Background and tandem-pore potassium channels in magnocellular neurosecretory cells of the rat supraoptic nucleus

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

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

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

Neurogenesis and brain injury: managing a renewable resource for repair

The brain shows limited ability to repair itself, but neurogenesis in certain areas of the adult brain suggests that neural stem cells may be used for structural brain repair. It will be necessary to understand how neurogenesis in the adult brain is regulated to develop strategies that harness neural stem cells for therapeutic use

Desensitization of mechano-gated K(2P) channels

The neuronal mechano-gated K(2P) channels TREK-1 and TRAAK show pronounced desensitization within 100 ms of membrane stretch. Desensitization persists in the presence of cytoskeleton disrupting agents, upon patch excision, and when channels are expressed in membrane blebs. Mechanosensitive currents evoked with a variety of complex stimulus protocols were globally fit to a four-state cyclic kinetic model in detailed balance, without the need to introduce adaptation of the stimulus. However, we show that patch stress can be a complex function of time and stimulation history. The kinetic model couples desensitization to activation, so that gentle conditioning stimuli do not cause desensitization. Prestressing the channels with pressure, amphipaths, intracellular acidosis, or the E306A mutation reduces the peak-to-steady-state ratio by changing the preexponential terms of the rate constants, increasing the steady-state current amplitude. The mechanical responsivity can be accounted for by a change of in-plane area of ≈2 nm(2) between the closed and open conformations. Desensitization and its regulation by chemical messengers is predicted to condition the physiological role of K(2P) channels

Properties of single two-pore domain TREK-2 channels expressed in mammalian cells

TREK-2 (K2P10.1), a member of the two-pore domain K+ (K2P) channel family, provides the background K+ conductance in many cell types, and is a target of neurotransmitters that act on receptors coupled to Gs and Gq. We report here that TREK-2 exhibits small (TREK-2S) and large (TREK-2L) conductance phenotypes when expressed in mammalian cell lines (COS-7, HEK293, HeLa) and in Xenopus oocytes. TREK-2S phenotype shows a noisy open state with a mean conductance of 54 pS (+40 mV). TREK-2L phenotype shows a full open state (202 pS) with several short-lived sub-conductance levels. Both phenotypes were strongly activated by arachidonic acid, membrane stretch (−40 mmHg) and intracellular acidification (pH 6.4). Phosphorylation of TREK-2 produced by treatment of cells with activators of protein kinases A and C, and okadaic acid (a serine/threonine phosphatase inhibitor) decreased the current contributed by TREK-2S and TREK-2L, and caused partial switching of conductance levels from those of TREK-2S and TREK-2L to more intermediate values. Under this condition, TREK-2 exhibited six conducting levels and one closed level. TREK-2 mutants in which putative protein kinases A and C phosphorylation sites were mutated to alanines (S326A, S359A, S326A/S359A) displayed mostly TREK-2S and TREK-2L phenotypes. However, S326D and S359D mutants (as well as the double mutants) that mimic the phosphorylated state showed all six conducting levels and low channel activity. The S326A and S359A mutants did not significantly affect the intrinsic voltage dependence of TREK-2 in Mg2+-free solution. Phenotypes resembling TREK-2S and TREK-2L were also observed in cerebellar granule neurons that express TREK-2 mRNA. These results show that TREK-2 exhibits two primary modes of gating that give rise to two channel phenotypes under dephosphorylated conditions, and that its phosphorylation shifts the gating mode to include intermediate conducting levels. This represents a novel mechanism by which receptor agonists modulate the function of a K+ channel to alter cell excitability

Control of the single channel conductance of K2P10.1 (TREK-2) by the amino-terminus: role of alternative translation initiation

TREK-2 expressed in mammalian cells exhibits small (∼52 pS) and large (∼220 pS) unitary conductance levels. Here we tested the role of the N-terminus (69 amino acids long) in the control of the unitary conductance, and role of the alternative translation initiation as a mechanism that produces isoforms of TREK-2 that show different conductance levels. Deletion of the first half (Δ1–36) of the N-terminus had no effect. However, deletion of most of the N-terminus (Δ1–66) resulted in the appearance of only the large-conductance channel (∼220 pS). In support of the critical function of the distal half of the N-terminus, the deletion mutants Δ1–44 and Δ1–54 produced ∼90 pS and 188 pS channels, respectively. In Western blot analysis, TREK-2 antibody detected two immunoreactive bands at ∼54 kDa and ∼60 kDa from cells expressing wild-type TREK-2 that has three potential translation initiation sites (designated M1M2M3) within the N-terminus. Mutation of the second and third initiation sites from Met to Leu (M1L2L3) produced only the ∼60 kDa isoform and the small-conductance channel (∼52 pS). Mutants designed to produce translation from the second (M2L3) or third (M3) initiation site produced the ∼54 kDa isoform, and the large conductance channel (∼185–224 pS). M1L2L3, M2L3 and M3 were relatively selectively permeable to K+, as judged by the 51–55 mV shifts in reversal potential following a 10-fold change in [K+]o. PNa/PK values were also similar for M1L2L3 (∼0.02), M2L3 (∼0.02) and M3 (∼0.03). Arachidonic acid, proton and membrane stretch activated, whereas dibutyryl-cAMP inhibited all three isoforms of TREK-2, indicating that deletion of the N-terminus does not abolish modulation. These results show that the small and large conductance TREK-2 channels are produced as a result of alternative translation initiation, producing isoforms with long and short N-termini, and that the distal half of the N-terminus controls the unitary conductance