Voltage-activated Ca2+ currents in insulin-secreting cells

Membrane voltage and voltage-clamped membrane currents have been investigated with the whole-cell patch clamp method in the insulin-secreting cell line RINm5F. The mean resting membrane potential of RINm5F cells was found to be -52 mV. Overshooting spike potentials could be evoked by depolarising voltage steps in the absence of a secretagogue. Inward membrane currents evoked by depolarising voltage steps were dependent upon extracellular Ca2+ and blocked by Co2+, nifedipine and verapamil. Outward membrane currents which were evoked by depolarising voltage steps to positive membrane potentials were reduced when Ca2+ entry was prevented. It is concluded that the voltage-activated Ca2+ currents underlie the voltage-activated spike potentials recorded from insulin-secreting cells.     

Voltage-activation of high-conductance K+ channel in the insulin-secreting cell line RINm5F is dependent on local extracellular Ca2+ concentration

Patch-clamp single-channel current recording experiments have been carried out on intact insulin-secreting RINm5F cells. Voltage-activation of high-conductance K+ channels were studied by selectively depolarizing the electrically isolated patch membrane under conditions with normal Ca2+ concentration in the bath solution but with or without Ca2+ in the patch pipette solution. When Ca2+ was present in the pipette, 40 mV to 120 mV depolarizing pulses (100 ms) from the normal resting potential (-70 mV) regularly evoked tetraethylammonium-sensitive large outward single-channel currents and the average open state probability during the pulses varied from about 0.015 (40 mV pulses) to 0.1 (120 mV pulses). In the absence of Ca2+ in the pipette solution the same protocol resulted in fewer and shorter K+ channel openings and the open-state probability varied from about 0.0015 (40 mV pulses) to about 0.03 (120 mV pulses). It is concluded that Ca2+ entering voltage-gated channels raises [Ca2+]i locally and thereby markedly enhances the open-state probability of tetraethylammonium-sensitive voltage-gated high-conductance K+ channels.     

Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINm5F) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101 +/- 5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7 +/- 0.8 min. In contrast to the action of K+, exposure of the RINm5F cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity. This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in beta-cells. Unlike the case in normal beta-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINm5F cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINm5F cells exposed to glucose.     

Block by calcium of ATP-activated channels in pheochromocytoma cells

We have investigated the effects of Ca2+ on Na+ influx through ATP- activated channels in pheochromocytoma PC12 cells using single channel current recordings. Under cell-attached patch-clamp conditions with 150 mM Na+ and 2 mM Ca2+ in the pipette, the unitary current activity showed an open level of about -4.3 pA at -150 mV. The channel opening was interrupted by flickery noise as well as occasional transition to a subconducting state of about -1.7 pA at -150 mV. The open level was decreased with increased external Ca2+, suggesting that external Ca2+ blocks Na+ permeation. We assessed the block by Ca2+ as the mean amplitude obtained with heavy filtration according to Pietrobon et al. (Pietrobon, D., B. Prod'hom, and P. Hess, 1989. J. Gen. Physiol. 94:1- 21). The block was concentration dependent with a Hill coefficient of 1 and a half-maximal concentration of approximately 6 mM. A similar block was observed with other divalent cations, and the order of potency was Cd2+ > Mn2+ > Mg2+ not equal to Ca2+ > Ba2+. High Ca2+, Mg2+ and Ba2+ did not block completely, probably because they can carry current in the channel. The block by external Ca2+ did not exhibit voltage dependence between -100 and -210 mV. In the inside-out patch-clamp configuration, the amplitude of inward channel current obtained with 150 mM external Na+ was reduced by increased internal Ca2+. The reduction was observed at lower concentrations than that by external Ca2+. Internal Ba2+ and Cd2+ induced similar reduction in current amplitude. This inhibitory effect of internal Ca2+ was voltage dependent; the inhibition was relieved with hyperpolarization. The results suggest that both external and internal Ca2+ can block Na+ influx through the ATP-activated channel. A simple one-binding site model with symmetric energy barriers is not sufficient to explain the Ca2+ block from both sides.     

Single-channel recordings of two types of calcium channels in rat pancreatic beta-cells.

Using the cell-attached configuration of the patch clamp technique, we have identified two different types of Ca channels in rat pancreatic beta-cell membranes. The two channels differ in single channel conductance, voltage dependence, and inactivation properties. The single-channel conductance, measured with 100 mM Ba2+ in the pipette, was 21.8 pS for the large channel and 6.4 pS for the small channel. The large-conductance channel is similar to the fast deactivating or L-type Ca channel described in other preparations. It is voltage dependent, has a threshold for activation around -30 mV, and can be activated from a holding potential of -40 mV. On the other hand, the small-conductance Ca channel is similar to the SD or T type Ca channel; it has a lower activation threshold, around -50 mV, and it can be inactivated by holding the membrane potential at -40 mV.     

External barium influences the gating charge movement of Shaker potassium channels.

External Ba2+ speeds the OFF gating currents (IgOFF) of Shaker K+ channels but only upon repolarization from potentials that are expected to open the channel pore. To study this effect we used a nonconducting and noninactivating mutant of the Shaker K+ channel, ShH4-IR (W434F). External Ba2+ slightly decreases the quantity of ON gating charge (QON) upon depolarization to potentials near -30 mV but has little effect on the quantity of charge upon stepping to more hyperpolarized or depolarized potentials. More strikingly, Ba2+ significantly increases the decay rate of IgOFF upon repolarization to -90 mV from potentials positive to approximately -55 mV. For Ba2+ to have this effect, the depolarizing command must be maintained for a duration that is dependent on the depolarizing potential (> 4 ms at -30 mV and > 1 ms at 0 mV). The actions of Ba2+ on the gating current are dose-dependent (EC50 approximately 0.2 mM) and are not produced by either Ca2+ or Mg2+ (2 mM). The results suggest that Ba2+ binds to a specific site on the Shaker K+ channel that destabilizes the open conformation and thus facilitates the return of gating charge upon repolarization.     

Protein kinase C decreases the apparent affinity of the inositol 1,4,5-trisphosphate receptor type 3 in RINm5F cells

In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R) is an intracellular Ca2+ channel which plays a major role in Ca2+ signalling. Three isoforms of IP3R have been identified (IP3R-1, IP3R-2 and IP3R-3) and most cell types express different proportions of each isoform. The differences between the pharmacological and functional properties of the various isoforms of IP3R are poorly known. RINm5F cells who express almost exclusively (approximately 90%) the IP3R-3, represent an interesting model to study this particular isoform. Here, we investigated a regulatory mechanism by which protein kinase C (PKC) may influence IP3R-3-mediated Ca2+ release. With an immunoprecipitation approach we confirmed that RINm5F cells express almost exclusively the IP3R-3 isoform. With an in vitro phosphorylation approach, we showed that the immunopurified IP3R-3 was efficiently phosphorylated by exogenous PKC. With a direct in cellulo approach and an indirect in cellulo back-phosphorylation approach we showed that phorbol-12-myristate-13-acetate (PMA) causes the phosphorylation of IP3R-3 in intact RINm5F cells. In saponin-permeabilized RINm5F cells, 3-induced Ca2+ release was reduced after a pre-treatment with PMA. PMA also reduced the Ca2+ response of intact RINm5F cells stimulated with carbachol and EGF, two agonists that use different receptor types to activate phospholipase C. These results suggest the existence of a negative feedback mechanism involving two components of the Ca2+ signalling cascade, whereby activated PKC dampens IP3R-3 activity.     

32P, 86Rb+ and 45Ca2+ handling by tumoral insulin-secreting cells (RINm5F line)

In perifused tumoral islet cells (RINm5F line), which were prelabelled with either [32P]orthophosphate, 86Rb+ or 45Ca2+, the administration of D-glucose (1.4, 2.8 or 16.7 mM) increased the efflux of 32P, decreased the outflow of 86Rb, increased slightly the efflux of 45Ca from cells perifused in the presence of Ca2+, and decreased modestly the outflow of 45Ca from cells perifused in the absence of Ca2+. D-glucose also stimulated the net uptake of 45Ca2+. When Ba2+ (2 mM) was used, in the absence of Ca2+, instead of D-glucose as an insulin secretagogue, the efflux of 32P was little affected, but the outflow of 45Ca was dramatically increased. These changes are qualitatively similar to those occurring in normal islet cells. Nevertheless, the ionic response to D-glucose appeared, as a rule, less marked in tumoral than normal islet cells. Moreover, the concentration-response relationship was shifted to a lower range of hexose concentrations in the RINm5F cells.     

Multi-ion occupancy alters gating in high-conductance, Ca(2+)-activated K+ channels

In this study, single-channel recordings of high-conductance Ca(2+)-activated K+ channels from rat skeletal muscle inserted into planar lipid bilayer were used to analyze the effects of two ionic blockers, Ba2+ and Na+, on the channel's gating reactions. The gating equilibrium of the Ba(2+)-blocked channel was investigated through the kinetics of the discrete blockade induced by Ba2+ ions. Gating properties of Na(+)-blocked channels could be directly characterized due to the very high rates of Na+ blocking/unblocking reactions. While in the presence of K+ (5 mM) in the external solution Ba2+ is known to stabilize the open state of the blocked channel (Miller, C., R. Latorre, and I. Reisin. 1987. J. Gen. Physiol. 90:427-449), we show that the divalent blocker stabilizes the closed-blocked state if permeant ions are removed from the external solution (K+ less than 10 microM). Ionic substitutions in the outer solution induce changes in the gating equilibrium of the Ba(2+)-blocked channel that are tightly correlated to the inhibition of Ba2+ dissociation by external monovalent cations. In permeant ion-free external solutions, blockade of the channel by internal Na+ induces a shift (around 15 mV) in the open probability--voltage curve toward more depolarized potentials, indicating that Na+ induces a stabilization of the closed-blocked state, as does Ba2+ under the same conditions. A kinetic analysis of the Na(+)-blocked channel indicates that the closed-blocked state is favored mainly by a decrease in opening rate. Addition of 1 mM external K+ completely inhibits the shift in the activation curve without affecting the Na(+)-induced reduction in the apparent single-channel amplitude. The results suggest that in the absence of external permeant ions internal blockers regulate the permeant ion occupancy of a site near the outer end of the channel. Occupancy of this site appears to modulate gating primarily by speeding the rate of channel opening.     

Kinetics of Ca2+-activated K+ channels from rabbit muscle incorporated into planar bilayers. Evidence for a Ca2+ and Ba2+ blockade

The interaction of Ca2+ and Ba2+ with a Ca2+-activated K+ channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca2+] greater than or equal to 100 microM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca2+], whereas the mean time for the quiescent (closed) periods is independent of [Ca2+]. The number of quiescent periods is reduced by increasing [K+]. Micromolar amounts of cis Ba2+ do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba2+] and the mean closed time is independent of [Ba2+]. Raising [K+] either symmetrically or only in the trans side relieved the Ba2+ effect. trans Ba2+ also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba2+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2+ and Ba2+ suggest that Ca2+ blocks the channel by binding to the same site. However, binding of Ca2+ to the site is 10(5)- fold weaker.     

Phorbol ester-stimulated insulin secretion by RINm5F insulinoma cells is linked with membrane depolarization and an increase in cytosolic free Ca2+ concentration

In studying the regulation of insulin secretion by phorbol esters, we examined their effects on the cytosolic free Ca2+ concentration ([Ca2+]i), using the Ca2+ indicator fura-2 in the rat insulin-secreting beta-cell line RINm5F. [Ca2+]i was measured in parallel with the rate of insulin release. 50 nM 12-O-tetradecanoylphorbol-13-acetate (TPA), which may act via protein kinase C, stimulated insulin release and caused an increase in [Ca2+]i. Ca2+-free conditions eliminated the increase in [Ca2+]i and resulted in a reduced stimulation of insulin release by TPA. The Ca2+ channel blocker nitrendipine (300 nM) inhibited both the increase in [Ca2+]i and the increased rate of insulin secretion. Another phorbol ester, 4 beta-phorbol 12,13-didecanoate, which activates protein kinase C, also induced an increase in [Ca2+]i and in the rate of insulin release, while 4 alpha-phorbol 12,13-didecanoate, which fails to stimulate protein kinase C, was without effect. Further studies with bis-oxonol as an indicator of membrane potential showed that TPA depolarized the beta-cell plasma membrane. From these results, it is concluded that TPA depolarizes the plasma membrane, induces the opening of Ca2+ channels in the RINm5F beta-cell plasma membrane, increases [Ca2+]i, and results in insulin secretion. The action of TPA was next compared with that of a depolarizing concentration of KC1 (25 mM), which stimulates insulin secretion simply by opening Ca2+ channels. TPA consistently elicited less depolarization, a smaller rise of [Ca2+]i, but a greater release of insulin than KC1. Therefore an additional action of TPA is suggested, which potentiates the action of the elevated [Ca2+]i on insulin secretion.     

Stimulation of insulin release by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate in the clonal cell line RINm5F despite a lowering of the free cytoplasmic Ca2+ concentration

The effects of 12-O-tetradecanoylphorbol 13-acetate (TPA) on the handling of Ca2+ and insulin release were investigated in the clonal insulin-producing cell line RINm5F. The presence of the phorbol ester lowered the free cytoplasmic Ca2+ and suppressed the increase obtained by depolarization with high concentrations of K+. Despite the lowering in cytoplasmic Ca2+ by TPA, there was a concomitant stimulation of insulin release indicating that one feature of protein kinase C activation is to make the secretory system more sensitive to Ca2+. Furthermore, there was no interaction of TPA with the mechanisms responsible for inositol 1,4,5-tris(phosphate) induced Ca2+ release or Ca2+ uptake in permeabilized cells. Although TPA slightly depolarized the RINm5F cells there was no interference with K+-induced depolarization. It is suggested that an additional effect of protein kinase C activation in these cells, is to stimulate the extrusion of Ca2+ over the plasma membrane.     

Permeation and gating properties of the L-type calcium channel in mouse pancreatic beta cells

Ba2+ currents through L-type Ca2+ channels were recorded from cell- attached patches on mouse pancreatic beta cells. In 10 mM Ba2+, single- channel currents were recorded at -70 mV, the beta cell resting membrane potential. This suggests that Ca2+ influx at negative membrane potentials may contribute to the resting intracellular Ca2+ concentration and thus to basal insulin release. Increasing external Ba2+ increased the single-channel current amplitude and shifted the current-voltage relation to more positive potentials. This voltage shift could be modeled by assuming that divalent cations both screen and bind to surface charges located at the channel mouth. The single- channel conductance was related to the bulk Ba2+ concentration by a Langmuir isotherm with a dissociation constant (Kd(gamma)) of 5.5 mM and a maximum single-channel conductance (gamma max) of 22 pS. A closer fit to the data was obtained when the barium concentration at the membrane surface was used (Kd(gamma) = 200 mM and gamma max = 47 pS), which suggests that saturation of the concentration-conductance curve may be due to saturation of the surface Ba2+ concentration. Increasing external Ba2+ also shifted the voltage dependence of ensemble currents to positive potentials, consistent with Ba2+ screening and binding to membrane surface charge associated with gating. Ensemble currents recorded with 10 mM Ca2+ activated at more positive potentials than in 10 mM Ba2+, suggesting that external Ca2+ binds more tightly to membrane surface charge associated with gating. The perforated-patch technique was used to record whole-cell currents flowing through L-type Ca2+ channels. Inward currents in 10 mM Ba2+ had a similar voltage dependence to those recorded at a physiological Ca2+ concentration (2.6 mM). BAY-K 8644 (1 microM) increased the amplitude of the ensemble and whole-cell currents but did not alter their voltage dependence. Our results suggest that the high divalent cation solutions usually used to record single L-type Ca2+ channel activity produce a positive shift in the voltage dependence of activation (approximately 32 mV in 100 mM Ba2+).     

Quinine inhibits Ca2+-independent K+ channels whereas tetraethylammonium inhibits Ca2+-activated K+ channels in insulin-secreting cells

The effects of quinine and tetraethylammonium (TEA) on single-channel K+ currents recorded from excised membrane patches of the insulin-secreting cell line RINm5F were investigated. When 100 microM quinine was applied to the external membrane surface K+ current flow through inward rectifier channels was abolished, while a separate voltage-activated high-conductance K+ channel was not significantly affected. On the other hand, 2 mM TEA abolished current flow through voltage-activated high-conductance K+ channels without influencing the inward rectifier K+ channel. Quinine is therefore not a specific inhibitor of Ca2+-activated K+ channels, but instead a good blocker of the Ca2+-independent K+ inward rectifier channel whereas TEA specifically inhibits the high-conductance voltage-activated K+ channel which is also Ca2+-activated.     

Calcium entry through receptor-operated channels in bovine pulmonary artery endothelial cells

The activation of endothelial cells by endothelium-dependent vasodilators has been investigated using bioassay, patch clamp and 45Ca flux methods. Cultured pulmonary artery endothelial cells have been demonstrated to release EDRF in response to thrombin, bradykinin, ATP and the calcium ionophore A23187. The resting membrane potential of the endothelial cells was -56 mV and the cells were depolarized by increasing extracellular K+ or by the addition of (0.1-1.0 mM)Ba2+ to the bathing solution. The electrophysiological properties of the cultured endothelial cells suggest that the membrane potential is maintained by an inward rectifying K+ channel with a mean single channel conductance of 35.6 pS. The absence of a depolarization-activated inward current and the reduction of 45Ca influx with high K+ solution suggests that there are no functional voltage-dependent calcium or sodium channels. Thrombin and bradykinin were shown to evoke not only an inward current (carried by Na+ and Ca2+) but also an increase in 45Ca influx suggesting that the increase in intracellular calcium necessary for EDRF release is mediated by an opening of a receptor operated channel. High doses of thrombin and bradykinin induced intracellular calcium release, however, at low doses of thrombin no intracellular calcium release was observed. We propose that the increased cytosolic calcium concentration in endothelial cells induced by endothelium dependent vasodilators is due to the influx of Ca2+ through a receptor operated ion channel and to a lesser degree to intracellular release of calcium from a yet undefined intracellular store.     

Multiple channels mediate calcium leakage in the A7r5 smooth muscle-derived cell line.

Ca2+ entry under resting conditions may be important for contraction of vascular smooth muscle, but little is known about the mechanisms involved. Ca2+ leakage was studied in the A7r5 smooth muscle-derived cell line by patch-clamp techniques. Two channels that could mediate calcium influx at resting membrane potentials were characterized. In 110 mM Ba2+, one channel had a slope conductance of 6.0 +/- 0.6 pS and an extrapolated reversal potential of +41 +/- 13 mV (mean +/- SD, n = 8). The current rectified strongly, with no detectable outward current, even at +90 mV. Channel gating was voltage independent. A second type of channel had a linear current-voltage relationship, a slope conductance of 17.0 +/- 3.2 pS, and a reversal potential of +7 +/- 4 mV (n = 9). The open probability increased e-fold per 44 +/- 10 mV depolarization (n = 5). Both channels were also observed in 110 mM Ca2+. Noise analysis of whole-cell currents indicates that approximately 100 6-pS channels and 30 17-pS channels are open per cell. These 6-pS and 17-pS channels may contribute to resting calcium entry in vascular smooth muscle cells.     

Calcium-dependent potentiation of store-operated calcium channels in T lymphocytes

The depletion of intracellular Ca2+ stores triggers the opening of Ca2+ release-activated Ca2+ (CRAC) channels in the plasma membrane of T lymphocytes. We have investigated the additional role of extracellular Ca2+ (Ca02+) in promoting CRAC channel activation in Jurkat leukemic T cells. Ca2+ stores were depleted with 1 microM thapsigargin in the nominal absence of Ca02+ with 12 mM EGTA or BAPTA in the recording pipette. Subsequent application of Ca02+ caused ICRAC to appear in two phases. The initial phase was complete within 1 s and reflects channels that were open in the absence of Ca02+. The second phase consisted of a severalfold exponential increase in current amplitude with a time constant of 5-10 s; we call this increase Ca(2+)-dependent potentiation, or CDP. The shape of the current-voltage relation and the inferred single-channel current amplitude are unchanged during CDP, indicating that CDP reflects an alteration in channel gating rather than permeation. The extent of CDP is modulated by voltage, increasing from approximately 50% at +50 mV to approximately 350% at -75 mV in the presence of 2 mM Ca02+. The voltage dependence of CDP also causes ICRAC to increase slowly during prolonged hyperpolarizations in the constant presence of Ca02+. CDP is not affected by exogenous intracellular Ca2+ buffers, and Ni2+, a CRAC channel blocker, can cause potentiation. Thus, the underlying Ca2+ binding site is not intracellular. Ba2+ has little or no ability to potentiate CRAC channels. These results demonstrate that the store-depletion signal by itself triggers only a small fraction of capacitative Ca2+ entry and establish Ca2+ as a potent cofactor in this process. CDP confers a previously unrecognized voltage dependence and slow time dependence on CRAC channel activation that may contribute to the dynamic behavior of ICRAC.     

Vasopressin directly closes ATP-sensitive potassium channels evoking membrane depolarization and an increase in the free intracellular Ca2+ concentration in insulin-secreting cells.

The effects of arginine-vasopressin (AVP) (0.01-1 microM) on membrane potential, [Ca2+]i and ATP-sensitive potassium channels have been studied in the insulin-secreting cell line RINm5F. In whole cells, with an average spontaneous cellular transmembrane potential of -64 +/- 3 mV (n = 33) and an average basal [Ca2+]i of 102 +/- 6 nM (n = 40), AVP evoked: (i) membrane depolarization, (ii) voltage-dependent Ca2+ spike-potentials and (iii) a sharp rise in [Ca2+]i. Single-channel current events recorded from excised outside-out membrane patches show that AVP closes potassium channels that are also closed by tolbutamide (100 microM) and opened by diazoxide (100 microM). AVP acts on KATP channels specifically from the outside of the membrane and a soluble cytosolic messenger appears not to be involved, since there is no channel activation in cell-attached membrane patches when the peptide is added to the bath solution.     

Inactivation of single cardiac Na+ channels in three different gating modes.

In small cell-attached patches containing one and only one Na+ channel, inactivation was studied in three different gating modes, namely, the fast-inactivating F mode and the more slowly inactivating S mode and P mode with similar inactivation kinetics. In each of these modes, ensemble-averaged currents could be fitted with a Hodgkin-Huxley-type model with a single exponential for inactivation (tauh). tauh declined from 1.0 ms at -60 mV to 0.1 ms at 0 mV in the F mode, from 4.6 ms at -40 mV to 1.1 ms at 0 mV in the S mode, and from 4.5 ms at -40 mV to 0.8 ms at +20 mV in the P mode, respectively. The probability of non-empty traces (net), the mean number of openings per non-empty trace (op/tr), and the mean open probability per trace (popen) were evaluated at 4-ms test pulses. net inclined from 30% at -60 mV to 63% at 0 mV in the F mode, from 4% at -90 mV to 90% at 0 mV in the S mode, and from 2% at -60 mV to 79% at +20 mV in the P mode. op/tr declined from 1.4 at -60 mV to 1.1 at 0 mV in the F mode, from 4.0 at -60 mV to 1.2 at 0 mV in the S mode, and from 2.9 at -40 mV to 1.6 at +20 mV in the P mode. popen was bell-shaped with a maximum of 5% at -30 mV in the F mode, 48% at -50 mV in the S mode, and 16% at 0 mV in the P mode. It is concluded that 1) a switch between F and S modes may reflect a functional change of inactivation, 2) a switch between S and P modes may reflect a functional change of activation, 3) tauh is mainly determined by the latency until the first channel opening in the F mode and by the number of reopenings in the S and P modes, 4) at least in the S and P modes, inactivation is independent of pore opening, and 5) in the S mode, mainly open channels inactivate, and in the P mode, mainly closed channels inactivate.     

Activators of protein kinase C depolarize insulin-secreting cells by closing K+ channels.

Carbohydrate stimuli of insulin secretion depolarize the pancreatic B cell and the B-cell line RINm5F by inhibiting ATP-sensitive K+ channels. We examined the possibility that this effect is mediated by activation of protein kinase C. In RINm5F cells, the triose D-glyceraldehyde evoked a rapid increase of the mass of 1,2-diacylglycerol, the endogenous activator of protein kinase C. This effect is mainly due to de novo synthesis of the lipid from glycolytic intermediates, as glyceraldehyde carbon was incorporated into 1,2-diacylglycerol within 1 min of exposure to 14C-labelled glyceraldehyde. The effects of two exogenous activators of kinase C, 4-beta-12-phorbol-myristate 13-acetate (PMA) and 1,2-didecanoylglycerol (DC10) on single K+ channel currents were examined in RINm5F cell-attached membrane patches. Both PMA and DC10 depolarized the cells and decreased the open-state probability of the ATP-sensitive K+ channels. These actions were not due to changes in cellular ATP content, since PMA, like glyceraldehyde, failed to alter cellular ATP. As is the case for glyceraldehyde, PMA and DC10 raised cytosolic free Ca2+ [( Ca2+]i) and stimulated insulin secretion. Both of these effects are inhibited in the absence of external Ca2+. This, and the attenuation of the [Ca2+]i rise by verapamil, suggest that all three stimuli raise [Ca2+]i by promoting Ca2+ influx through voltage-gated channels in turn leading to insulin secretion. As the exogenous activators of protein kinase C mimic the effects of glyceraldehyde, it is proposed that the carbohydrate-mediated production of 1,2-diacylglycerol constitutes the link between metabolism and membrane depolarization.(ABSTRACT TRUNCATED AT 250 WORDS)