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. 2002 Jul 15;542(Pt 2):431-44.
doi: 10.1113/jphysiol.2002.017590.

Characterization of four types of background potassium channels in rat cerebellar granule neurons

Jaehee Han  1 , Jeffrey Truell, Carmen Gnatenco, Donghee Kim
Affiliations

Characterization of four types of background potassium channels in rat cerebellar granule neurons

Jaehee Han et al. J Physiol. .

Abstract

Cerebellar granule neurons express a standing outward (background) K+ current (I(K,SO)) that regulates the resting membrane potential and cell excitability. As several tandem-pore (2P) K+ channel mRNAs are highly expressed in cerebellar granule cells, we studied whether, and which, 2P K+ channels contribute to I(K,SO). I(K,SO) was highly sensitive to changes in extracellular pH and was partially inhibited by acetylcholine, as reported previously. In cell-attached patches from cultured cerebellar granule neurons, four types of K+ channels were found to be active when membrane potential was held at -50 mV or +50 mV in symmetrical 140 mM KCl. Based on single-channel conductances, gating kinetics and modulation by pharmacological agents and pH, three K+ channels could be considered as functional correlates of TASK-1, TASK-3 and TREK-2, which are members of the 2P K+ channel family. The fourth K+ channel (Type 4) has not been described previously and its molecular correlate is not yet known. Based on the measurement of channel current densities, the Type 2 (TASK-3) and the Type 4 K+ channels were determined to be the major sources of I(K,SO) in cultured cerebellar granule neurons. The Type 1 (TASK-1) and Type 3 (TREK-2) activities were relatively low throughout cell growth in culture (1-10 days). Similar to TASK-1 and TASK-3, the Type 4 K+ channel was highly sensitive to changes in extracellular pH, showing a 78 % inhibition by changing the extracellular pH from 7.3 to 6.3. The results of this study show that three 2P K+ channels and an additional pH-sensing K+ channel (Type 4) comprise the I(K,SO) in cultured cerebellar granule neurons. Our results also show that the high sensitivity of I(K,SO) to extracellular pH comes from the high sensitivity of Type 2 (TASK-3) and Type 4 K+ channels.

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Figures

Figure 1
Figure 1
The standing outward K+ current in cerebellar granule neurons A, whole-cell current was activated by a step pulse protocol as shown. After holding the cell for 4 s at −20 mV, membrane potential was changed to −80 mV and then back to −20 mV. ACh (10 μm) was applied to the extracellular perfusion solution. GTP (100 μm) and ATP (1 mm) were present in the pipette solution for all whole-cell experiments. B, whole-cell current was activated by a 1 s duration ramp pulse protocol (-100 mV to −20 mV). ACh (10 μm) was applied to the extracellular perfusion solution. C, whole-cell currents were recorded at different extracellular pH values. The ramp protocol was the same as that in B. D, the bar graph shows the effect of extracellular pH on the whole-cell current measured at −20 mV. Each bar is the mean ± s.d. of five determinations. *Significant difference from the corresponding control value observed at pH 7.3 (P < 0.05).
Figure 2
Figure 2
Single-channel currents recorded from cell-attached patches A, four types of channels with distinct opening kinetics are shown (Type 1-Type 4). Dotted lines indicate the open states. B, cell-attached patches show a mixture of channels, as indicated. The cell membrane potential was held at −50 mV. Pipette and bath solutions contained 140 mm KCl.
Figure 3
Figure 3
Changes K+ channel activity during culture A, whole-cell currents were activated by a ramp pulse protocol (-100 to 0 mV) similar to that in Fig. 1B, and the peak currents at 0 mV determined from cells grown in culture for day 1 to day 10. B, approximately 40 patches per day were analysed for four types of K+ channels during cell growth in culture. The channel activity was determined from each cell-attached patch and averaged to give channel activity per patch. Each point is the mean ± s.d. of 35–42 determinations. Cell membrane potential was held at −50 mV. C, same experiment as B except that the membrane potential was at +50 mV to measure outward currents.
Figure 4
Figure 4
Electrophysiological properties of the Type 1 K+ channel in cerebellar granule neurons A, openings of the Type 1 K+ channel in neurons and TASK-1 expressed on COS-7 cells are shown at three different membrane potentials in cell-attached patches. The pipette solution contained 140 mm KCl. B, amplitude histograms of Type 1 channel openings in a CG neuron and TASK-1 expressed in a COS-7 cell show single peaks of 1.2 pA (-60 mV). C, duration histograms of Type 1 K+ channel openings in a CG neuron and TASK-1 expressed in a COS-7 cell are shown. D, current amplitudes were determined at each membrane potential and used to plot the current-voltage relationships. Each point is the mean ± s.d. of three determinations. E, outside-out patches from CG neurons were formed and the effect of pHo determined at three pH values. Tracings are inward currents observed at −50 mV in symmetrical 140 mm KCl. Same experiment was done using COS-7 cells expressing TASK-1 (tracings not shown). Channel activity was determined at each pH value and plotted (n = 3). *Significant difference from the corresponding control value observed at pH 7.3 (P < 0.05).
Figure 5
Figure 5
Electrophysiological properties of the Type 2 K+ channel in cerebellar granule neurons A, openings of the Type 2 K+ channel in neurons and TASK-3 expressed on COS-7 cells are shown at five different membrane potentials in cell-attached patches in symmetrical 140 mm KCl. B, amplitude histograms of Type 2 channel openings in a CG neuron and TASK-3 expressed in a COS-7 cell show single major peaks of 2.0 pA (-60 mV). C, duration histograms of channel openings in a granule cell and a COS-7 cell are shown. D, current amplitudes were determined at each membrane potential and used to plot the current-voltage relationships for Type 2 channel, TASK-3. Current-voltage relationship for TASK-1 is also shown for comparison (n = 3). E, outside-out patches from CG neurons were formed and the effect of pHo determined at four pHo values. Inward currents observed at −50 mV in symmetrical 140 mm KCl are shown. Same experiment was done using COS-7 cells expressing TASK-3 (traces not shown). Channel activity was determined at each pH value and plotted. Each bar represents the mean ± s.d. of five determinations. *Significant difference from the corresponding control value observed at pH 7.3 (P < 0.05).
Figure 6
Figure 6
Electrophysiological properties of the Type 3 K+ channel in cerebellar granule neurons A, openings of the Type 3 K+ channel in neurons and TREK-2 expressed on COS-7 cells are shown at three different membrane potentials in cell-attached patches in symmetrical 140 mm KCl. B, duration histograms of channel openings in a granule cell and a COS-7 cell are shown. C, current amplitudes were determined at each membrane potential and used to plot the current-voltage relationships for the Type 3 channel and TREK-2. Each point is the mean ± s.d. of three determinations. D, inside-out patches were formed from CG neurons and the effects of negative pressure, low pHi and arachidonic acid were examined. Cell membrane potential was held at −30 mV in symmetrical 140 mm KCl.
Figure 7
Figure 7
Electrophysiological properties of the Type 4 K+ channel in cerebellar granule neurons A, channel openings in a cell-attached patch at different membrane potentials are shown. Pipette and bath solution contained 140 mm KCl. B, open-time histograms are obtained from channel openings at −50 mV and +50 mV. C, amplitude histograms are obtained from channel openings at −50 mV (1.7 pA) and +50 mV (1.8 pA). D, current amplitudes were determined at each membrane potential and used to plot the current-voltage relationship. Each point is the mean ± s.d. of three values. E, channel activity is plotted as a function of membrane potential. Each point is the mean ± s.d. of four values.
Figure 8
Figure 8
Effect of pH on the Type 4 K+ channel current in cerebellar granule neurons A, openings of the Type 4 K+ channels in an outside-out patch are shown at different pHo values. Amplitude histograms of the first open level are shown on the right. B, a bar graph shows the relative channel currents at different pHo values. Each bar is the mean ± s.d. of four determinations. *Significant difference from the control value observed at pH 7.3 (P < 0.05)
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