Showing papers by "Thomas Baukrowitz published in 2012"

State-independent intracellular access of quaternary ammonium blockers to the pore of TREK-1

Abstract: We previously reported that TREK-1 gating by internal pH and pressure occurs close to or within the selectivity filter. These conclusions were based upon kinetic measurements of high-affinity block by quaternary ammonium (QA) ions that appeared to exhibit state-independent accessibility to their binding site within the pore. Intriguingly, recent crystal structures of two related K2P potassium channels were also both found to be open at the helix bundle crossing. However, this did not exclude the possibility of gating at the bundle crossing and it was suggested that side-fenestrations within these structures might allow state-independent access of QA ions to their binding site. In this addendum to our original study we demonstrate that even hydrophobic QA ions do not access the TREK-1 pore via these fenestrations. Furthermore, by using a chemically reactive QA ion immobilized within the pore via covalent cysteine modification we provide additional evidence that the QA binding site remains accessible to the...

Identification of the muscarinic pathway underlying cessation of sleep-related burst activity in rat thalamocortical relay neurons

Abstract: Modulation of the standing outward current (I SO) by muscarinic acetylcholine (ACh) receptor (MAChR) stimulation is fundamental for the state-dependent change in activity mode of thalamocortical relay (TC) neurons. Here, we probe the contribution of MAChR subtypes, G proteins, phospholipase C (PLC), and two pore domain K+ (K2P) channels to this signaling cascade. By the use of spadin and A293 as specific blockers, we identify TWIK-related K+ (TREK)-1 channel as new targets and confirm TWIK-related acid-sensitve K+ (TASK)-1 channels as known effectors of muscarinic signaling in TC neurons. These findings were confirmed using a high affinity blocker of TASK-3 and TREK-1, namely, tetrahexylammonium chloride. It was found that the effect of muscarinic stimulation was inhibited by M1AChR-(pirenzepine, MT-7) and M3AChR-specific (4-DAMP) antagonists, phosphoinositide-specific PLCβ (PI-PLC) inhibitors (U73122, ET-18-OCH3), but not the phosphatidylcholine-specific PLC (PC-PLC) blocker D609. By comparison, depleting guanosine-5′-triphosphate (GTP) in the intracellular milieu nearly completely abolished the effect of MAChR stimulation. The block of TASK and TREK channels was accompanied by a reduction of the muscarinic effect on I SO. Current-clamp recordings revealed a membrane depolarization following MAChR stimulation, which was sufficient to switch TC neurons from burst to tonic firing under control conditions but not during block of M1AChR/M3AChR and in the absence of intracellular GTP. These findings point to a critical role of G proteins and PLC as well as TASK and TREK channels in the muscarinic modulation of thalamic activity modes.