Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets

Abstract

1. The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin-secreting B-cells and 65 % non-B-cells (A- and D-cells). 2. Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mM glucose and had the electrophysiological characteristics of isolated B-cells maintained in tissue culture. 3. The Ca2+ current recorded from B-cells in situ was 80 % larger than that of isolated B-cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4. Voltage-dependent K+ currents were observed during depolarisations to membrane potentials above -20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between -90 and -30 mV. 5. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP-regulated K+ (KATP) channels, amounted to approximately 4 nS. Glucose produced a concentration-dependent reduction of KATP channel conductance with half-maximal inhibition observed with 5 mM glucose. 6. Combining voltage- and current-clamp recording allowed the estimation of the gap junction conductance between different B-cells. These experiments indicated that the input conductance of the B-cell at stimulatory glucose concentrations ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.

Figure 1. Experimental design

A, confocal image of superficial cells in a mouse pancreatic islet. The cells have been stained with anti-insulin (red), anti-glucagon (green) and anti-somatostatin (blue) antibodies. Note that all islet cell types are accessible on the surface. B, schematic diagram of the experimental system. The islet is held in place by gentle suction applied to the interior of a wide-bore holding pipette. Patch-clamp experiments are then performed using standard patch electrodes placed on the opposite pole of the islet.

Figure 2. Two types of islet cell can be identified from electrophysiological properties

A, the majority of superficial cells had a stable and negative membrane potential even in the presence of 10 mm glucose. B, voltage-clamp depolarisation of these cells (5 ms to voltages between −40 and 0 mV) elicited a spiky inward current and a rapidly activating outward current. The glucose concentration was 10 mm. C, electrical activity recorded from a fraction of the cells in the presence of 10 mm glucose. D, voltage-clamp depolarisation of cells of this type (same protocol as in B) elicited smaller currents with slower activation kinetics. The voltage-clamp records were obtained after lowering the glucose concentration to 5 mm to suppress regenerative electrical activity.

Figure 3. Characterisation of the Ca²⁺ current

A, oscillations in holding current due to bursts of action potentials in neighbouring B-cells recorded in the presence of 10 mm glucose. B, Ca²⁺ currents elicited by voltage-clamp depolarisations to membrane potentials between −40 and −10 mV from a holding potential of −70 mV. C, peak Ca²⁺ current amplitudes recorded during depolarisations to voltages between −40 and +40 mV from B-cells in intact islets (▴) and isolated B-cells maintained in tissue culture for 2–4 days (▪). Data are mean values ±s.e.m. of 15 (▴) and 8 (▪) experiments. * P < 0.05 vs. current amplitude recorded at the same voltage in B-cells of intact islets.

Figure 6. Delayed outward K⁺ currents recorded from B-cells in situ

A, K⁺ currents evoked by depolarisation (100 ms applied at a frequency of 0.25 Hz) to membrane potentials between −50 and +60 mV from a holding potential of −70 mV. B, I–V relationship of peak outward current. Data are mean values ±s.e.m. of the peak K⁺ current recorded under control conditions (▪) and after removal of extracellular Ca²⁺ (▴), obtained from 7 and 4 experiments, respectively. C, outward K⁺ current in a B-cell was independent of variations of the holding potential between −90 and −30 mV. Prior to the test pulse to −10 mV (200 ms), a 200 ms conditioning pulse to −90 or −30 mV was applied. Between the conditioning pulse and the test pulse there was an interval of 20 ms during which the cell was held at −70 mV. Note that the current elicited during the test pulse was sustained and that its amplitude was the same irrespective of whether the conditioning pulse was to −90 or −30 mV. D, activation of the K⁺ current can be described by n⁴ kinetics. The fitted function (smooth curve) has been superimposed on the current evoked by a voltage-clamp depolarisation to 0 mV. E, relationship between time constant of activation (τ_n) and membrane potential (V). Data are mean values ±s.e.m. from 4 experiments.

Figure 9. Electrical coupling between different B-cells

A, glucose-induced electrical activity recorded with 10 mm glucose. Note that the membrane potential undergoes regular oscillations. The maximally negative interburst potential (V_i) and the plateau potential (V_p) are indicated by dotted lines. B, variations of the holding current recorded from the same cell. The variations of the holding current reflect bursts of action potentials in a neighbouring cell. The current levels corresponding to the repolarisation of the burst of action potentials (I_i) and the plateau (I_p) are indicated by dotted lines. C, stable holding current recorded at −70 mV in a non-B-cell in an intact islet exposed to 10 mm glucose.

Figure 4. Voltage dependence of Ca²⁺ current activation and inactivation

A, Ca²⁺ currents evoked by membrane depolarisations to −30 mV (trace with superimposed blue curve) and −10 mV (trace with superimposed red curve). The blue and red curves were derived assuming m² kinetics. The values of the time constant of activation (τ_m) were 0.56 ms at −30 mV and 0.33 ms at −10 mV. The value in parentheses for the scale bar refers to the trace with superimposed blue curve. B, τ_m measured at different membrane potentials (V). C, inactivation of the Ca²⁺ currents elicited by depolarisations to −30 mV (trace with superimposed blue curve) and −10 mV (trace with superimposed red curve). To facilitate comparison of the inactivation, the current amplitudes have been normalised. The value in parentheses for the scale bar refers to the trace with superimposed blue curve. The blue and red curves represent an exponential function fitted to the current using time constants of inactivation (τ_h) of 19.5 and 12.3 ms at −30 and −10 mV, respectively. D, τ_h displayed against voltage during the depolarisation (V). Note the U-shaped relationship with a minimum at 0 mV. Data are mean values ±s.e.m. from 6 experiments.

Figure 5. Steady-state inactivation of the Na⁺ current in pancreatic B-cells

A, the B-cell was subjected to a conditioning pulse (100 ms) to voltages between −180 and −50 mV prior to the 5 ms test pulse to 0 mV. Between the conditioning pulse and the test pulse the cell was held at −70 mV for 1 ms. B, relationship between conditioning voltage (V) and relative current amplitude (h_∞=I/I_max). The current response elicited by depolarisation to 0 mV following a conditioning pulse to −180 mV was taken as unity. Mean values ±s.e.m. from 4 experiments. The curve was obtained by fitting the Boltzmann equation (eqn (3)) to the data points. C, currents elicited by a 5 ms depolarisation to 0 mV following a 100 ms conditioning pulse to −150 mV under control conditions and after addition of tetrodotoxin (TTX, 0.1 μg ml⁻¹).

Figure 7. Whole-cell K_ATP conductance in B-cells in situ

A, membrane potential recordings from a B-cell in an intact islet at 10, 5, 0 and 20 mm glucose. Note that varying the glucose concentration (as indicated above the membrane potential trace) influenced the B-cell membrane potential and electrical activity. B, recordings of resting conductance from the same cell. The conductance was monitored by application of 500 ms voltage pulses to −80 and −60 mV from the holding potential of −70 mV (bottom). The associated current responses (top) were recorded as indicated by letters a-d in A.

Figure 8. Modulation of whole-cell K_ATP conductance

A, relationship between glucose concentration and measured whole-cell conductance calculated from current responses elicited by ±10 mV voltage excursions in Fig. 7B. Values are means ±s.e.m. of the indicated (within parentheses) number of experiments. The rightmost data point indicates the input resistance measured in the simultaneous presence of > 10 mm glucose and 0.1 mm tolbutamide. Note that the whole-cell conductance does not fall below ≈1 nS. B, block of whole-cell K_ATP conductance by Co²⁺ (5 mm) applied in the absence of glucose.