The response of the tandem pore potassium channel TASK-3 (K2P9.1) to voltage: gating at the cytoplasmic mouth (original) (raw)
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Journal of Biological Chemistry, 2013
Background: TASK-3 is gated cooperatively by extracellular pH. Results: Mutual electrostatic interaction between K ϩ ions and two pH-sensing histidines occurs in a recently discovered extracellular ion pathway. Conclusion: Channel opening requires neutralization of both sensing histidines, with neutralization of the second sensor becoming favored by an electrostatic effect K ϩ ions. Significance: The work suggests a central role for the extracellular ion pathway in the gating of K 2P K ϩ channels. Proton-gated TASK-3 K ؉ channel belongs to the K 2P family of proteins that underlie the K ؉ leak setting the membrane potential in all cells. TASK-3 is under cooperative gating control by extracellular [H ؉ ]. Use of recently solved K 2P structures allows us to explore the molecular mechanism of TASK-3 cooperative pH gating. Tunnel-like side portals define an extracellular ion pathway to the selectivity filter. We use a combination of molecular modeling and functional assays to show that pH-sensing histidine residues and K ؉ ions mutually interact electrostatically in the confines of the extracellular ion pathway. K ؉ ions modulate the pK a of sensing histidine side chains whose charge states in turn determine the open/closed transition of the channel pore. Cooperativity, and therefore steep dependence of TASK-3 K ؉ channel activity on extracellular pH, is dependent on an effect of the permeant ion on the channel pH o sensors. K ϩ leak or background conductances are responsible for setting the resting membrane potential (1) and play crucial roles in the regulation of neuronal firing, muscle contraction, hormone, neurotransmitter and enzyme secretion, and transepithelial transport (2). K ϩ channels of a family known as K 2P have been found to be the molecular counterparts of these widespread background K ϩ conductances. Best known families of K ϩ channels are membrane proteins with six (K v) or two (K ir) transmembrane ␣-helices (TMs) 5 and one highly conserved P-domain that forms the K ϩ selectivity filter in a tetrameric * This work was supported by Fondecyt Grants 1090478 (to M. I. N.) and 11100373 (to W. G.). □ S This article contains supplemental Tables 1-4 and Figs. S1-S6.
Molecular and functional properties of two-pore-domain potassium channels
American Journal of Physiology-Renal Physiology, 2000
The two-pore-domain K+ channels, or K2P channels, constitute a novel class of K+channel subunits. They have four transmembrane segments and are active as dimers. The tissue distribution of these channels is widespread, and they are found in both excitable and nonexcitable cells. K2P channels produce currents with unusual characteristics. They are quasi-instantaneous and noninactivating, and they are active at all membrane potentials and insensitive to the classic K+ channel blockers. These properties designate them as background K+ channels. They are expected to play a major role in setting the resting membrane potential in many cell types. Another salient feature of K2P channels is the diversity of their regulatory mechanisms. The weak inward rectifiers TWIK-1 and TWIK-2 are stimulated by activators of protein kinase C and decreased by internal acidification, the baseline TWIK-related acid-sensitive K+ (TASK)-1 and TASK-2 channels are sensitive to external pH changes in a narrow ra...
GYGD pore motifs in neighbouring potassium channel subunits interact to determine ion selectivity
The Journal of Physiology, 2001
1. Cells maintain a negative resting membrane potential through the constitutive activity of background K + channels. A novel multigene family of such K + channels has recently been identified. A unique characteristic of these K + channels is the presence of two homologous, subunit-like domains, each containing a pore-forming region. Sequence co-variations in the GYGD signature motifs of the two pore regions suggested an interaction between neighbouring pore domains. 2. Mutations of the GYGD motif in the rat drk1 (Kv2.1) K + channel showed that the tyrosine (Y) position was important for K + selectivity and single channel conductance, whereas the aspartate (D) position was a critical determinant of open state stability. 3. Tandem constructs engineered to mimic the GYGx-GxGD pattern seen in two-domain K + channels delineated a cooperative intersubunit interaction between the Y and D positions, which determined ion selectivity, conductance and gating. 4. In the bacterial KcsA K + channel crystal structure, the equivalent aspartate residue (D80) does not directly interact with permeating K + ions. However, the data presented here show that the D position is able to fine-tune ion selectivity through a functional interaction with the Y position in the neighbouring subunit. 5. These data indicate a physiological basis for the extensive sequence variation seen in the GYGD motifs of two-domain K + channels. It is suggested that a cell can precisely regulate its resting membrane potential by selectively expressing a complement of two-domain K + channels.
Gating the pore of potassium leak channels
European Biophysics Journal, 2009
A key feature of potassium channel function is the ability to switch between conducting and non-conducting states by undergoing conformational changes in response to cellular or extracellular signals. Such switching is facilitated by the mechanical coupling of gating domain movements to pore opening and closing. Two-pore domain potassium channels (K 2P) conduct leak or background potassium-selective currents that are mostly time-and voltage-independent. These channels play a signiWcant role in setting the cell resting membrane potential and, therefore modulate cell responsiveness and excitability. Thus, K 2P channels are key players in numerous physiological processes and were recently shown to also be involved in human pathologies. It is well established that K 2P channel conductance, open probability and cell surface expression are signiWcantly modulated by various physical and chemical stimuli. However, in understanding how such signals are translated into conformational changes that open or close the channels gate, there remain more open questions than answers. A growing line of evidence suggests that the outer pore area assumes a critical role in gating K 2P channels, in a manner reminiscent of C-type inactivation of voltage-gated potassium channels. In some K 2P channels, this gating mechanism is facilitated in response to external pH levels. Recently, it was suggested that K 2P channels also possess a lower activation gate that is positively coupled to the outer pore gate. The purpose of this review is to present an up-to-date summary of research describing the conformational changes and gating events that take place at the K 2P channel ion-conducting pathway during the channel regulation.
Journal of Biological Chemistry, 2012
Background: Two-pore domain K ϩ channels mediate background K ϩ conductance and regulate cellular function. Results: Low extracellular pH (pH o) significantly increases the Na ϩ to K ϩ relative permeability of TWIK-1, TASK-1, and TASK-3 K ϩ channels. Conclusion: TWIK-1, TASK-1, and TASK-3 channels change ion selectivity in lowered pH o. Significance: The findings provide insights on the mechanism of regulation of acid-sensitive K2P channels by low pH o. Two-pore domain K ؉ channels (K2P) mediate background K ؉ conductance and play a key role in a variety of cellular functions. Among the 15 mammalian K2P isoforms, TWIK-1, TASK-1, and TASK-3 K ؉ channels are sensitive to extracellular acidification. Lowered or acidic extracellular pH (pH o) strongly inhibits outward currents through these K2P channels. However, the mechanism of how low pH o affects these acid-sensitive K2P channels is not well understood. Here we show that in Na ؉based bath solutions with physiological K ؉ gradients, lowered pH o largely shifts the reversal potential of TWIK-1, TASK-1, and TASK-3 K ؉ channels, which are heterologously expressed in Chinese hamster ovary cells, into the depolarizing direction and significantly increases their Na ؉ to K ؉ relative permeability. Low pH o-induced inhibitions in these acid-sensitive K2P channels are more profound in Na ؉-based bath solutions than in channel-impermeable N-methyl-D-glucamine-based bath solutions, consistent with increases in the Na ؉ to K ؉ relative permeability and decreases in electrochemical driving forces of outward K ؉ currents of the channels. These findings indicate that TWIK-1, TASK-1, and TASK-3 K ؉ channels change ion selectivity in response to lowered pH o , provide insights on the understanding of how extracellular acidification modulates acid-sensitive K2P channels, and imply that these acid-sensitive K2P channels may regulate cellular function with dynamic changes in their ion selectivity.
The Journal of biological chemistry, 1991
Extracellular tetraethylammonium (TEA) inhibits currents in Xenopus oocytes that have been injected with mRNAs encoding voltage-dependent potassium channels. Concentration-response curves were used to measure the affinity of TEA; this differed up to 700-fold among channels RBK1 (KD 0.3 mM), RGK5 (KD 11 mM), and RBK2 (KD greater than 200 mM). Studies in which chimeric channels were expressed localized TEA binding to the putative extracellular loop between trans-membrane domains S5 and S6. Site-directed mutagenesis of residues in this region identified the residue Tyr379 of RBK1 as a crucial determinant of TEA sensitivity; substitution of Tyr in the equivalent positions of RBK2 (Val381) and RGK5 (His401) made these channels as sensitive to TEA as RBK1. Nonionic forces are involved in TEA binding because (i) substitution of the Phe for Tyr379 in RBK1 increased its affinity, (ii) protonation of His401 in RGK5 selectively reduced its affinity, and (iii) the affinity of TEA was unaffected...
Studies of the pore-forming domain of a voltage-gated potassium channel protein
"Protein Engineering, Design and Selection", 1994
Recent mutagenesis studies nave identified a stretch of amino acid residues which form the ion-selective pore of the voltagegated potassium channel. It has been suggested that this sequence of amino acids forms a /3-barrel structure making up the structure of the ion-selective pore [
Elucidating the Structural Basis of the Intracellular pH Sensing Mechanism of TASK-2 K2P Channels
International Journal of Molecular Sciences, 2020
Two-pore domain potassium (K2P) channels maintain the cell’s background conductance by stabilizing the resting membrane potential. They assemble as dimers possessing four transmembrane helices in each subunit. K2P channels were crystallized in “up” and “down” states. The movements of the pore-lining transmembrane TM4 helix produce the aperture or closure of side fenestrations that connect the lipid membrane with the central cavity. When the TM4 helix is in the up-state, the fenestrations are closed, while they are open in the down-state. It is thought that the fenestration states are related to the activity of K2P channels and the opening of the channels preferentially occurs from the up-state. TASK-2, a member of the TALK subfamily of K2P channels, is opened by intracellular alkalization leading the deprotonation of the K245 residue at the end of the TM4 helix. This charge neutralization of K245 could be sensitive or coupled to the fenestration state. Here, we describe the relationship between the states of the intramembrane fenestrations and K245 residue in TASK-2 channel. By using molecular modeling and simulations, we show that the protonated state of K245 (K245+) favors the open fenestration state and, symmetrically, that the open fenestration state favors the protonated state of the lysine residue. We show that the channel can be completely blocked by Prozac, which is known to induce fenestration opening in TREK-2. K245 protonation and fenestration aperture have an additive effect on the conductance of the channel. The opening of the fenestrations with K245+ increases the entrance of lipids into the selectivity filter, blocking the channel. At the same time, the protonation of K245 introduces electrostatic potential energy barriers to ion entrance. We computed the free energy profiles of ion penetration into the channel in different fenestration and K245 protonation states, to show that the effects of the two transformations are summed up, leading to maximum channel blocking. Estimated rates of ion transport are in qualitative agreement with experimental results and support the hypothesis that the most important barrier for ion transport under K245+ and open fenestration conditions is the entrance of the ions into the channel.