Single K ATP channel opening in response to action potential firing in mouse dentate granule neurons

Abstract

ATP-sensitive potassium channels (K(ATP) channels) are important sensors of cellular metabolic state that link metabolism and excitability in neuroendocrine cells, but their role in nonglucosensing central neurons is less well understood. To examine a possible role for K(ATP) channels in modulating excitability in hippocampal circuits, we recorded the activity of single K(ATP) channels in cell-attached patches of granule cells in the mouse dentate gyrus during bursts of action potentials generated by antidromic stimulation of the mossy fibers. Ensemble averages of the open probability (p(open)) of single K(ATP) channels over repeated trials of stimulated spike activity showed a transient increase in p(open) in response to action potential firing. Channel currents were identified as K(ATP) channels through blockade with glibenclamide and by comparison with recordings from Kir6.2 knock-out mice. The transient elevation in K(ATP) p(open) may arise from submembrane ATP depletion by the Na(+)-K(+) ATPase, as the pump blocker strophanthidin reduced the magnitude of the elevation. Both the steady-state and stimulus-elevated p(open) of the recorded channels were higher in the presence of the ketone body R-β-hydroxybutyrate, consistent with earlier findings that ketone bodies can affect K(ATP) activity. Using perforated-patch recording, we also found that K(ATP) channels contribute to the slow afterhyperpolarization following an evoked burst of action potentials. We propose that activity-dependent opening of K(ATP) channels may help granule cells act as a seizure gate in the hippocampus and that ketone-body-mediated augmentation of the activity-dependent opening could in part explain the effect of the ketogenic diet in reducing epileptic seizures.

Figure 1.

Single K_ATP channels in unstimulated dentate gyrus granule cells. A, Single-channel currents recorded from a DGN cell-attached patch at various apparent membrane voltages (V_m). V_m is based on the applied pipette potential and an estimated membrane potential of V_rest = −80 mV (V_m = V_rest − V_command). These current traces were selected to show bursts of channel openings and are thus not representative of typical open probability. Arrows indicate the zero current level. B, Plot of single-channel current (mean ± SEM) against apparent membrane voltage for eight patches in which a response to NN414 was observed. Linear fit corresponds to a single-channel conductance of 25 pS. C, Continuous records of single-channel currents from a cell-attached patch recorded with no applied voltage in the absence (left) or presence (right) of the K_ATP opener NN414 (5 μm). Single-channel openings in this and all subsequent figures are represented by upward deflections of the current trace.

Figure 2.

Stimulated spike activity increases channel p_open. A, Five examples of cell-attached records from a DGN with five stimuli delivered at 20 Hz, showing channel openings just for the period during and after action potential stimulation. The red shading indicates the period of stimulus artifact and action potential. B, Top, Histogram of channel activity (ensemble average open probability) over all traces from a representative five-stimulus experiment. The blue line shows a 320 ms moving average value. A prominent elevation in channel p_open can be observed during and after the stimulus period (indicated by the stimulus icon beneath the time axis). Bottom, A second histogram for the same patch for traces during which no stimuli were applied. Channel activity for all experiments is reported as N·p_open to acknowledge the possibility that more than one channel is active in a given patch. C, Summary for all trials where cells were stimulated to fire five spikes at 20 Hz versus all trials where no stimulus was delivered. The peak stimulus-period p_opens for five stimuli are significantly different from those for the equivalent time period in trials where no spikes were stimulated to fire (p < 0.01; Mann–Whitney U test). Gray squares, Mean N·p_open for all five-stimulus trials (n = 35); black circles, mean N·p_open for all trials conducted without stimulation (n = 13). Data points are presented as mean channel p_open ± SEM.

Figure 3.

Both the firing-induced and basal channel openings are mainly K_ATP channels. A, Average effect of glibenclamide wash-in on channel open probability after recording of baseline activity in ACSF. The K_ATP blocking drug glibenclamide (100 nm) attenuates the spike-elevated opening of channels compared with paired five-stimulus trials recorded in ACSF alone. Mean ± SEM; n = 6. B, Estimate of the fractional blockade by glibenclamide. Δp_open (final − initial) is plotted against the initial p_open (in ACSF), with each symbol representing a separate experiment with glibenclamide (black squares) or DMSO (gray circles) wash-in. Linear fits through the origin, for each dataset, represent a fixed fractional reduction in current produced by the blocker. The two graphs use different time samples: full-trace effects (bottom) simply used the full-trace average p_open, while peak-weighted effects (top) were weighted with a function that was constant during the stimulus interval and decayed with a time constant of 0.3 s thereafter. Dotted lines mark the 95% confidence limits for the linear fits. For glibenclamide, the fitted slopes were (peak) −0.81 ± 0.02 and (full) −0.87 ± 0.02, consistent with ∼80–90% inhibition. For DMSO, the fitted slopes were (peak) −0.33 ± 0.06 and (full) −0.31 ± 0.08 (n = 7). C, Channel activity is reduced in DGNs from Kir6.2 knock-out mice. Summary for experiments with five-stimuli in wild-type animals (gray symbols, mean ± SEM, n = 35), and Kir6.2^−/− (black symbols, mean ± SEM, n = 11). Most experiments were performed and analyzed blind to genotype.

Figure 4.

The Na⁺-K⁺ pump blocker strophanthidin blocks both the firing-induced and basal channel opening. A, B, Channel activity was recorded first in ACSF, then in strophanthidin; histograms are shown for a representative experiment (A) and for the ensemble average (B, n = 13). Experiments were performed and analyzed with the experimenters blind to the identity of the washed-in solution. C, The effects of strophanthidin (or 0.1% DMSO) are summarized (as in Fig. 3B). For strophanthidin (Stroph), the fitted slopes were (peak) −0.71 ± 0.15 and (full) −0.75 ± 0.14, consistent with ∼70–75% inhibition. For DMSO, the fitted slopes were (peak) 0.96 ± 0.06 and (full) 1.35 ± 0.31 (n = 11).

Figure 5.

Channel open probability is elevated with the ketone body R-β-hydroxybutyrate. A, Top, Set of five current traces recorded in ACSF supplemented with 2 mm R-βHB. Bottom, Set of five current traces recorded in ACSF alone. Left, First second of recording period. Right, Last second. B, Summary of all experiments in R-βHB (black diamonds; n = 8) versus all in ACSF (gray squares; n = 35). Note that the y-axis is on a larger scale than in previous sets of summary data.

Figure 6.

A glibenclamide-sensitive, long, slow AHP following a burst of evoked action potentials. Representative perforated patch voltage recordings from DGNs, with a burst of five action potentials evoked by five brief current pulses just before the indicated t = 0. A total of eight traces are overlaid, with a single trace shown in black. In recordings with glibenclamide, AHPs were smaller (ΔV = −5.7 ± 0.7 mV vs −8.8 ± 1.1 mV for control; n = 5, p < 0.05) and spontaneous action potential firing resumed after a shorter latency (4.1 ± 0.6 s vs 6.5 ± 0.7 s for control; n = 5, p < 0.05).