Electrophysiological characterization of rat β -cells

Cell identification

Functional identification of pancreatic ^β-cells by electrophysiological means is com-monly performed by elevating the external glucose concentration. Spiking electrical activity elicited in cells by this stimulation regime is generally regarded as a ^β -cell-exclusive hallmark (Dean and Matthews, 1968). Cells that have been functionally identified in this way exhibit a sizable inward Na⁺ current (Plant,1988). In contrast to cells not showing electrical activity in high glucose, this current is already fully inac-tivated at the normalβ-cell resting membrane potential of−70 to−90 mV (Ashcroft and Rorsman,1989;Göpel et al.,1999) in mouse but also in ratβ-cells (Plant,1988;

Hiriart and Matteson,1988). Accordingly, the steady-state inactivation properties of INa⁺ have repeatedly been used as a quick way to identify^β-cells in primary cell cultures (Yang et al.,2004;Speier et al.,2005) as well as in more intact preparations (Göpel et al.,1999;Speier and Rupnik,2003).

Using a standard protocol of conditioning prepulses ranging from very negative to more positive membrane potentials (⁻159 to⁻9 mV) followed by a test pulse to

−9 mV the inactivation properties of rat islet cells in tissue slice preparation were assessed (see fig. 3.3). The average leak corrected peak inward Na⁺ current in these cells was 1102±95.3 pA, (n= 31). Of 78 cells tested in total, only 8 cells showed half-maximal inactivation at conditioning potentials (V1/2) more positive than−60 mV.

The remaining cells had an average V1/2 of−98±1.4 mV. This value lies between the

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Figure 3.3: Identification of rat^β-cells based on the steady-state inactivation charac-teristics of voltage-gated Na⁺ currents. (A) The cells were subjected to conditioning prepulses (50 ms) ranging from⁻159 to ⁻9 mV to achieve steady state inactivation.

After a delay of 3 ms at a holding potential of⁻89 mV a test pulse to⁻9 mV of 5 ms duration elicited inward Na⁺ currents (left panels). If plotted against the prepulse potential and fitted with the Boltzmann equation (eqn. 2.5), normalized Na⁺ currents (I/Imax) of 31 cells (intracellular solution: IS-2) show half-maximal inactivation (V1/2) at a potential of−97±1.3 mV (upper right panel, dotted lines). The histogram of V1/2

values of 78 cells (including cells measured without leak correction and cells patched with IS-1) shows normal distribution for V1/2 centered atx¯ = −98 mV (eqn. 2.6). (B) β- and non-β-cells identified by V1/2 being more negative (β) or more positive (non-β) than the mean V1/2 plus double standard deviation (i.e.⁻75 mV).

frequently reported⁻110 mV for mouse^β-cells (Plant,1988;Göpel et al.,1999) and the⁻90 mV recently demonstrated for isolated rat^β-cells (Lou et al.,2003). All cells showing a V1/2 more positive than−75 mV—i.e. double standard deviation (11.5 mV) of the average V1/2—were considered to be non-β-cells. Upon establishment of the whole-cell configuration, Wistar rat β-cells identified accordingly did not show any electrical activity in the absence of stimulatory glucose concentrations. Non-β-cells, however, occasionally fired single action potentials in 3 mM external glucose (see fig.

3.3).

A

15 mM glucose + 100 µM tolbutamide 3 mM glucose + 100 µM tolbutamide

Figure 3.4: Electrical activity in rat β-cells. (A) Spiking activity of three different β-cells preincubated at different glucose concentrations and monitored immediately after establishment of the whole-cell configuration (*). (B) Electrical activity of two cells after prolonged periods of whole-cell dialysis. (i) Bursting electrical activity after a recording duration of 6 min and 1.5 min perifusion with 15 mM glucose. (ii, upper panel) Bursting measured after 25 min of whole-cell dialysis and 10 min with 100µM tolbutamide. (ii, lower panel) High frequent bursting in the same cell 35 min after the beginning of the recording and 8 min after the glucose concentration was increased to 15 mM in the continued presence of 100 µM tolbutamide.

Electrical activity in rat β-cells

As stated above, Wistar ratβ-cells were electrically silent at an external glucose concentra-tion of 3 mM. The resting Vmof^β-cells is primarily dependent on the K⁺conductance of the KATP channels (see section1.3.1). Therefore, both the intracellular ATP concen-tration as well as the K⁺ equilibrium potential (EK⁺) govern the resting Vmof these cells.

Because during prolonged whole-cell recording the intracellular ATP- as well as K⁺ -concentrations are determined by the composition of the pipette solution, undisturbed measurements of Vm can only be obtained right in the beginning of a current-clamp

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recording before whole-cell dialysis is complete (Pusch and Neher,1988). Measured accordingly, ratβ-cells showed a resting Vmof−77±2.5 mV (n= 80). This value is in reasonable agreement with the previously reported resting Vmof −50 mV measured with intracellular microelectrodes in isolated rat islet β-cells (Antunes et al.,2000), considering that different external K⁺-concentrations have been used (5 mM [K⁺]ovs.

current study: 2.5 mM [K⁺]o). This would account for a 18 mV more positive EK⁺ in the microelectrode recordings if similar cytosolic K⁺concentrations are assumed.

Further microelectrode measurements have shown that rat ^β-cells in intact islets depolarize upon glucose stimulation and exhibit single Ca²⁺-dependent action potentials (Antunes et al.,2000). In the current study, rat ^β-cells in tissue slices rarely showed spiking activity after prolonged periods of whole-cell dialysis. However, if preincubated in glucose concentrations higher than 3 mM (5–20 mM glucose) for∼10 min, 14 out of 20 β-cells fired action potentials at the start of a recording (see fig.3.4 A). This activity usually presented itself as single spikes of an amplitude of∼20 mV. In contrast to previous reports, however, short bursts of ∼2–20 action potentials could also be observed. Usually, the spiking activity ceased in the first minute of whole-cell recording (see fig.3.4A, lower trace). Nevertheless, in some cases^β-cells showed electrical activity even after prolonged recording durations (see fig.3.4B). In the cell shown in fig.3.4Bii, closing of KATP channels by extracellular application of the KATP channel antagonist tolbutamide (100µM) led to clear oscillatory bursting electrical activity. An increase in the extracellular glucose concentration further depolarized the cell and decreased the inter-burst duration and the number of spikes per burst.

Electrical coupling

Action potential firing and oscillatory changes in Vm observed after prolonged cell dialysis usually coincided with fluctuations of the holding current. After switching from the current- to the voltage-clamp mode during periods of oscillatory activity, Im

frequently showed oscillations as well. These Im changes were in phase with the Vm

oscillations but were of opposing sign (see fig.3.5). The activity patterns shown in the β-cells of fig.3.4B are therefore most likely secondary to electrical activity generated in one ore more neighboring^β-cells. A depolarization in an adjacent cell would result

Vm [mV]

-70 -60 -50

I [pA]

-30 -20 -10

1 s voltage-clamp (10 mM glucose) current-clamp (10 mM glucose)

1 s

A B

Figure 3.5:Electrical coupling between^β-cells. (A) Whole-cell current-clamp recording of glucose-induced (10 mM) Vmoscillations of a^β-cell. Immediately afterwards, record-ing was continued in the voltage-clamp mode (B). Note the oscillations of identical period but opposing sign in the holding current.

in an injection of current into the patched^β-cell via gap junctions. These changes in Imand the corresponding Vmcan be used to estimate the gap junctional conductance (Mears et al.,1995;Göpel et al.,1999). The peak-to-trough amplitudes of Vm- and Im-changes (∆Vm,∆Im) in the cell shown in fig. 3.5range from ∼5–10 mV and∼5–

10 pA, respectively. According to Ohm’s law (Gj = ^∆Im/∆Vm) this corresponds to a gap junctional conductance (Gj) of∼1 nS.

To further quantify electrical coupling of rat ^β-cells in our preparation we took advantage of a feature innate to more intact islet preparations. Upon inhibition of KATP

channels by sulphonylureas like tolbutamide, single cultured ^β-cells show a very small remaining membrane conductance (Speier et al., 2005). Contrariwise, in^β-cells of isolated mouse islets and even more in tissue slice preparation a high residual conduc-tance of 1–1.7 nS remains even if KATP channels are fully blocked. This conductance is commonly attributed to gap junctional coupling (Göpel et al.,1999;Speier et al.,2005).

We measured Im in rat β-cells in response to ramp depolarizations (−110 to 60 mV, 100 ms duration: 1.7 mV s^-1, see fig. 3.6). At potentials of up to −50 mV, Im was a linear function of Vm. Then voltage-dependent outward currents activated, most likely attributable to the activation of voltage-gated K⁺channels (Gillis et al.,1989;Ashcroft and Rorsman,1989). Therefore, the resting membrane conductance was determined from the slope of the IV curves between⁻100 and⁻60 mV. The cell shown in fig.3.6A had an initial conductance of 3.6 nS immediately after the whole-cell configuration was

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Figure 3.6: Whole cell conductance inβ-cells before and after KATPchannel inhibition.

(A) Current-voltage (IV) relations right after recording begin (solid trace) and after inhibition of KATPchannels by extracellular application of 100µM tolbutamide (dashed trace). Current was measured in response to ramp depolarizations from−110 to 60 mV of 100 ms duration (1.7 mV s^-1). No leak subtraction was performed since the KATP

as well as gap junction current amplitudes depend effectively linearly on the applied voltage and accordingly would be subtracted as well. (B) Comparison of initial (open bar) and residual conductance after KATP channel inhibition (gray bar) as determined from the slopes of IV curves between ⁻100 and ⁻60 mV (dotted lines in A). ***, p₆0.001, unpaired t-test. Here and in the following figures error bars represent... and numbers on bars indicate the number of cells tested.

established. After application of tolbutamide 0.9 nS residual conductance remained. As expected, membrane conductance was prominently decreased by tolbutamide applica-tion in all cells tested (see fig.3.6). The residual conductance of 0.6^±0.07 nS (n= 28), however, was about 50% lower than the values observed in mouse^β-cells (Göpel et al., 1999;Speier et al.,2005), suggesting that rat^β-cells are significantly less coupled than mice.