Regulation of cloned ATP-sensitive K channels by adenine nucleotides and sulfonylureas: interactions between SUR1 and positively charged domains on Kir6.2.

K(ATP) channels, comprised of the pore-forming protein Kir6.x and the sulfonylurea receptor SURx, are regulated in an interdependent manner by adenine nucleotides, PIP2, and sulfonylureas. To gain insight into these interactions, we investigated the effects of mutating positively charged residues in Kir6.2, previously implicated in the response to PIP2, on channel regulation by adenine nucleotides and the sulfonylurea glyburide. Our data show that the Kir6.2 "PIP2-insensitive" mutants R176C and R177C are not reactivated by MgADP after ATP-induced inhibition and are also insensitive to glyburide. These results suggest that R176 and R177 are required for functional coupling to SUR1, which confers MgADP and sulfonylurea sensitivity to the K(ATP) channel. In contrast, the R301C and R314C mutants, which are also "PIP2-insensitive," remained sensitive to stimulation by MgADP in the absence of ATP and were inhibited by glyburide. Based on these findings, as well as previous data, we propose a model of the K(ATP) channel whereby in the presence of ATP, the R176 and R177 residues on Kir6.2 form a specific site that interacts with NBF1 bound to ATP on SUR1, promoting channel opening by counteracting the inhibition by ATP. This interaction is facilitated by binding of MgADP to NBF2 and blocked by binding of sulfonylureas to SUR1. In the absence of ATP, since K(ATP) channels are not blocked by ATP, they do not require the counteracting effect of NBF1 interacting with R176 and R177 to open. Nevertheless, channels in this state remain activated by MgADP. This effect may be explained by a direct stimulatory interaction of NBF2/MgADP moiety with another region of Kir6.2 (perhaps the NH2 terminus), or by NBF2/MgADP still promoting a weak interaction between NBF1 and Kir6.2 in the absence of ATP. The region delimited by R301 and R314 is not involved in the interaction with NBF1 or NBF2, but confers additional PIP2 sensitivity.

Regulation of cloned ATP-sensitive K channels by phosphorylation, MgADP, and phosphatidylinositol bisphosphate (PIP(2)): a study of channel rundown and reactivation.

Kir6.2 channels linked to the green fluorescent protein (GFP) (Kir6. 2-GFP) have been expressed alone or with the sulfonylurea receptor SUR1 in HEK293 cells to study the regulation of K(ATP) channels by adenine nucleotides, phosphatidylinositol bisphosphate (PIP(2)), and phosphorylation. Upon excision of inside-out patches into a Ca(2+)- and MgATP-free solution, the activity of Kir6.2-GFP+SUR1 channels spontaneously ran down, first quickly within a minute, and then more slowly over tens of minutes. In contrast, under the same conditions, the activity of Kir6.2-GFP alone exhibited only slow rundown. Thus, fast rundown is specific to Kir6.2-GFP+SUR1 and involves SUR1, while slow rundown is a property of both Kir6.2-GFP and Kir6.2-GFP+SUR1 channels and is due, at least in part, to Kir6.2 alone. Kir6. 2-GFP+SUR1 fast phase of rundown was of variable amplitude and led to increased ATP sensitivity. Excising patches into a solution containing MgADP prevented this phenomenon, suggesting that fast rundown involves loss of MgADP-dependent stimulation conferred by SUR1. With both Kir6.2-GFP and Kir6.2-GFP+SUR1, the slow phase of rundown led to further increase in ATP sensitivity. Ca(2+) accelerated this process, suggesting a role for PIP(2) hydrolysis mediated by a Ca(2+)-dependent phospholipase C. PIP(2) could reactivate channel activity after a brief exposure to Ca(2+), but not after prolonged exposure. However, in both cases, PIP(2) reversed the increase in ATP sensitivity, indicating that PIP(2) lowers the ATP sensitivity by increasing P(o) as well as by decreasing the channel affinity for ATP. With Kir6.2-GFP+SUR1, slow rundown also caused loss of MgADP stimulation and sulfonylurea inhibition, suggesting functional uncoupling of SUR1 from Kir6.2-GFP. Ca(2+) facilitated the loss of sensitivity to MgADP, and thus uncoupling of the two subunits. The nonselective protein kinase inhibitor H-7 and the selective PKC inhibitor peptide 19-36 evoked, within 5-15 min, increased ATP sensitivity and loss of reactivation by PIP(2) and MgADP. Phosphorylation of Kir6.2 may thus be required for the channel to remain PIP(2) responsive, while phosphorylation of Kir6.2 and/or SUR1 is required for functional coupling. In summary, short-term regulation of Kir6.2+SUR1 channels involves MgADP, while long-term regulation requires PIP(2) and phosphorylation.

The sulphonylurea receptor SUR1 regulates ATP-sensitive mouse Kir6.2 K+ channels linked to the green fluorescent protein in human embryonic kidney cells (HEK 293).

1. Using a chimeric protein comprising the green fluorescent protein (GFP) linked to the C-terminus of the K+ channel protein mouse Kir6.2 (Kir6.2-C-GFP), the interactions between the sulphonylurea receptor SUR1 and Kir6.2 were investigated in transfected human embryonic kidney cells (HEK 293) by combined imaging and patch clamp techniques. 2. HEK 293 cells transfected with mouse Kir6.2-C-GFP and wild-type Kir6.2 exhibited functional K+ channels independently of SUR1. These channels were inhibited by ATP (IC50 = 150 microM), but were not responsive to stimulation by ADP or inhibition by sulphonylureas. Typically 15 +/- 7 active channels were found in an excised patch. 3. The distribution of Kir6.2-C-GFP protein was investigated by imaging of GFP fluorescence. There was a lamellar pattern of fluorescence labelling inside the cytoplasm (presumably associated with the endoplasmic reticulum and the Golgi apparatus) and intense punctate labelling near the cell membrane, but little fluorescence was associated with the plasma membrane. 4. In contrast, cells co-transfected with Kir6.2-C-GFP and SUR1 exhibited intense uniform plasma membrane labelling, and the lamellar and punctate labelling seen without SUR1 was no longer prominent. 5. In cells co-transfected with Kir6.2-C-GFP and SUR1, strong membrane labelling was associated with very high channel activity, with 484 +/- 311 active channels per excised patch. These K+ channels were sensitive to inhibition by ATP (IC50 = 17 microM), stimulated by ADP and inhibited by sulphonylureas. 6. We conclude that co-expression of SUR1 and Kir6.2 generates channels with the properties of native KATP channels. In addition, SUR1 promotes uniform insertion of Kir6.2-C-GFP into the plasma membrane and a 35-fold increase in channel activity, suggesting that SUR1 facilitates protein trafficking of Kir6.2 into the plasma membrane.

Sulfonylurea binding to a low-affinity site inhibits the Na/K-ATPase and the KATP channel in insulin-secreting cells.

We have used hamster insulinoma tumor (HIT) cells, an insulin-secreting tumor cell line, to investigate modulation of the Na/K-ATPase and of the ATP-sensitive K channel (K(ATP)) by the sulfonylurea glyburide. Membrane proteins from cells cultured in RPMI with 11 mM glucose have at least two glyburide receptor populations, as evidenced by high and low binding affinity constants, (K(d) = 0.96 and 91 nM, respectively). In these cells K(ATP) channel activity was blocked by low glyburide concentrations, IC(50) = 5.4 nM. At 12.5 nM glyburide the inhibition developed slowly, tau = 380 s, and caused reduction of channel activity by 75 percent. At higher concentrations, however, inhibition occurred at a fast rate, tau = 42 s at 100 nM, and was almost complete. Na/K-ATPase activity measured enzymatically and electrophysiologically was also suppressed by glyburide, but higher concentrations were needed, IC(50) = 20-40 nM. Inhibition occurred rapidly, tau = 30 s at 50 nM, when maximum, activity was reduced by 40 percent. By contrast, cells cultured in RPMI supplemented with 25 mM glucose exhibit a single receptor population binding glyburide with low affinity, K(d)= 68 nM. In these cells inhibition of the Na/K-ATPase by the sulfonylurea was similar to that observed in cells cultured in 11 mM glucose, but K(ATP) channel inhibition was markedly altered. Inhibition occurred only at high concentrations of glyburide and at a fast rate; maximum inhibition was observed at 100 nM. Based on these data, we propose that glyburide binding to the high affinity site affects primarily K(ATP) channel activity, while interaction with the low affinity site inhibits both Na/K-ATPase and K(ATP) channel activities. The latter observation suggests possible functional interactions between the Na/K-ATPase and the K(ATP) channel.

Characterization of the G protein coupling of SRIF and beta-adrenergic receptors to the maxi KCa channel in insulin-secreting cells.

Modulation of the Ca- and voltage-dependent K channel--KCa--by receptors coupled to the G proteins G(i)/G(o) and Gs has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches somatostatin (somatotropin-releasing inhibitory factor; SRIF) caused concentration-dependent inhibition of the KCa channel, an effect that was prevented by pertussis toxin (PTX). In inside-out patches, exogenous alpha subunits of either G(i)- or G(o)-type G proteins also inhibited the KCa channel (IC50 5.9 and 5.7 pM, respectively). These data indicate that SRIF suppresses KCa channel activity via a membrane-delimited pathway that involves the alpha subunits of PTX-sensitive G proteins G(i) and/or G(o). In outside-out patches, activation of Gs either by beta-agonists or with cholera toxin (CTX) increased KCa channel activity, consistent with a membrane-delimited stimulatory pathway linking the beta-adrenergic receptor to the KCa channel via Gs. In outside-out patches, channel inhibition by SRIF suppressed the stimulatory effect of beta-agonists but not that of CTX, while in inside-out patches CTX reversed channel inhibition induced by exogenous alpha i or alpha o. Taken together these data suggest that KCa channel activity is enhanced by activation of Gs and blocked by activated G(i) and/or G(o). Further, KCa channel stimulation by activated Gs may be "direct," while inhibition by G(i)/G alpha may involve deactivation of Gs. In inside-out patches KCa channel activity was reduced by an activator of protein kinase C (PKC) and enhanced by inhibitors of PKC, indicating that PKC also acts to inhibit the KCa channel via a membrane delimited pathway. In outside-out patches, chelerythrine, a membrane permeant inhibitor of PKC prevented the inhibitory effect of SRIF, and in inside-out patches PKC inhibitors prevented the inhibitory effect of exogenous alpha i or alpha o. These data indicate that PKC facilitates the inhibitory effect of the PTX-sensitive G proteins which are activated by coupling to SRIF receptors. To account for these results a mechanism is proposed whereby PKC may be involved in G(i)/G(o)-induced deactivation of Gs.

Characterization of the G protein coupling of a somatostatin receptor to the K+ATP channel in insulin-secreting mammalian HIT and RIN cell lines.

1. The G protein-mediated coupling of a somatostatin (somatotropin-releasing inhibitory factor; SRIF) receptor to the ATP-dependent K+ channel (K+ATP channel) has been studied in insulin-secreting cells using the patch clamp technique. 2. In excised outside-out patches, the concentration-dependent stimulation of the K+ATP channel by SRIF was biphasic. Stimulation reached a maximum at 15 nM (EC50 = 5.5 nM), then decayed to a minimum at 50 nM and returned to maximum stimulation at 500 nM. 3. In cell-attached patches, bath-applied SRIF caused K+ATP channel stimulation in most experiments. In a few cases, however, SRIF suppressed channel activity, a response that was reversed by addition of dibutyryl cyclic AMP (DBcAMP). Channel stimulation by SRIF or by DBcAMP did not occur in the presence of glucose. 4. In excised inside-out patches, the alpha-subunits of Gi or G(o)-type G proteins stimulated the K+ATP channel (EC50 = 29 and 42 pM, respectively). The K+ATP channel stimulation by alpha i- or alpha o-subunits had no effect on the concentration-dependent inhibition by ATP. 5. In excised inside-out patches, K+ATP channel activity was reduced by inhibitors of protein kinase C (PKC) and stimulated by a PKC activator. The stimulatory effect of PKC was unaffected by the presence of pertussis toxin, but stimulation by exogenous alpha-subunits of the G protein Gi or G(o) was prevented by PKC inhibitors. 6. From these data we deduce that SRIF can affect K+ATP channel activity directly via a membrane-delimited pathway or indirectly via a pathway requiring diffusible messengers. In the former case, alpha i/alpha o may either enhance PLC activity, stimulating PKC and thus inducing K+ATP channel phosphorylation with consequent increase of activity, or channel phosphorylation by PKC may facilitate a direct stimulation of the channel by alpha i/alpha o. In the latter case, an alpha i/alpha o-induced fall in cAMP contributes to reduced PKA-mediated phosphorylation and suppression of channel activity.

Characterization of the G protein coupling of a glucagon receptor to the KATP channel in insulin-secreting cells.

The G-protein-mediated coupling of a glucagon receptor to ATP-dependent K channels--KATP--has been studied in insulin-secreting cells using the patch clamp technique. In excised outside-out patches, KATP channel activity was inhibited by low concentrations of glucagon (IC50 = 2.4 nM); the inhibitory effect vanished at concentrations greater than 50 nM. In cell-attached patches, inhibition by bath-applied glucagon was seen most often, although stimulation was observed in a few cases. A dual action of the hormone is proposed to resolve these apparently divergent results. In excised inside-out patches, KATP channel activity was inhibited by addition of beta gamma subunits purified from either erythrocyte or retina (IC50 = 50 pM and 1 nM, respectively). Subsequent exposure of the patch to alpha i or alpha o reversed this effect. In excised inside-out patches, increasing Mg2+ in the bath stimulated the channel activity between 0 and 0.5 nM, but blocked it at higher concentrations (IC50 = 2.55 mM). In most cases (70%), GTP had a stimulatory effect at concentrations up to 100 microns. However, in three cases, similar GTP levels had clear inhibitory effects. In excised inside-out patches, cholera toxin (CTX) caused channel inhibition. Although the effect could not be reversed by removal of the toxin, the activity was restored by subsequent addition of purified alpha i or alpha o. These results are compatible with a model whereby channel inhibition by activated Gs-coupled receptors occurs, at least in part, via association of the beta gamma subunits of Gs with alpha i/alpha o subunits and deactivation of the alpha i/alpha o-dependent stimulatory pathway. On the basis of this hypothesis, a model is developed to describe the effects of G proteins on the KATP channel, as well as to account for the concentration-dependent stimulation and inhibition of KATP channel by Mg2+. An interpretation of the ability of glucagon to potentiate, but not initiate, insulin release is also given in terms of this model and the effects of ATP on KATP channels.

ATP mediates both activation and inhibition of K(ATP) channel activity via cAMP-dependent protein kinase in insulin-secreting cell lines.

The single-channel recording technique was employed to investigate the mechanism conferring ATP sensitivity to a metabolite-sensitive K channel in insulin-secreting cells. ATP stimulated channel activity in the 0-10 microM range, but depressed it at higher concentrations. In inside-out patches, addition of the cAMP-dependent protein kinase inhibitor (PKI) reduced channel activity, suggesting that the stimulatory effect of ATP occurs via cAMP-dependent protein kinase-mediated phosphorylation. Raising ATP between 10 and 500 microM in the presence of exogenous PKI progressively reduced the channel activity; it is proposed that this inactivation results from a reduction in kinase activity owing to an ATP-dependent binding of PKI or a protein with similar inhibitory properties to the kinase. A model describing the effects of ATP was developed, incorporating these two separate roles for the nucleotide. Assuming that the efficacy of ATP in controlling the channel activity depends upon the relative concentrations of inhibitor and catalytic subunit associated with the membrane, our model predicts that the channel sensitivity to ATP will vary when the ratio of these two modulators is altered. Based upon this, it is shown that the apparent discrepancy existing between the sensitivity of the channel to low ATP concentrations in the excised patch and the elevated intracellular level of ATP may be explained by postulating a change in the inhibitor/kinase ratio from 1:1 to 3:2 owing to the loss of protein kinase after patch excision. At a low concentration of ATP (10-20 microM), a nonhydrolyzable ATP analogue, AMP-PNP, enhanced the channel activity when present below 10 microM, whereas the analogue blocked the channel activity at higher concentrations. It is postulated that AMP-PNP inhibits the formation of the kinase-inhibitor complex in the former case, and prevents phosphate transfer in the latter. A similar mechanism would explain the interaction between ATP and ADP which is characterized by enhanced activity at low ADP concentrations and blocking at higher concentrations.

Comparative study of K channel behavior in beta cell lines with different secretory responses to glucose.

The patch-clamp technique was used to identify and investigate two K channels in the cell membrane of the HIT cell, an insulin secreting cell line with glucose-sensitive secretion. Channel characteristics were compared with those of glucose-modulated K channels in the RINm5F cell, an insulin secreting cell line in which secretion is largely glucose insensitive. A 65.7 pS channel, identified with the ATP-sensitive K channel was seen in HIT cell-attached patches. Channel activity was dose-dependently inhibited by glucose, by 50 and 100% at 450 microM and 8 mM glucose, respectively, similar to the values previously reported for RIN cells. In inside-out patches channel activity was 50% inhibited by 56 microM ATP and completely blocked between 500 microM and 1 mM, again, similar to the values reported for RIN cells. As in RIN cells a second, considerably larger (184 pS), K channel was glucose sensitive; the glucose sensitivity was similar to that in RIN cells with 50 and 100% channel inhibition at 7.5 and 25 mM, respectively. After patch excision the mean channel conductance increased from 184 to 215 pS. Under these conditions activity was strongly calcium dependent in the range pCa 5-7, identifying this as a calcium- and voltage-dependent K (K(Ca, V] channel; the calcium sensitivity was similar to that of the adult rat beta cell K(Ca, V) channel. In inside-out RIN cell patches, the large K channel was less abundant but displayed a similar conductance (223 pS). However, its calcium sensitivity was more than 10 times lower than in HIT cells, similar to that of the K(Ca, V) channel in the neonatal rat beta cell, which also displays a reduced secretory response to glucose. Based on these observations, it is proposed that the low calcium sensitivity of the K(Ca, V) channel may be causally associated with secretory deficiency in RIN cells and the immature secretory response of the neonatal beta cell.

Metabolic regulation of the K(ATP) and a maxi-K(V) channel in the insulin-secreting RINm5F cell.

K channels in the cell membrane of the insulin-secreting RINm5F cell line were studied using the patch-clamp technique in cell-attached patch mode. With 140 mM K in the pipette, two channels displaying different conductive and kinetics properties were observed. A voltage-independent, inward-rectifying, 55-pS channel was active at rest (no glucose, -70 mV), but was almost completely inhibited by 5 mM glucose. A 140-pS channel was seen in the absence of glucose only after cell membrane depolarization with high (30 mM) K. This channel was voltage dependent, with a linear slope conductance between -60 and +60 mV, and was completely inhibited only by greater than 15 mM glucose. The former channel we identify as an ATP-sensitive channel previously described in excised patches and refer to it as the K(ATP) channel. The latter, because of its large conductance and voltage-dependent kinetics, will be referred to as the maxi-K(V) channel, adopting a nomenclature previously used to classify highly conductive K channels (Latorre, R., and C. Miller, 1983, Journal of Membrane Biology, 71:11-30). In addition to glucose, mannose and 2-ketoisocaproate, which also initiate insulin secretion and electrical activity in the islet beta cell, reduced the activity of both the K(ATP) and the maxi-K(V) channel. Lactate and arginine, which potentiate but do not initiate insulin secretion or beta cell electrical activity in normal islets, each caused a large reduction in maxi-K(V) channel activity, without consistently affecting the activity of K(ATP) channels. Another agonist that potentiates insulin secretion and electrical activity in normal cells, the tumor-promoting phorbol ester TPA, blocked maxi-K(V) channel activity while stimulating the activity of the K(ATP) channel, thereby implicating phosphorylation in the control of channel activity. These results indicate that metabolic substrates that initiate electrical activity and insulin secretion in normal beta cells reduce the activity of both the K(ATP) and the maxi-K(V) channel, while potentiating agents reduce only the maxi-K(V) channel. The possible role of these two channels in the processes of initiation and potentiation of the beta cell response is discussed.

Ion permeation and rectification in ATP-sensitive channels from insulin-secreting cells (RINm5F): effects of K+, Na+ and Mg2+.

Patch-clamp techniques were used to study the permeability to ions of an ATP-sensitive channel in membranes from the pancreatic B-cell line (RINm5F). With patches in the outside-out configuration, the I-V curves for different Na+-K+ mixtures in the bath and 140 mM K+ in the pipette were almost linear, and crossed the zero-current axis at voltages that indicated a variable permeability ratio. When K+ was added symmetrically, the plot of the conductance vs. K+ activity exhibited saturation, with a Gmax of about 160 pS and a half-maximal activity of 216 mM. The I-V behavior for different K+-Na+ mixtures in the bath could be accurately described with a model based on Eyring theory, assuming two sites and one-ion occupancy. For K+, the dissociation constants (KK) of the two sites were 290 and 850 mM, the lower value pertaining to the site close to the intracellular medium. In experiments with inside-out patches, both Na+ and Mg2+, when present in the bath, induced a voltage-dependent block of the outward current. Fitting the data with the model suggested that for these ions only one of the two sites binds significantly, the corresponding dissociation constants being (mM): 46 for Na+ and 34 for Mg2+. Blocking by Na+ and Mg2+ may account for the low outward current seen in intact cells. This hypothesis is consistent with the observation that such current is further reduced by addition of 2,4-DNP, since metabolism inhibitors are expected to lower the ATP level, thereby liberating Mg2+ from the Mg2+-ATP complex, as well as inducing accumulation of Na+ by decreasing the rate of the Na+-K+ pump.

Regulation by cell metabolism and adenine nucleotides of a K channel in insulin-secreting B cells (RIN m5F).

A potassium channel in membranes of the insulin-secreting B-cell line, RIN m5F, was studied using the patch-clamp technique. In cell-attached patches, and for pipette solutions containing 140 mM KCl, the I-V curves exhibited a pronounced rectification, with the conductance being higher when the current flowed from the electrode into the cell (50 pS). Addition of glucose (5-7 mM) to the bath was sufficient to abolish the channel activity, while low doses of the metabolic inhibitor 2,4-dinitrophenyl enhanced it, indicating a link between channel activity and cell metabolism. In excised inside-out patches, the activity of a channel with similar conductive properties was inhibited by ATP in a dose-dependent fashion, the half-maximal concentration being approximately 70 microM. ADP also acted as an inhibitor, but with much lower potency. However, when ADP was present, the blocking effectiveness of ATP was reduced, suggesting a nonadditive interaction of the two nucleotides with the channel. In conclusion, complete closure of the K channel by glucose (5-7 mM) suggests that this channel may be responsible for the early phase of the B-cell depolarization, which is seen in normal cells for glucose increments up to similar values. In addition, since ATP and ADP are both present in the cells, the reduction by ADP of the blocking efficacy of ATP in excised patches may help one to understand why, in intact cells, the channel is still functional for intracellular ATP concentrations that in excised patches would completely suppress its activity.

Caffeine potentiation of calcium release in frog skeletal muscle fibres.

The effects of caffeine at concentrations up to 3 mM were studied on Ca signals obtained using the metallochromic Ca indicator dyes Arsenazo III and Antipyrylazo III in cut frog skeletal muscle fibres mounted in a triple Vaseline-gap chamber and stimulated by voltage clamp or action potential. The peak amplitude of the transient absorbance change due to Ca2+ release following action potential stimulation is potentiated by an amount dependent on caffeine concentration up to 0.5 mM, and by a concentration-independent amount between 0.5 and 2 mM. At 3 mM-caffeine, the potentiation is reduced, and the Ca signal can have a smaller amplitude than under the control condition. The time course of the rising phase of the Ca signal is preserved by the potentiating effect of caffeine; however, the decay rate of the Ca signal is increasingly slowed at caffeine concentrations greater than 0.5 mM. No substantial change was found in the resting myoplasmic Ca2+ level at caffeine concentrations near 0.5 mM. Even if the free Ca2+ concentration in the presence of this level of caffeine were to increase by 0.04 microM (the threshold of detectability), the calculated potentiation of the Ca signal due to increased partial saturation of intracellular Ca2+ buffers would amount to only about 7%. This value is significantly less than the amount of potentiation observed (up to 40%) following action potentials at caffeine levels of 0.5 mM and above. Experiments made with the impermeant potentiometric dye NK2367 show no alteration by caffeine of the electrical properties of the tubular system. Caffeine at up to moderate concentrations causes a substantial increase in the maximal Ca2+ release obtained following large depolarizations. The voltage dependence of the Ca2+ release is characterized by a caffeine concentration-dependent shift towards more negative membrane potentials. The potentiation of Ca2+ release by caffeine was found to be independent of the external free Ca2+ level. The potentiation of the Ca2+ release process by caffeine is likely to occur at a step subsequent to the depolarization of the transverse tubule membrane, and suggests the presence of an intermediate step, or second messenger, in the excitation-contraction coupling process.

Effects of sodium on beta-cell electrical activity.

The present studies, designed to evaluate the contribution of Na+ to the mouse pancreatic beta-cell membrane potential, were performed utilizing intracellular microelectrodes. Complete removal of external sodium, in the presence of glucose, did not significantly affect spike peak potential. However, it caused a negative shift of the resting membrane potential, both in the presence and absence of glucose. After this initial hyperpolarization, the membrane gradually depolarized, the rate of depolarization being slower in the absence of glucose. This two-phase hyperpolarization-depolarization pattern remained when ouabain was added, both in the presence and absence of glucose. An increase of input resistance was associated with the slow depolarization. During this depolarization the maximum rate of rise (dV/dtmax) of the action potential ("spike") decreased. There was no direct relationship between dV/dtmax and [Na]0. Readdition of low [Na]0 (14 mM) to a glucose medium reactivated the postburst hyperpolarization (PBH), even in the presence of ouabain. These observations indicate that there is a significant resting sodium permeability (PNa). However, the action potential (spike) is not generated by activation of a voltage-dependent (gated) sodium channel. The membrane depolarization after Na+ removal reflects concomitant inhibition of the Na+-K+ pump and decrease of potassium permeability (PK). The blockage of PBH in the absence of Na+ is not related to the inhibition of an oscillatory Na+-K+ pump but to the inactivation of a PK. Aside from its effect on the Na+-K+ pump, ouabain may stimulate PNa.

Effects of divalent cations on beta-cell electrical activity.

The effects of various divalent cations, added to the external medium, upon beta-cell action potential were studied using intracellular microelectrodes. Changes of spike peak potential, as a function of external cation concentration, indicate that Sr2+ or Ba2+ may substitute for Ca2+ as a charge carrier. Complete blockage by Mn2+ of electrical activity elicited by Sr2+, Ba2+, or Ca2+ suggests that these cations penetrate the membrane though the same Ca2+ channel. The increase of maximum rate of depolarization, dV(d)/dtmax, and decrease of maximum rate of repolarization, dV(r)/dtmax, when Sr2+ is substituted for Ca2+ suggest that Sr2+ penetrates more readily the Ca2+ channel but is less effective than Ca2+ in activating K permeability. Reversal of these effects by addition of equimolar Ca2+ to Sr2+ indicates that Ca2+ has a greater affinity than Sr2+ for the receptor site. The blockage of electrical activity by Ba2+ at a depolarized membrane level suggests that Ba2+ markedly reduces all K+ permeabilities. Analysis of dV(d)/dtmax at various Ca2+ concentrations, in the presence of nonpermeant divalent cations (Co2+, Mn2+, and Mg2+), shown that these cations bind competitively at the same receptor site with differing dissociation constants, For all of these divalent cations, the order of binding would be Co2+ greater than Mn2+ greater than Ca2+ greater than Sr2+, Mg2+.

Calcium action potentials and potassium permeability activation in pancreatic beta-cells.

Ionic control mechanisms of mouse pancreatic beta-cell action potentials ("spikes"), in response to glucose, were studied by measuring membrane potentials with intracellular microelectrodes. The curve relating the peaks of the spikes to the log of the external calcium concentration above 10 mM has a slope of 25 mV/10-fold increase of Ca2+. This approaches the value predicted by the Nernst equation for a pure Ca2+ electrode. Increasing the external [Ca2+]o from 0 to 42.5 mM caused an increase in rates of spike depolarization and repolarization. Lowering [Ca2+]o or applying Ca2+ conductance blockers, including Co2+ (1.25 mM), Mn2+ (2mM), and D-600 (2 X 10(-4) M), caused a decrease in rates of spikes depolarization and repolarization, with an increase of [Ca2+]o reversing this effect. Higher concentrations of these Ca2+-conductance blockers eliminated the spike activity. Quinidine at a high concentration (10(-3) M) blocked spike repolarization. Tetraethylammonium (TEA, 25 mM) increased spike amplitude and duration. Therefore, it is concluded that Ca2+ entry during the spike affects potassium permeability, which is inhibited by TEA. Also, there is a competitive binding between Co2+, Mn2+, Mg2+, and Ca2+, the charge carrier. These cations may have an additional action of substituting for Ca2+ to "stabilize" the membrane.

Beta-cell electrical activity in response to high glucose concentration.

Glucose-induced beta-cell electrical activity, recorded with glass microelectrodes, is characterized by trains of fast action potentials ("spikes"). The membrane depolarizes before each train of spikes and then repolarizes. This pattern is termed a "burst." There is a characteristic biphasic response to a square wave of 11.1 mM glucose. Pulses at higher glucose concentrations (22.2 mM or more) evoke transient, constant spike activity. The duration of this activity is lengthened and the lag period shortened in proportion to the concentration and length of the glucose pulse. The lag period between removal of glucose and the cessation of spike activity is also proportional to the glucose concentration.

Cyclic variation of K+ conductance in pancreatic beta-cells: Ca2+ and voltage dependence.

Pulses of hyperpolarizing current were injected through the microelectrode recording the electrical activity of beta-cells in order to measure input resistance. Increase in resistance during depolarization of the slow oscillation ("burst") indicates inactivation of an outward current, probably K+. Decrease in resistance as the plateau commences suggests that the previous depolarization causes activation of an inward current, probably calcium. The postburst hyperpolarization, caused by a late activation of potassium permeability (PK), would result from the increase of intracellular free calcium. An intracellular buffering system may control this intracellular free calcium level. By restoring the silent phases, in the presence of ouabain or high potassium, injection of hyperpolarizing current shows also a voltage dependency of the PK involved in the postburst hyperpolarization. Glucose, by stimulating intracellular binding of calcium, would cause membrane depolarization at glucose levels below threshold and elongation of the plateau phase at higher concentrations.