that small GTP-binding proteins are involved in regulating exocytosis in beta cells. The identification and characterisation of the proteins involved in exocytosis in beta cells and clarification of the mechanism(s) of action of Ca2+ is clearly an important goal for the future.

Animals↗
PubMed1993

Modification of K-ATP channels in pancreatic beta-cells by trypsin.

The inside-out configuration of the patch-clamp method was used to study the effects of trypsin on the activity of ATP-sensitive potassium (K-ATP) channels from isolated mouse pancreatic beta-cells. Trypsin (20 micrograms/ml) irreversibly enhanced channel activity around twofold by reducing the interburst intervals without altering the burst kinetics. No effect on the single channel conductance or the inward rectification produced by internal Mg2+ was observed: however, the protease did reduce the inhibitory effect of Mg2+ on channel activity. Trypsin both prevented rundown of K-ATP channel activity and reactivated the channels after complete rundown. These effects of trypsin were absent in the presence of trypsin inhibitor. The protease also reduced the inhibitory effect of ATP on channel activity, increasing the dissociation constant from 7 to 49 microM. Trypsin removed the activating effect of ADP (0.1 mmol/l) on channel activity and reduced the inhibitory effect of tolbutamide (0.5 mmol/l). Carboxypeptidase A did not activate K-ATP channels in excised patches, although it was able to slightly reactivate channels after complete rundown, whereas chymotrypsin increased K-ATP channel activity but it did not produce reactivation. The effects of papain were similar to those of trypsin.

Adenosine Diphosphate↗
PubMed1992

Comparison of 9-aminoacridine and atebrine induced changes in optical, electrical and mechanical characteristics of lipid bilayers.

The effects of fluorescent probes 9-aminoacridine (9AA) and atebrine (AT) on physical properties of liposomes and planar bilayer lipid membranes (BLM) were studied. The method of fluorescence spectroscopy and the electrostriction method based on measurement of higher current harmonics were used. At low concentrations (10(-5)-5 x 10(-5) mol/l), 9AA increased fluorescence intensity, while in liposomes from soybean phosphatidylcholine fluorescence quenching occurred at higher probe concentration. Fluorescence quenching occurred over the entire concentration range tested (10(-5)-10(-4) mol/l) in liposomes made from a mixture of egg phosphatidylcholine and cardiolipin. In contrast to 9AA, AT, thanks to its hydrophobic chain, penetrates deeper into the hydrophobic membrane moiety; thus, immobilization of the molecule and an increase in fluorescence intensity was always observed. Probes adsorbed to membranes, leaving their electric capacitance effectively unchanged. Adsorption of charged dye particles induced small changes in transmembrane potential. In the presence of 10(-5) mol/l AT, the modulus of elasticity E perpendicular increased somewhat for soft membranes (E perpendicular approximately 2.5 x 10(7) Pa), whereas it decreased for hard membranes (E perpendicular approximately 5 x 10(7) Pa). pH gradient present on the membrane affected the ability of the dyes to incorporate into the membranes. Our results provide evidence against the proposed model of the quenching mechanism introduced by Rottenberg and Lee (1975).

Aminacrine↗
PubMed1991

Low-conductance chloride channel from crayfish skeletal muscle incorporated into planar lipid bilayers.

Low-conductance chloride channel from skeletal muscle SR vesicles of the crayfish Astacus fluviatilis was incorporated into planar lipid bilayers and its basic characteristics were investigated. The channel has a relatively low unitary conductance of 26 pS in symmetrical 160 mmol/l choline-chloride. The dependence of the channel conductance on Cl- concentration shows saturating behavior with a maximum conductance of 37 pS and an ionic activity for half-maximum conductance Km = 75 mmol/l. The channel exhibits a complex kinetics with several modes of activity. Open state probability slightly decreases with the increasing absolute value of voltage. The channel activity does not appear to be dependent on the presence of Ca2+ ions. The channel is effectively inhibited by DIDS, a stilbene derivative. The permeability properties of the channel are similar to the specific behavior of the "double-barrelled" channel from Torpedo electroplax described by Miller and White (1984).

Animals↗