Cerebellar granule neurons express a standing outward (background) K+ current (IK,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 IK,SO. IK,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 IK,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 IK,SO in cultured cerebellar granule neurons. Our results also show that the high sensitivity of IK,SO to extracellular pH comes from the high sensitivity of Type 2 (TASK‐3) and Type 4 K+ channels.

TRAAK is a member of the tandem-pore K+ channel family, and is expressed mainly in the brain. Using rat TRAAK (rTRAAK), we studied its single-channel kinetics, interactive modes of activation, and the role of the C-terminus on its pressure-, fatty-acid- and pH-sensitivity. When expressed in COS-7 cells, rTRAAK showed a mildly inwardly rectifying single-channel current/voltage relationship in symmetrical 140 mM KCl. Unlike TREK-1 and TREK-2, which are activated by acidic conditions, rTRAAK was activated by alkali conditions, such that a change in intracellular pH from 7.3 to 8.3 and 8.8 increased channel activity 5- and 14-fold, respectively. Pressure and alkali produced a strong synergistic activation, and pressure and arachidonic acid (AA) produced a mild synergistic activation. The only additive effect was observed with alkali and AA. Replacing the C-terminus of rTRAAK with that of TASK-1 or TASK-3 did not affect the response to pressure, AA or alkali. In contrast, replacing the C-terminus of TREK-2 with that of TASK-3 abolished the sensitivity to AA and acid, but not to pressure. These results show that rTRAAK is an alkali-sensing K+ channel that shows synergistic activation with pressure, and that the mechanism of activation of rTRAAK and TREK by free fatty acids are different.

TREK-2, a member of the tandem-pore K+ channel family, is activated by membrane stretch, unsaturated free fatty acids and acidic conditions, and exhibits unique open channel kinetics. To identify the regions responsible for these properties, we studied the role of the cytoplasmic regions of TREK-2. Deletion of the N-terminus had no effect on any aspect of TREK-2 function. Deletion of the C-terminus or its substitution with that of TASK-3 abolished the sensitivity to free fatty acids and intracellular pH (pHi), and reduced the sensitivity to pressure. The regions that allow activation by free fatty acids and low pHi were localized to 25- and 10-amino-acid domains, respectively, close to the fourth transmembrane segment. Substitution of KKTKEE, a charged region near the proximal C-terminus, with uncharged amino acids produced little change in TREK-2 function; however, its deletion abolished sensitivity to fatty acids and low pH, indicating that this region is structurally very important. The TREK-2 C-terminus was also found to be critical for its channel opening in bursts as well as for its increased basal activity. Thus, the C-terminus endows TREK-2 with unique channel kinetics and the ability to be gated by free fatty acids and low pHi, and with increased mechanosensitivity.

Magnocellular neurosecretory cells (MNCs) were isolated from the supraoptic nucleus of rat hypothalamus, and properties of K+ channels that may regulate the resting membrane potential and the excitability of MNCs were studied. MNCs showed large transient outward currents, typical of vasopressin‐ and oxytocin‐releasing neurons. K+ channels in MNCs were identified by recording K+ channels that were open at rest in cell‐attached and inside‐out patches in symmetrical 150 mm KCl. Eight different K+ channels were identified and could be distinguished unambiguously by their single‐channel kinetics and voltage‐dependent rectification. Two K+ channels could be considered functional correlates of TASK‐1 and TASK‐3, as judged by their single‐channel kinetics and high sensitivity to pHo. Three K+ channels showed properties similar to TREK‐type tandem‐pore K+ channels (TREK‐1, TREK‐2 and a novel TREK), as judged by their activation by membrane stretch, intracellular acidosis and arachidonic acid. One K+ channel was activated by application of pressure, arachidonic acid and alkaline pHi, and showed single‐channel kinetics indistinguishable from those of TRAAK. One K+ channel showed strong inward rectification and single‐channel conductance similar to those of a classical inward rectifier, IRK3. Finally, a K+ channel whose cloned counterpart has not yet been identified was highly sensitive to extracellular pH near the physiological range similar to those of TASK channels, and was the most active among all K+ channels. Our results show that in MNCs at rest, eight different types of K+ channels can be found and six of them belong to the tandem‐pore K+ channel family. Various physiological and pathophysiological conditions may modulate these K+ channels and regulate the excitability of MNCs.

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