Deletion Analysis of the Mechanosensitive TREK-1 Channel

Abstract

TREK-1, the first functionally characterized mechanosensitive K channel from the two-pore family (K2P) is involved in protective regulation of resting potential in CNS neurons and many other tissues. The structural basis of TREK-1 sensitivity to stretch and other factors such as arachidonic acid (AA) and anesthetics remains unknown. Attempts to use existing K channel structures as templates for TREK-1 modeling have identified several motifs that are not present in canonical K channels, which include divergent cytoplasmic N- and C-termini, and a characteristic 50-residue extracellular loop in the first homologous repeat. To characterize functional roles of these domains, we analyzed TREK-1 deletion and cysteine mutants in patch-clamp experiments. In response to steps of suction, the control TREK-1-EGFP fusion protein expressed in HEK-293 cells produced transient currents in cell-attached patches and non-inactivating sustained currents upon patch excision. Responses in both configurations were augmented by AA. Deletion of the extracellular loop (Δ76-124) reduced functional surface expression of channels and increased background activity, but the activation by tension augmented by AA was fully retained. Further deletion of the C-terminal end (Δ76-124, Δ334-411) produced no additional effect. In an attempt to generate cysteine-free version of the channel, we additionally mutated two cysteines in the transmembrane domain. C219A did not compromise channel activity, whereas C159A/S was essentially inactive. Experiments in the presence of mercaptoethanol suggested that none of these cysteines form functionally-important disulfides. The functional deletion mutant without C219 is now topologically closer to other K channels and makes an amenable system for homology modeling and testing by disulfide cross-linking.