MlotiK1 is a prokaryotic homolog of cyclic-nucleotide-dependent ion channels that contains an intracellular C-terminal cyclic nucleotide binding (CNB) domain. X-ray structures of the CNB domain have been solved in the absence of ligand and bound to cAMP. Both the full-length channel and CNB domain fragment are easily expressed and purified, making MlotiK1 a useful model system for dissecting activation by ligand binding. We have used X-ray crystallography to determine three new MlotiK1 CNB domain structures: a second apo configuration, a cGMP-bound structure, and a second cAMP-bound structure. In combination, the five MlotiK1 CNB domain structures provide a unique opportunity for analyzing, within a single protein, the structural differences between the apo state and the bound state, and the structural variability within each state. With this analysis as a guide, we have probed the nucleotide selectivity and importance of specific residue side chains in ligand binding and channel activation. These data help to identify ligand-protein interactions that are important for ligand dependence in MlotiK1 and, more globally, in the class of nucleotide-dependent proteins.
Cyclic nucleotide-regulated ion channels are present in bacteria, plants, vertebrates, and humans. In higher organisms, they are closely involved in signaling networks of vision and olfaction. Binding of cAMP or cGMP favors the activation of these ion channels. Despite a wealth of structural and studies, there is a lack of structural data describing the gating process in a full-length cyclic nucleotideregulated channel. We used high-resolution atomic force microscopy (AFM) to directly observe the conformational change of the membrane embedded bacterial cyclic nucleotide-regulated channel MlotiK1. In the nucleotide-bound conformation, the cytoplasmic cyclic nucleotide-binding (CNB) domains of MlotiK1 are disposed in a fourfold symmetric arrangement forming a pore-like vestibule. Upon nucleotide-unbinding, the four CNB domains undergo a large rearrangement, stand up by ∼1.7 nm, and adopt a structurally variable grouped conformation that closes the cytoplasmic vestibule. This fully reversible conformational change provides insight into how CNB domains rearrange when regulating the potassium channel.conformational changes | cyclic nucleotide gating | membrane protein | MloK1 | single-molecule imaging P otassium channels are tetrameric membrane proteins that facilitate the permeation of potassium ions through the membrane with high specificity and high-throughput rates. These channels are central to the electrical activity of cells in humans and are, therefore, of fundamental importance for the function of nervous and muscular systems. The major mode of functional regulation in potassium channels is gating, a conformational change that occurs on the intracellular regions of the ion pore domain and involves an iris-like movement of the C-terminal transmembrane helices and a widening of the intracellular pore. Gating in potassium channels is induced by a variety of stimuli, including membrane voltage, intracellular calcium concentration, and cyclic nucleotide levels (1). These stimuli are sensed by a separate domain from the ion pore domain, inducing a conformational change that is then propagated to the gate of the channel.The MlotiK1 potassium channel, from the bacterium Mesorhizobium loti, belongs to the family of channels that is regulated by cyclic nucleotides and includes eukaryotic cyclic nucleotide-gated (CNG) and hyperpolarization activated cyclic nucleotide-gated (HCN) channels (2, 3). These channels have C-terminal cytoplasmic cyclic nucleotide-binding (CNB) domains and upon binding of cAMP or cGMP, these domains undergo a conformational change that favors the opening of the gate of the channel. The major difference between the MlotiK1 channel and the CNG or HCN channels is the linker that connects the gate to the CNB domains. This helical linker (C linker) is roughly 80 residues long in CNG and HCN channels and only ∼20 residues long in the MlotiK1 channel.The MlotiK1 channel has been the focus of structural and functional studies with the aim of understanding channel regulation by cyclic nucleotides. X-ray...
Airway epithelial cells are the site of Cl-secretion through the cystic fibrosis transmembrane regulator (CFTR). Cystic fibrosis (CF) is a fatal genetic disease caused by mutations in CFTR. The most frequent mutation consists of a deletion of the phenylalanine at position 508 (DF508-CFTR) that impairs protein maturation and alters channel gating. In the last years, several small molecules were identified by high throughput screening that could restore DF508-CFTR function. Compounds addressing DF508-CFTR gating defects are referred to as potentiators and have been documented to increase the activity of DF508-CFTR to a level similar to wild-type CFTR. The basolateral Kþ channel KCa3.1 has been documented to play a prominent role in establishing a suitable driving force for CFTR-mediated Cl-secretion in airway epithelial cells. Thus, in a global approach of transepithelial transport, the research for physiologically relevant DF508-CFTR potentiators should also consider their effects on the KCa3.1 channel. A characterization of the effect of different DF508-CFTR potentiators on the KCa3.1 channel was undertaken using inside-out patch clamp measurements on cDNA injected xenopus oocytes and on transformed HEK-293 cells that express the KCa3.1 channel. In this work we present preliminary results on the effects of different DF508-CFTR potentiators on KCa3.1. Our inside-out patch-clamp measurements show that VRT-532 has a state independent inhibitory effect on KCa3.1, but very little action on the V282G mutant of KCa3.1, which is constitutively active. In contrast, CBIQ succeded to activate KCa3.1, through a mechanism likely to involve an action on the channel gate. These effects were observed at concentrations known to activate DF508-CFTR. Supported by CCFF.
confirmed a small conductance of~1.5 pS but observed a pronounced cooperative gating of multiple channels. We expressed HCN2 channels in Xenopus oocytes and studied single channel currents in insideout patches. Our results confirm a small conductance (~2 pS) and do not provide any evidence for a cooperative gating between the channels, enabling recording from patches containing one and only one channel. The activating effect of cAMP, applied to the bath solution, is mediated by an increase of the open probability. In conclusion, HCN2 channels expressed in Xenopus oocytes develop an only small single-channel conductance, gate as individual channels, and are activated by cAMP via an increase of the open probability. These results are of importance for modelling single-channel properties from macroscopic currents.
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