Biomedical subjects
Publications and source records attributed to G Trube.
Two types of exogenous K channel were identified in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin-secreting cell line HIT T15. One of these was the ATP-regulated K channel (G channel) as evidenced by its conductance and inhibition by tolbutamide. The other resembled the Ca-activated K channel from beta-cells.
The effects of intracellular purine nucleotides on tolbutamide-induced block of ATP-dependent K+ channels from mouse pancreatic B-cells were studied using the patch-clamp technique. When applied to the inside of excised patches, tolbutamide alone blocked channel activity half-maximally at 55 microM and the concentration-response curve for the inhibition of K+ channels by tolbutamide was flat. ADP (1 mM), but not other nucleotides (AMP, GTP or GDP) increased the steepness of the concentration-response curve and decreased the half-maximally effective tolbutamide concentration to 4.2 microM. It is suggested that the ATP-dependent K+ channel or a closely related structure contains a receptor which is accessible for cytosolic ADP and controls the sensitivity to tolbutamide.
The influence of the hypoglycemic drugs tolbutamide, meglitinide, glipizide and glibenclamide on ATP-dependent K+ currents of mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. In the absence of albumin, tolbutamide blocked the currents half maximally at 4.1 mumol/l. In the presence of 2 mg/ml albumin half maximal inhibition of the currents was observed at 2.1 mumol/l meglitinide, 6.4 nmol/l glipizide and 4.0 nmol/l glibenclamide. The hyperglycemic sulfonamide diazoxide opened ATP-dependent K+ channels. Half maximally effective concentrations of diazoxide were 20 mumol/l with 0.3 mmol/l ATP and 102 mumol/l with 1 mmol/l ATP in the recording pipette. Thus, the action of diazoxide was dependent on the presence of ATP in the recording pipette. The free concentrations of the drugs which influenced ATP-dependent K+ currents were comparable with the free plasma concentrations in humans and the free concentrations which affected insulin secretion in vitro. The results support the view that the target for the actions of sulfonylureas and of diazoxide is the ATP-dependent K+ channel of the pancreatic B-cell or a structure closely related to this channel.
K+ channels in the membrane of murine pancreatic beta-cells were studied using the patch-clamp technique. The delayed outward current was activated in whole-cell experiments by depolarizing voltage pulses to potentials between -30 mV and 0 mV. Forskolin blocked the current rapidly (less than 5 s) and reversibly with 50% inhibition at 13 microM. The inhibition did not depend on a stimulation of the adenylate cyclase since it occurred even in presence of 1 mM cAMP in the pipette solution which replaced the cytoplasm. Membrane permeant cAMP analogues and phosphodiesterase inhibitors did not influence the delayed outward current. In experiments on outside-out patches forskolin (100 microM) shortened the openings of a channel of about 10 pS conductance at 0 mV and a time course of activation and inactivation similar to the whole-cell current. Another smaller, slowly activating channel and the Ca2+- and ATP-dependent K+ channels were influenced only weakly or not at all. It is therefore concluded that the 10-pS channel generates most of the delayed outward K+ current in murine pancreatic beta-cells. The Ca2+-independent part of the delayed outward current in bovine adrenal chromaffin cells was also blocked by forskolin (100 microM).
Barium currents flowing through single Ca2+ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca2+ channels was observed. In 110 mM Ba2+, the single channel conductance was 24pS (at negative membrane potentials) and the current amplitude at 0 mV was -0.7 pA. Channel openings were activated by depolarisations more positive than -30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd2+. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca2+ channels in other tissues. It is suggested that this Ca2+ channel participates in the generation of the B-cell action potential and mediates the increase in Ca2+ influx required for insulin secretion.
Clinically potent skeletal muscle relaxants are used primarily for their effects on the central nervous system. But they also have direct effects on muscle contraction that possibly involve Ca2+ channels. We compared the effects of dantrolene, an agent known to have a direct action on vertebrate skeletal muscle, with other substances used as (1) relaxants and (2) antagonists of Ca-dependent excitation-contraction coupling. Isolated intact frog muscle cells were injected with the photoprotein aequorin, and membrane potential changes, intracellular Ca2+ transients, and contractile force were measured. Dantrolene (10(-8) to 10(-5) M) decreased the amplitude of Ca2+ transients, did not affect their rates of decay, and reduced contractile force. We also used an integrated digital-imaging system to record microscopic changes, namely, active shortening in myofibrils and changes in striation spacing. Dantrolene did not increase the time between contraction in myofibrils near the surface compared with myofibrils near the center of a cell. Hence dantrolene does not suppress Ca2+ transients by disturbing current flow in the transverse tubular system. Each of the following actually increased Ca2+ transients and contractile force evoked by action potentials: baclofen (10(-7) to 10(-5) M) less than flordipine (10(-6) M) less than meprobamate (10(-7) to 10(-3) M) less than chlordiazepoxide (10(-5) X 10(-4) M) less than procaine (10(-5) to 5 X 10(-4] less than GABA (10(-5) M) less than D-600 (10(-6) M) less than nylidrin (10(-5) M)--in order of increasing potency. Ca2+ channels in the sarcoplasmic reticulum of intact skeletal muscle are evidently inhibited by dantrolene but not by Ca2+ antagonists.
K+ currents through ATP-dependent channels were recorded from inside-out patches of beta-cell membrane as previously described (Rorsman and Trube 1985). Channels were opened by removing ATP from the intracellular side of the membrane. The open probability and/or the number of active channels declined spontaneously ("run-down") when ATP was absent for periods longer than about 30 s. Channels subject to the run-down could be activated again after applying a blocking concentration (greater than 0.1 mM) of ATP in presence of 1 mM MgCl2 for at least 2 min. ATP in absence of Mg and the ATP-analogues AMP-PNP, AMP-PCP and ATP gamma S were ineffective in reactivating the channels. This suggests that phosphorylation of the channels or associated proteins or hydrolysis of ATP may be necessary for keeping the channels available. In contrast to the differential effects on the run-down, ATP in presence and absence of Mg and the ATP analogues were similarly effective in blocking the channels at concentrations above 0.1 mM. Using an experimental protocol avoiding the run-down the dose-inhibition curve for ATP was found to reach 50% at 18 microM.
Single isolated myocytes were obtained from the ventricles of adult guinea pig hearts. The whole-cell recording configuration of the patch-clamp technique was used to measure membrane currents. A decrease (run-down) of the Ca2+ inward current and an increase of a time-independent K+ outward current were observed during long lasting (1-3h) recordings. The time at which the outward current developed depended on the intracellular ATP concentration in the pipette, suggesting that this current is identical to the ATP-dependent K+ current described by Noma and Shibasaki (1985). However, the maximum outward current reached in the experiments was independent of the ATP concentration indicating a limited diffusion of ATP in the cell interior. In single-channel experiments on isolated patches of cell membrane and in whole-cell recordings the ATP-dependent K+ current could be blocked by the hypoglycaemic sulphonylurea tolbutamide. The IC50 of 0.38 mM was about 50 times higher than that reported for pancreatic beta-cells (Trube et al. 1986). The Ca2+ inward current and the inwardly rectifying K+ current were not affected by tolbutamide (3 mM).
Patch-clamp techniques were used to study the ATP-dependent K+ channel in single ventricular myocytes from guinea pig hearts. During long lasting whole-cell recordings an increase of a time-independent K+ outward current could be observed. This outward current could be inhibited by the intracellular application of ATP (3-20 mM), but not by the Ca2+-chelator EGTA (20 mM). The blocking effect of ATP was also observed in single-channel experiments. The hypoglycaemic sulphonylurea tolbutamide specifically inhibited the ATP-dependent K+ current in whole-cell recordings, inside-out and outside-out patches (IC50 = 0.4 mM).
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The influence of the antidiabetic sulphonylurea tolbutamide on K+ channels of mouse pancreatic beta-cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K+ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of beta-cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 microM. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K+ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca2+-dependent K+ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K+ channels.
Pancreatic islets of NMRI mice were dissociated into single cells which were kept in tissue culture for 1-3 days. The whole-cell configuration of the patch-clamp technique was used to study inward and delayed outward currents of beta-cells under voltage-clamp conditions at 20-22 degrees C. Outward currents were suppressed by substituting the impermeant cation N-methyl-D-glucamine for intracellular K+. The remaining inward current had a V-shaped current-voltage relation reaching a peak value of 39 +/- 4 pA (mean +/- S.E. of mean) around -15 mV. It was identified as a Ca2+ current, because the peak amplitude was increased 1.6 times by increasing external [Ca2+] ([Ca2+]o) from 2.6 mM to 10 mM and it was blocked by Co2+ (5 mM) or nifedipine (5 microM) but not by TTX (20 microM). The activation time constant of the inward current at -10 mV was 1.28 +/- 0.08 ms. The relation between the degree of activation (estimated from the size of the tail currents) and membrane potential V followed the sigmoidal function f = 1/(1 + exp [(Vh-V)/k]) with half-maximal activation potential, Vh = 4 +/- 1 mV and slope factor, k = 14 +/- 1 mV (for [Ca2+]o 10 mM). The inward current inactivated only weakly during depolarizing pulses of 0.1-1 s duration. The delayed outward current (in experiments with 155 mM-internal [K+] ([K+]i)) had a linear voltage dependence at potentials above -20 mV; its amplitude at -10 mV was 210 +/- 30 pA. Tail currents related to the activation of the outward current had K+-dependent reversal potentials. The current was blocked by extracellularly applied tetraethylammonium (20 mM) and 4-aminopyridine (2 mM). It was not affected by glibenclamide (3 microM), tolbutamide (0.2 mM) and alterations of intracellular [Ca2+] (1 nM-1 microM). The activation time constant of the outward current at -10 mV was 21 +/- 3 ms. The voltage dependence of activation could be described by the sigmoidal function (see above) with Vh = 19 +/- 1 mV and k = 5.6 +/- 0.4 mV. The outward current inactivated during long (15 s) depolarizing pre-pulses (time constant at -10 mV: 2.6 +/- 0.6 s). 50% inactivation occurred at Vh = -36 +/- 2 mV, k was -4.1 +/- 0.2 mV. Inward and outward currents during depolarizing voltage pulses in beta-cells are similar to Ca2+ and delayed K+ currents in other cell types. These currents seem sufficient to generate the action potentials of the beta-cell.
Single-channel currents in ventricular cells of guinea-pig hearts were recorded by the patch-clamp technique. An inwardly rectifying K+-current was found in cell-attached membrane patches. Patches could be isolated from the cell exposing the cytoplasmic face of the membrane directly to the bathing solution. After isolation, the same current as in cell-attached patches was seen if the bath contained 4 mM ATP. Without ATP, this current disappeared and another channel of larger conductance and different kinetics was activated. Currents through the latter channel were also seen in cell-attached patches after poisoning the cells by DNP. It is suggested that the ATP-dependence of the observed membrane channels mediates the increase of potassium conductance after metabolic inhibition.
The resting conductance of cultured beta-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (greater than 20 G omega per cell or greater than 100 k omega X cm2), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K+ conductance. In cell-attached patches single K+-channels were observed in the absence of glucose. Addition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K+]0 = 145 mM, T = 21 degrees C) and kinetics (at -70 mV: tau open = 2.2 ms and 1.8 ms, tau closed = 0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the beta-cell by increasing the cytoplasmic concentration of ATP.
Inward rectifying potassium single-channel currents were studied in the membrane of guinea pig cardiac myocytes. In isolated inside-out patches two different channels were observed: a channel of 25 pS conductance ([K+]o = 147 mM, T = 21 degrees C), if the solution at the cytoplasmic face of the patch contained 4 mM ATP and a channel of 80 pS conductance without ATP. The 25-pS-channel was also regularly seen in cell-attached patches (Sakmann and Trube 1984a,b), but the 80-pS-channel appeared only after inhibiting cellular metabolism by DNP. The percentage of time which the 25-pS-channel spent in the open state was 3.3 times larger in isolated patches compared to cell-attached patches. However, both types of single channel currents disappeared several minutes after the isolation of the patches. In contrast to the 25-pS-channel, the 80-pS-channel, which is activated by the lack of ATP, carried measurable outward currents saturating at 1.5 pA (inward rectification). It is suggested that the 80-pS-channel mediates part of the increase in potassium current during metabolic inhibition. The openings of this channel appeared in bursts. The mean open time was 1.6 ms and the mean duration of the gaps within bursts 0.33 ms at -80 mV.
Single myocytes from adult rat hearts were prepared following the method of Powell and co-workers (9, 10, 11). Low resting potentials (Em) could be improved by three techniques. (i) Elevation of Cao to 7.2 mM which, however, mostly resulted in spontaneity and irreversible contracture. (ii) Pre-incubation in a "KB medium" (6). (iii) Use of suction pipettes instead of tapered microelectrodes for intracellular recordings (2). It is concluded that low Em measured previously (11) were due to membrane damage upon microelectrode impalement accompanied by insufficient healing of the membrane around the electrode insertion.
Single ventricular cells were enzymatically isolated from adult guinea-pig hearts (Isenberg & Klöckner, 1982). The patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981) was used to examine the conductance properties of an inward-rectifying K+ channel present in their sarcolemmal membrane. When the K+ concentration on the extracellular side of the patch was between 10.8 and 300 mM, inward current steps were observed at potentials more negative than the K+ equilibrium potential (EK). At more positive potentials no current steps were detectable, demonstrating the strong rectification of the channel. The zero-current potential extrapolated from the voltage dependence of the inward currents depends on the external K4 concentration [K+]o in a fashion expected for a predominantly K+-selective ion channel. It is shifted by 49 mV for a tenfold change in [K+]o. The conductance of the channel depends on the square root of [K+]o. In approximately symmetrical transmembrane K+ concentrations (145 mM-external K+), the single-channel conductance is 27 pS (at 19-23 degrees C). In normal Tyrode solution (5.4 mM-external K+) we calculate a single-channel conductance of 3.6 pS. The size of inward current steps at a fixed negative membrane potential V increases with [K+]o. The relation between step size and [K+]o shows saturation. Assuming a Michaelis-Menten scheme for binding of permeating K+ to the channel, an apparent binding constant of 210 mM is calculated for a membrane potential of -100 mV. For this potential the current at saturating [K+]o is estimated as 6.5 pA. The rectification of the single-channel conductance at membrane potentials positive to EK occurs within 1.5 ms of stepping the membrane potential from a potential of high conductance to one of low conductance. In addition to the main conductance state, the channel can adopt several substates of conductance. The main state could be the result of the simultaneous opening of four conducting subunits, each of which has a conductance of about 7 pS in 145 mM-external K+. The density of the inward-rectifying K+ channels in the ventricular sarcolemma is 0-10 channel/10 micron2 of surface membrane; the average of twenty-eight patches was 1 channel/1.8 micron2. It is concluded that the inward-rectifying K+ channels mediate the resting K+ conductance of ventricular heart muscle and the current termed IK1 in conventional voltage-clamp experiments.
Inward currents through single K+ channels in isolated ventricular heart cells of the guinea-pig were recorded using the patch-clamp technique (Hamill, Marty, Neher, Sakmann & Sigworth, 1981). The voltage-dependent gating properties of the channels were examined in the potential range between 0 and -120 mV with 145 mM-KCl on the extracellular side of the membrane patch, i.e. with approximately symmetrical transmembrane K+ concentrations. When voltage pulses from 0 mV to negative test potentials were applied to patches containing several channels, more channels were open at the beginning of the pulses than in the steady state. Averages of many current responses showed inactivation of the mean current in response to the hyperpolarizing voltage pulses. The inactivation was stronger and faster at larger hyperpolarization. The lifetimes of the open and closed states of the channel and the probability of the open state p were estimated from records of the elementary currents at various constant potentials. As indicated by the inactivation of the averaged currents, the value of p was smaller at more negative potentials, approximately 0.15 at -50 mV and 0.02 at -110 mV. This caused a negative slope in the current-voltage relation of the time-averaged current at potentials more negative than -50 mV. The channel openings were grouped in complex bursts. At least three exponentials were needed to fit the frequency histogram of the lifetimes of all closed states (time constants at -50 mV: 1.1 ms, 16 ms and 3.2 s). The lifetimes of the individual openings were exponentially distributed (time constant: 70 ms). The kinetics of the channel were interpreted by two different models involving three states of a channel (closed-closed-open or closed-open-closed). The rate constants and their voltage dependence were estimated for both models. Both models describe the data equally well; the reason for this ambiguity is discussed. The channels are blocked by Cs+ or Ba2+. Cs+ (0.1 mM) caused frequent and short interruptions of the individual channel openings. Ba2+ (0.5 mM) also shortened the openings and in addition decreased the number of openings per burst. The results suggest that the inward-rectifying current IK1 in heart ventricular cells is partially inactivated by hyperpolarization. The inactivation could account for part of the time-dependent decrease in the whole-cell current previously ascribed to depletion of K+.