Slow oscillations of KATP conductance in mouse pancreatic islets provide support for electrical bursting driven by metabolic oscillations

Abstract

We used the patch clamp technique in situ to test the hypothesis that slow oscillations in metabolism mediate slow electrical oscillations in mouse pancreatic islets by causing oscillations in KATP channel activity. Total conductance was measured over the course of slow bursting oscillations in surface β-cells of islets exposed to 11.1 mM glucose by either switching from current clamp to voltage clamp at different phases of the bursting cycle or by clamping the cells to -60 mV and running two-second voltage ramps from -120 to -50 mV every 20 s. The membrane conductance, calculated from the slopes of the ramp current-voltage curves, oscillated and was larger during the silent phase than during the active phase of the burst. The ramp conductance was sensitive to diazoxide, and the oscillatory component was reduced by sulfonylureas or by lowering extracellular glucose to 2.8 mM, suggesting that the oscillatory total conductance is due to oscillatory KATP channel conductance. We demonstrate that these results are consistent with the Dual Oscillator model, in which glycolytic oscillations drive slow electrical bursting, but not with other models in which metabolic oscillations are secondary to calcium oscillations. The simulations also confirm that oscillations in membrane conductance can be well estimated from measurements of slope conductance and distinguished from gap junction conductance. Furthermore, the oscillatory conductance was blocked by tolbutamide in isolated β-cells. The data, combined with insights from mathematical models, support a mechanism of slow (∼5 min) bursting driven by oscillations in metabolism, rather than by oscillations in the intracellular free calcium concentration.

Keywords: ATP-sensitive potassium channels; insulin; islets; oscillations.

Fig. 1.

Recordings of peripheral islet cells in current clamp and voltage clamp and slope conductances measured under different conditions. A: patch-clamped cells in current-clamp mode were identified as β-cells by their characteristic bursting pattern. B: when the recording mode was switched to voltage clamp, inverted current bursts were seen, reflecting current from neighboring cells entering via gap junctions. The ratio of voltage differential to current differential yields an estimate of 1 nS coupling for the islet shown; the mean for 15 islets was 0.96 ± 0.01 nS. C: interrupted voltage ramp (IVR) protocol was used to generate current-voltage (I-V) curves in which the potential of the clamped cell was ramped from −100 to −60 mV. Invading action currents were taken as an indication that the rest of the islet was in its active phase (red); silent-phase trace indicated in black. Slope was estimated by linear curve fitting. D: in the presence of 11.1 mM glucose, conductance in the active phase was reduced compared with the silent phase (active-phase) conductance. E: representative I-V recording traces during voltage ramps. Voltage ramps were applied to islets exposed to saline containing 2.8 mM glucose or in the presence of 100 μM diazoxide in saline containing 11.1 mM glucose. Different slopes indicate that conductance is different under these conditions. F: islets exposed to 2.8 mM glucose have higher conductance than silent or active phases of islets in 11.1 mM glucose. G: conductance in the presence of diazoxide is higher than in silent or active phases in 11.1 mM glucose. *Significant difference from active phase; #significant difference from silent phase.

Fig. 2.

Islet conductance oscillates in 11.1 mM glucose. A: representative recording trace showing oscillatory membrane potential of an islet exposed to 11.1 mM glucose. B: oscillatory conductance values obtained using voltage ramps from the same β-cell held at −60 mV between ramps. The oscillation period was similar for voltage and conductance. Recordings were sequential, with conductance measured after establishing the presence of membrane potential oscillations. C: silent- (○) and active- (●) phase durations for all 11 islets tested.

Fig. 3.

Conductance oscillations are abolished by tolbutamide or 2.8 mM glucose. A and B: in the presence of K_ATP blocker tolbutamide (200 μM), oscillations in membrane potential and conductance that were previously observed in a solution containing 11.1 mM glucose were subsequently abolished. Membrane potential after sulfonylurea exposure remained continuously depolarized, with fast spiking superimposed, and conductance declined to a lower level without oscillations under corresponding conditions. C: when exposed to 2.8 mM glucose, islets were hyperpolarized and did not burst (top). D: in low glucose, conductance was much larger than in 11.1 mM glucose and exhibited no oscillations.

Fig. 4.

Conductance oscillations are abolished in recordings using glyburide-filled electrodes. A and B: membrane potential and slope conductance oscillations recorded using the holding potential ramp (HPR) protocol in 11.1 mM glucose with a standard electrode. C and D: membrane potential and slope conductance recorded in the same islet and glucose concentration as shown in A but using a glyburide-filled electrode (200 μM); electrical oscillations were preserved, but conductance oscillations were abolished in 5 of 6 islets tested. Membrane potential oscillations that occur in neighboring cells are presumed to be transmitted to the voltage-clamped cell through gap junctional coupling. The hypothesis that conductance oscillation amplitude was lower in glyburide than in control was significant by unpaired t-test with P = 0.03 (0.118 ± 0.04 vs. 0.051 ± 0.06 nS).

Fig. 5.

K_ATP conductance (gK_ATP) oscillations present in isolated β-cells were abolished by tolbutamide. Ramp voltage commands elicited oscillatory slope conductances in 3 of 4 isolated β-cells, detected using the Cluster algorithm (*, detected peaks). Tolbutamide (100 μM) blocked conductance oscillations and significantly decreased mean conductance (P < 0.05 with paired t-test). Data plotted are 2 point moving averages of raw conductance values.

Fig. 6.

Membrane potential in unclamped and clamped portions of the model islet and simulation of membrane current in the clamped cell. Simulation of an experiment where 2-s I-V ramps were applied once every 20 s. A: membrane potential in the unclamped supercell. B: membrane potential in the voltage clamped cell. C: current in the unclamped cell. Parameters for ionic currents and metabolism are the same as in Fig. 7, B and D (slow glycolytic bursting case), with the addition of diffusive coupling on V_m, FBP, and G6P. Coupling parameters in Eq. 1 in materials and methods were g_c = 10 pS and P = 0.01 for an effective coupling strength, as seen by the clamped cell equal to ∼500 pS. This agrees well with the empirical value estimated from the model using voltage and current differentials in A and C, respectively. Corresponding effective coupling strengths for FBP and G6P were 1.0 and 0.5 ms⁻¹, respectively.

Fig. 7.

Extraction of gK_ATP in the model islet. Values are taken from the holding potential ramp simulated in Fig. 4. A: I–V curves in the clamped cell during I–V ramps simulated in Fig. 5. Curves with oscillations were taken when the unclamped portion of the islet was in its active phase of bursting. B: linear curve fits to 2 representative I–V curves, 1 from a silent phase and 1 from an active phase (see boxed regions in Fig. 5C). C: actual gK_ATP in the clamped cell during each ramp (solid line); total slope conductance from linear fits as in B (○); and total conductance with coupling conductance removed (triangles). Filled symbols, active phase; open symbols, silent phase.

Fig. 8.

Symmetrical gK_ATP in Keizer-Magnus model vs. asymmetrical gK_ATP when glycolysis oscillates. A: Membrane potential (A) and gK_ATP (C) for the Dual Oscillator model (DOM) in Keizer-Magnus mode (i.e., no glycolytic oscillations). Membrane potential (B) and gK_ATP (D) for the DOM in glycolytic oscillation mode. See text for interpretation and details.