Inside-out Patch Recordings Research Articles

Potassium currents in human freshly isolated bronchial smooth muscle cells.

1. K+ currents were studied in smooth muscle cells enzymatically dissociated from human bronchi, by use of the patch-clamp technique. 2. In whole-cell recordings a depolarization-induced, 4-aminopyridine (4-AP)-sensitive current was observed in only 26 of 155 cells, and in 20 of these 26 cells its amplitude at a test potential of 0 mV was less than 100 pA. 3. In the majority of cells depolarization to -40 mV or more positive potentials induced a noisy outward current which activated within milliseconds and showed almost no inactivation even during a 5 s depolarizing voltage step. This current was insensitive to 4-AP (up to 5 mM) but was strongly inhibited in the presence of tetraethylammonium (TEA, 1 mM), charybdotoxin (ChTX, 100 nM) or iberiotoxin (IbTX, 50 nM) in the bath. The same current was also recorded by the nystatin-perforated patch technique. 4. Single channels with a conductance of about 210 pS were recorded in cell-attached patch, inside-out patch, outside-out patch and whole-cell recording configurations. Channel open state probability in inside-out patches was 0.5 at a membrane potential of 4 +/- 14 mV (mean +/- s.d., n = 13) mV even with a free Ca2+ concentration on the cytosolic side of the patch of less than 0.1 nM. Open state probability increased with depolarization and internal Ca2+ concentration. Single channels could be reversibly blocked by externally applied TEA, ChTX and IbTX. 5. In current-clamp recordings with 100 nM free Ca2+ in the intracellular solution both TEA and ChTX caused substantial concentration-dependent depolarization. 6. These results suggest that in human bronchial smooth muscle cells, in marked contrast to other species, the majority of the outward current induced by depolarization is not due to a delayed rectifier,but to the activity of a large conductance, ChTX-sensitive K+ channel. The Ca2+- and voltage-dependency of this channel may well allow a sufficiently high open state probability for it to play a partin the regulation of the resting membrane potential.

ADP exerts a protective effect against rundown of the Ca2+ current in bovine chromaffin cells.

In isolated chromaffin cells, the high-voltage-activated Ca2+ current, recorded using 5 mM Ca2+ as the divalent charge carrier, exhibits rundown within 10 min, which is delayed for 1 h at least by the addition of 1 mM adenosine 5'-triphosphate (ATP) to the pipette medium. The mechanism of this stabilizing action of ATP has been examined. ATP action is dose dependent; the rundown process, which was delayed at concentrations below 0.4 mM, was totally abolished at higher concentrations. The requirement for ATP was shown to be quite strict: 2 mM inosine 5'-triphosphate (ITP) could not replace ATP, whereas guanosine 5'-triphosphate (GTP) could, but at higher concentrations. This effect of ATP was shown to require the presence of MgCl2 and the liberation of a phosphate group since the ATP analogue 5'-adenylyl-imidodiphosphate (AMP-PNP) could not act as a substitute for ATP, suggesting an action through either adenosine 5'-diphosphate (ADP) or a phosphorylation step. ADP, in the presence of Mg2+ only, could replace ATP in the same concentration range. This effect was shown to be specific to ADP; it was maintained after blocking the pathways which convert ADP into ATP, and could not be mimicked by guanosine 5'-diphosphate (GDP). Similarly, ATP and ADP effects were abolished at an increased internal Ca2+ concentration (pCa 6 instead of pCa 7.7, where pCa = -log10[Ca2+]). Nevertheless, the presence of 1 mM Mg-ADP in the bathing solution did not prevent the rundown of the Ca2+ channels when going to the inside-out patch recording configuration.(ABSTRACT TRUNCATED AT 250 WORDS)

Na(+)-activated nonselective cation channels in primary olfactory neurons.

1. Excised inside-out patch recordings were used to describe a novel cation channel from cultured lobster olfactory receptor neurons that is activated by [Na+]i. 2. The channel is reversibly activated by intracellular Na+ as low as 5 mM. The half-effect concentration of intracellular Na+ is approximately 60 mM at -60 mV. The dependence of the channel open probability on [Na+]i is sigmoidal with a Hill coefficient of 3.1, indicating that more than one Na+ must bind to activate the channel. 3. The channel is equally permeable to Na+, K+, and Li+. In symmetrical 210 mM Na+, the open channel current-voltage relationship shows slight inward rectification at positive potentials. The slope conductance of the channel is 107 pS between -90 and 0 mV. 4. Although the channel is not activated by voltage in the absence of intracellular Na+, the gating of the channel is dependent on voltage as well as [Na+]i and [Na+]o. 5. Both intracellular Ca2+ and Mg2+ reversibly affect channel activity in a concentration-dependent manner starting at 1 microM. Ca2+ decreases both the open probability and the single channel amplitude, whereas Mg2+ decreases the open probability but has no effect on the single channel amplitude. Ba2+ (5 mM), but not 20 mM Cs+ and 100 microM amiloride, reversibly block the channel. 6. We speculate that this novel cation channel regulates neuronal excitability by accentuating the rate and/or the magnitude of depolarization of the cell to odors.

Properties of histamine-activated chloride channels in the large monopolar cells of the dipteran compound eye: a comparative study

The large monopolar cells (LMCs) of the first optic neuropil (lamina) in insects respond to the photoreceptor neurotransmitter histamine with an increase in chloride conductance. We have compared the properties of this conductance from a range of diptera from different visual environments: Tipula paludosa (slow flying, crepuscular), Drosophila melanogaster (slow-flying diurnal), and 3 fast-flying diurnal species Musca domestica, Calliphora vicina and Lucilia sericata. In whole-cell recordings of dissociated LMCs, histamine-induced currents were elicited using a multichannel parallel perfusion device, allowing rapid determination of the dose-response function, characterised by affinity (Kd) and Hill coefficient (n). Calliphora, Lucilia and Musca had the steepest dose response curves (n = 2.8) and the lowest affinity for histamine (Kd 35–50 μM); the crepuscular Tipula had a significantly higher affinity (Kd = 16 μM) and lower Hill coefficient (n = 1.8). Drosophila had a high affinity (Kd 24 μM), and a high Hill coefficient (n = 2.5). In excised inside-out patch recordings all species showed similar single channel properties (conductance 40–60 pS, mean open time < 1 ms). The low Hill coefficient in Tipula would be expected to result in lower synaptic gain. We suggest this may be an adaptation to prevent the LMC's response bandwidth being filled with the high levels of photon noise typical of photoreceptors adapted for low light levels. The lower affinity for histamine found in the more photopic species suggests that the concentration of histamine (and therefore presumably number of synaptic vesicles released from the photoreceptors) should be higher. This might improve signal-to-noise ratio by decreasing the contribution of the shot event noise introduced by stochastic release of synaptic vesicles.

A conductance maximum observed in an inward-rectifier potassium channel.

One prediction of a multi-ion pore is that its conductance should reach a maximum and then begin to decrease as the concentration of permeant ion is raised equally on both sides of the membrane. A conductance maximum has been observed at the single-channel level in gramicidin and in a Ca(2+)-activated K+ channel at extremely high ion concentration (> 1,000 mM) (Hladky, S. B., and D. A. Haydon. 1972. Biochimica et Biophysica Acta. 274:294-312; Eisenmam, G., J. Sandblom, and E. Neher. 1977. In Metal Ligand Interaction in Organic Chemistry and Biochemistry. 1-36; Finkelstein, P., and O. S. Andersen. 1981. Journal of Membrane Biology. 59:155-171; Villarroel, A., O. Alvarez, and G. Eisenman. 1988. Biophysical Journal. 53:259a. [Abstr.]). In the present study we examine the conductance-concentration relationship in an inward-rectifier K+ channel, ROMK1. Single channels, expressed in Xenopus oocytes, were studied using inside-out patch recording in the absence of internal Mg2+ to eliminate blockade of outward current. Potassium, at equal concentrations on both sides of the membrane, was varied from 10 to 1,000 mM. As K+ was raised from 10 mM, the conductance increased steeply and reached a maximum value (39 pS) at 300 mM. The single-channel conductance then became progressively smaller as K+ was raised beyond 300 mM. At 1000 mM K+, the conductance was reduced to approximately 75% of its maximum value. The shape of the conductance-concentration curve observed in the ROMK1 channel implies that it has multiple K(+)-occupied binding sites in its conduction pathway.

Phorbol myristate acetate and ATP-sensitive potassium channels in insulin-secreting cells.

The actions of 4 beta-phorbol 12-myristate 13-acetate (PMA; 10-100 nM) on the ATP-sensitive K+ channel have been studied in the RINm5F insulin-secreting cell line. These experiments were carried out using the inside-out patch and open-cell recording configurations of the patch-clamp technique. In the presence of intracellular ATP and ADP, PMA was found to have complex effects. Over short periods of time, i.e., less than 5 min, PMA promoted channel inhibition. However, in the sustained presence of the phorbol ester, this inhibition was only transient and was followed by a period of recovery and then stimulation of the channels. There were no effects of PMA on ATP-sensitive K+ (K+ATP) channels in the absence of intracellular ATP/ADP and in patches of membrane excised from cells that had been pretreated overnight with 1 microM PMA to downregulate endogenous C-kinase. The inactive phorbol ester 4 alpha-phorbol 12,13-didecanoate (10 nM-1 microM) was also found to have no actions on ATP-sensitive K+ channels. Overall, these data may suggest that through the modulation of protein kinase(s) associated with K+ATP channels, PMA is capable of causing both activation and inhibition of K+ channels in RINm5F cells.

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes.

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.

Characterization of the calcium-sensitive voltage-gated delayed rectifier potassium channel in isolated guinea pig hepatocytes.

The voltage-dependent K+ channel was examined in enzymatically isolated guinea pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. The resting membrane potential in isolated hepatocytes was -25.3 +/- 4.9 mV (n = 40). Under the whole-cell voltage-clamp, the time-dependent delayed rectifier outward current was observed at membrane potentials positive to -20 mV at physiological temperature (37 degrees C). The reversal potential of the current, as determined from tail current measurements, shifted by approximately 57 mV per 10-fold change in the external K+ concentration. In addition, the current did not appear when K+ was replaced with Cs+ in the internal and external solutions, indicating that the current was carried by K+ ions. The envelope test of the tails demonstrated that the growth of the tail current followed that of the current activation. The ratio between the activated current and the tail amplitude was constant during the depolarizing step. The time course of growth and deactivation of the tail current were best described by a double exponential function. The current was suppressed in Ca(2+)-free, 5 mM EGTA internal or external solution (pCa > 9). The activation curve (P infinity curve) was not shifted by changing the internal Ca2+ concentration ([Ca2+]i). The current was inhibited by bath application of 4-aminopyridine or apamin. alpha 1-Adrenergic stimulation with noradrenaline enhanced the current but beta-adrenergic stimulation with isoproterenol had no effect on the current. In single-channel recordings from outside-out patches, unitary current activity was observed by depolarizing voltage-clamp steps whose slope conductance was 9.5 +/- 2.2 pS (n = 10). The open time distribution was best described by a single exponential function with the mean open lifetime of 18.5 +/- 2.6 ms (n = 14), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 2.0 +/- 0.3 ms (n = 14) and that for the slow component of 47.7 +/- 5.9 ms (n = 14). Ensemble averaged current exhibited delayed rectifier nature which was consistent with whole-cell measurements. In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The concentration of Ca2+ at the half-maximal activation was 0.031 microM. These results suggest that guinea pig hepatocytes possess voltage-gated delayed rectifier K+ channels which are modified by intracellular Ca2+.

K channel activation by nucleotide diphosphates and its inhibition by glibenclamide in vascular smooth muscle cells.

1. Whole-cell and inside-out patch recordings were made from single smooth muscle cells that had been isolated enzymatically and mechanically from the rabbit portal vein. 2. In whole-cells the inclusion in the recording pipette solution of nucleotide diphosphates (NDPs), but not tri- or monophosphates, induced a K-current that developed gradually over 5 to 15 min. Intracellular 1 mM guanosine 5'-diphosphate (GDP) induced a slowly developing outward K-current at -37 mV that reached a maximum on average of 72 +/- 4 pA (n = 40). Half maximal effect was estimated to occur with about 0.2 mM GDP. Except for ADP, other NDPs had comparable effects. At 0.1 mM, ADP was equivalent to GDP but at higher concentration ADP was less effective. ADP induced its maximum effect at 1 mM but had almost no effect at 10 mM. 3. In 14% of inside-out patches exposed to 1 mM GDP at the intracellular surface, characteristic K channel activity was observed which showed long (> 1 s) bursts of openings separated by longer closed periods. The current-voltage relationship for the channel was linear in a 60 mM:130 mM K-gradient and the unitary conductance was 24 pS. 4. Glibenclamide applied via the extracellular solution was found to be a potent inhibitor of GDP-induced K-current (IK(GDP)) in the whole-cell. The Kd was 25 nM and the inhibition was fully reversible on wash-out. 5. IK(GDP) was not evoked if Mg ions were absent from the pipette solution. In contrast the omission of extracellular Mg ions had no effect on outward or inward IK(GDP). 6. Inclusion of 1 mM ATP in the recording pipette solution reduced IK(GDP) and also attenuated its decline during long (25 min) recordings. 7. When perforated-patch whole-cell recording was used, metabolic poisoning with cyanide and 2-deoxy-D-glucose induced a glibenclamide-sensitive K-current. This current was not observed when conventional whole-cell recording was used. Possible reasons for this difference are discussed. 8. These K channels appear similar to ATP-sensitive K channels but we refer to them as nucleotide diphosphate-dependent K channels (KNDP) to emphasise what seems to be a primary role for nucleotide diphosphates in their regulation.

Single channel and whole-cell K-currents evoked by levcromakalim in smooth muscle cells from the rabbit portal vein.

1. Single channel and whole-cell current recordings were made from single smooth muscle cells isolated from the rabbit portal vein. 2. Application of 10 microM levcromakalim ((-)-Ckm) to single cells held with pipettes containing 1 mM GDP induced a K-current (IK(Ckm)) which occurred in addition to the current caused by GDP alone (IK(GDP)) and averaged 135 pA at -37 mV. We have investigated whether the same K channels underlie the GDP- and Ckm-induced K-currents. 3. If 1 mM GDP was in the pipette but Mg ions were omitted the effect of GDP was absent and IK(Ckm) averaged only 10 pA, suggesting that the action of (-)-Ckm was Mg-dependent. 4. Intracellular ATP was not observed to have much effect on IK(-Ckm). Loading of cells with 10 mM ATP from the recording pipette had no significant effect and flash photolysis of caged-ATP loaded into cells from the pipette, estimated to release about 1 mM free ATP, also had no effect on IK(-Ckm). 5. Bath-applied glibenclamide inhibited IK(-Ckm) with an IC50 of 200 nM, a value 8 times higher than that found for inhibition of IK(GDP). The delayed rectifier K-current (IK(DR)) was also inhibited by glibenclamide but at higher concentrations (IC50 100 microM). Bath-applied tetraethylammonium ions (TEA) inhibited IK(-Ckm) and IK(GDP) to the same extent (IC50 about 7 mM). 6. In inside-out patch recordings (- )-Ckm (10 microM) applied to the intracellular surface of the membrane potentiated the opening of K channels already stimulated by I mM GDP and all of the channel activity was abolished by 10 microM glibenclamide. The unitary conductance of the channels was 24lpS in a 60 mM: 130 mM K-gradient.7. We suggest that (-)-Ckm may hyperpolarize and relax smooth muscle cells by opening KNDP, a class of small conductance K channels that are related to the ATP-sensitive K channels seen in other tissues.

Mastoparan blockade of currents through Ca 2+-activated K + channels in bovine chromaffin cells

The action of mastoparan (a wasp venom peptide) on “maxi” Ca 2+-activated K + channels was studied in excised inside-out patch recordings from cultured bovine chromaffin cells, under normal conditions (160 mM K + inside, 154 mM Na + outside). Mastoparan, when applied on the intracellular side of the membrane reduced the open channel probability in a concentration dependent manner. Changes in the channel kinetics were complex. The histograms of the open dwell times were all described by either one or two exponentials. Mastoparan shortened the mean duration of the major (long) component and to a lesser extent the minor (short) component. Closed dwell times, were described by three exponentials. While the short (major) component was prolonged by mastoparan, and the intermediate component was unaffected, the long component was shortened. Overall mean closed times were prolonged. The changes in channel kinetics could only partly be explained by a channel-blocking mechanism, even when assuming that mastoparan acts as both an intermediate and a slow channel blocker suggesting that it affects gating mechanism. The fact that mastoparan is a calmodulin inhibitor and a G-protein activator raises the possibility that in bovine chromaffin cells, either the membrane-bound calmodulin or a G-protein, plays a role in the modulation of Ca 2+-activated K + channels.

Chloride channels in cultured human skeletal muscle are regulated by G proteins.

The regulation of Cl- channels in human myoballs by G proteins was studied using whole-cell and inside-out patch recordings. After perfusion of the cell with 0.1 mM GTP[gamma S], the specific Cl- conductance, GCl, at standard resting potential (-85 mV) was increased from 5.9 microS/cm2 to 103 microS/cm2, and the kinetics upon stepping the potential to positive values was changed from an activating current with very slow inactivation to a fast inactivating current with no potential-dependent activation. These effects were not affected by the simultaneous blockade of several signal cascades involving G proteins. Addition of the protein kinase blockers PKI (25 microM), H8 (10 microM), or of the phospholipase-A2-blocking agent quinacrine (10 microM), had not much influence on these GTP[gamma S] effects. Buffering of the intracellular Ca2+ concentration (0.1 microM) or addition of the Ca2+/calmodulin antagonist trifluoperazine (50 microM) was also without effect. Pre-incubation of the cells with pertussis toxin or with cholera toxin did not change GCl. In excised inside-out patches voltage-clamped at -85 mV, application of GTP[gamma S] influenced the "intermediate" Cl- channel, the Cl- channel type having the highest density in these cells, by increasing the number of transitions in a half-conductance state. The probability of the channel being in one of the two conducting states rose from 0.015 to 0.67, and the kinetics of the single-channel currents was changed so that, on average, it was similar to the whole-cell current kinetics seen after application of GTP[gamma S]. It is concluded that a G protein is directly interacting with these channels.

Activation of nonselective cation channels by physiological cholecystokinin concentrations in mouse pancreatic acinar cells.

The activation of the nonselective cation channels in mouse pancreatic acinar cells has been assessed at low agonist concentrations using patch-clamp whole cell, cell-attached patch, and isolated inside-out patch recordings. Application of acetylcholine (ACh) (25-1,000 nM) and cholecystokinin (CCK) (2-10 pM) evoked oscillatory responses in both cation and chloride currents measured in whole cell experiments. In cell-attached patch experiments we demonstrate CCK and ACh evoked opening of single 25-pS cation channels in the basolateral membrane. Therefore, at least a component of the whole cell cation current is due to activation of cation channels in the basolateral acinar cell membrane. To further investigate the reported sensitivity of the cation channel to intracellular ATP and calcium we used excised inside-out patches. Micromolar Ca2+ concentrations were required for significant channel activation. Application of ATP and ADP to the intracellular surface of the patch blocked channel opening at concentrations between 0.2 and 4 mM. The nonmetabolizable ATP analogue, 5'-adenylylimidodiphosphate (AMP-PNP, 0.2-2 mM), also effectively blocked channel opening. The subsequent removal of ATP caused a transient increase in channel activity not seen with the removal of ADP or AMP-PNP. Patches isolated into solutions containing 2 mM ATP showed channel activation at micromolar Ca2+ concentrations. Our results show that ATP has two separate effects. The continuous presence of the nucleotide is required for operation of the cation channels and this action seems to depend on ATP hydrolysis. ATP can also close the channel and this effect can be demonstrated in excised inside-out patches when ATP is added to the bath after a period of exposure to an ATP-free solution. This action does not require ATP hydrolysis. Under physiological conditions hormonal stimulation can open the nonselective cation channels and this can be explained by the rise in the intracellular free Ca2+ concentration.

Analysis of single cyclic nucleotide-gated channels in olfactory receptor cells

In the accompanying article (Firestein et al., 1991b), we have demonstrated that odor- and cyclic nucleotide-sensitive channels exist at a low density in the dendritic membranes of isolated salamander olfactory receptor neurons. Here, we analyze the cyclic nucleotide sensitivity of these channels using the inside-out patch recording technique. Both cAMP and cGMP, at micromolar concentrations, are capable of inducing channel openings. The biophysical parameters of channel activity are nearly the same in response to either ligand. The unitary conductance is about 45 pS, the reversal potential of single-channel currents is +5 mV, and the I/V relation is linear over the range -80 to +80 mV. The channel activity shows no obvious voltage dependence in divalent cation-free symmetrical solutions. The channel shows no desensitization, even to agonist exposures lasting 15 sec. Mean open time is about 1.5 msec; the closed time distribution is best fit by two exponentials with a fast time constant in the submillisecond range (ca. 0.15 msec) and a slower time constant in the millisecond range (ca. 1.5 msec). The only clear difference in the activity of the two ligands is in their affinity constants. The K1/2 for cAMP is 20 microM; that for cGMP is 4 microM. In both cases, the Hill coefficient is greater than 2, suggesting that channel opening requires the cooperative action of three ligand molecules.

Inhibition of the muscarinic receptor-activated K+ current by N-ethylmaleimide in rabbit heart

The effects of N-ethylmaleimide (NEM), a sulfhydryl alkylating agent, on the ACh-activated K+ current were examined in single cells from rabbit hearts using whole-cell and single channel patch clamp techniques. Bath application of NEM (50 microM) or the muscarinic antagonist, atropine (1 microM) completely inhibited the ACh-activated K+ current in whole-cell recordings. In cell-attached patch conditions, the inhibitory effect of NEM was still observed; in contrast, atropine was ineffective when the agents were bath applied, indicating that the site of action of NEM is different from that of atropine. Inside-out patch recordings confirmed that GTP was required on the internal side of the membrane for activation of this K+ channel when ACh was present in the pipette. NEM abolished this GTP-activated K+ channel activity. GTP gamma S, a non-hydrolysable GTP analogue, was able to activate this K+ channel in the absence of a muscarinic agonist, an effect thought to be due to the direct activation of GTP-binding proteins. Pretreatment with NEM almost completely prevented this effect of GTP gamma S. In contrast, after the activation of the K+ channel by GTP gamma S had reached a steady-state, NEM failed to show a significant inhibitory effect. These results demonstrate that NEM prevents the activation of muscarinic receptor-regulated K+ channel and suggest an involvement of alkylation of the GTP-binding proteins which are coupled to this type of K+ channel.

Long-Term Culture of Neonatal Rat Pancreatic Endocrine Cells as Model for Insulin-Secretion and Ion-Channel Studies

So far, only freshly isolated cells or short-term cultures have been used to study ion-channel activity in pancreatic nontumor beta-cells. We report a procedure for the long-term cultivation of pancreatic endocrine cells to study the relationship between ion channels and insulin secretion. Using thimerosal to suppress fibroblastoid cell proliferation and a preliminary 2-day cell exposure to alternating normal (5.6 mM) and high (16.7 mM) glucose levels, we observed a significant secretory responsiveness of the cells to a glucose challenge for at least 4 wk in culture. Cells also responded to glucose or other secretagogues, such as quinine and the sulfonylurea glyburide, with membrane voltage oscillations. In the cell-attached configuration of the patch-clamp technique, a 65-pS-conductance K+ channel was observed, which was inhibited by glucose, quinine, and glyburide. In the inside-out configuration, the activity of this channel was suppressed by ATP applied to the cytoplasmic side of the membrane. A K+ channel with a conductance of 200 pS was also observed, which was activated by intracellular Ca2+. A 13-pS-conductance glucose-insensitive K+ channel was present in both cell-attached and inside-out patch recordings. Even after 3 wk, the characteristics of these currents and channels were comparable to those reported by other investigators with freshly dissociated or short-term-cultured beta-cells from neonatal and adult rats and adult mice. Therefore, the neonatal rat endocrine cell culture characterized herein provides an improved model for long-term investigations combining secretion and electrophysiological studies.

Selective modulation of the ATP-sensitive K+ channel by nicorandil in guinea-pig cardiac cell membrane

Effects of a vasodilator, nicorandil (2-nicotinamidoethyl nitrate) on four kinds for cardiac K+ channels were investigated in guinea pig ventricular and atrial cells using inside-out patch recording combined with "oil-gate" concentration jump method. Nicorandil of 300 mumols/l failed to affect the inward-rectifier K+ channel and the Na(+)-activated K+ channel. The open probability of the muscarinic K+ channel, when activated by the application of GTP, was not changed by the drug. Nicorandil selectively increased the open probability of the ATP-sensitive K+ channel that was partly suppressed by intracellular ATP. The median effective concentration (EC50) of nicorandil was 74 mumols/l and Hill coefficient was 1.32 in the concentration-open probability relationship. The closing rate of the K+ channel by ATP was markedly delayed by the drug, whereas the open rate on removal of ATP was scarcely affected. Nicorandil had only little effect on this channel after run-down. It was concluded that nicorandil selectively activates the ATP-sensitive K+ channel mainly by modulating the ATP-dependent gate.

ATP-dependent decay and recovery of K+ channels in guinea pig cardiac myocytes

ATP-dependent decay and recovery of the inward rectifier and ATP-sensitive K+ channels were investigated using inside-out patch recording in cardiac myocytes. The solution facing the inner side of the membrane was instantaneously changed with the oil-gate concentration jump method. Both channels were decayed by removing ATP and were recovered by reapplying ATP. The coexistence of Mg2+ was required for the recovery. 5'-Adenylylimidodiphosphate failed to reverse the ATP-dependent decay. The cumulative histograms of survival time and recovery time, obtained from the inward rectifier K+ channel, showed a single exponential distribution, time constants of which were 55 and 43 s, respectively. The time-dependent nature of decay and recovery was also confirmed in the ATP-sensitive K+ channel. The findings indicated that intracellular ATP is one of the factors that determines the activity of the K+ channels. It is most probable that phosphorylation of channel molecules is essential for maintaining the K+ channel in an operative state.

Quinine blockade of currents through Ca2+‐activated K+ channels in bovine chromaffin cells.

1. The blocking action of quinine of Ca2+-activated K+ channels was studied in excised inside-out patch recordings from cultured bovine chromaffin cells. Under normal ionic conditions (160 mM-K+ inside, 154 mM-Na+ outside), the unitary conductances measured between -20 and +40 mV were between 95 and 210 pS. 2. Quinine applied on the intracellular side of the membrane in micromolar concentrations chopped the unitary K+ currents into bursts of brief openings. These 'flickery bursts' appear to arise from discrete blocking and unblocking transitions. 3. Open and closed times within such flickery bursts were exponentially distributed suggesting a simple reaction between quinine and an open channel. 4. The blocking rate varied linearly with the concentration of quinine (rate coefficient 1.6 x 10(6) M-1 s-1). The unblocking rate was high (greater than 5000 s-1) and essentially independent of the quinine concentration. 5. The blocking rate was voltage dependent and increased with hyperpolarization which suggests that quinine acts predominantly or exclusively from the extracellular side of the membrane. The unblocking rate appeared to be independent of the membrane potential. 6. The probability of the open state was not altered by quinine indicating that quinine did not interfere directly with the gating process. 7. The total current carried by K+ ions was reduced by quinine only marginally (less than 10%) even at high quinine concentrations (1.5 mM). Moreover the reduction of the total current was also essentially voltage independent. 8. Quinine is clearly a very poor blocker. Poor block occurs, however, not only because of the brevity of the closed periods but presumably also because the channel can be closed only when it is not blocked. Only when the probability of channel opening (p) was high (greater than 0.85), i.e. when the channel's gate was predominantly open, did the flickery block appear to reduce the total current appreciably.