Two Identical Noninteracting Sites in an Ion Channel Revealed by Proton Transfer

Root, Michael J.; MacKinnon, Roderick

doi:10.1126/science.7522344

Cited by 150 publications

(148 citation statements)

References 30 publications

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“…Recently, on the basis of single-channel currents recorded from mutant AChRs, we proposed that only two of the four glutamates in the ring contribute to set the size of the unitary currents, and that these glutamates are deprotonated even at pH 6.0 (2). This is in stark contrast with the situation in voltage-dependent Ca 2+ channels (Ca V channels) and cyclicnucleotide-gated channels (CNG channels), for example, where all four selectivity-filter glutamates have been suggested to contribute (directly or indirectly) to the formation of one (in Ca V channels) or two (in CNG channels) proton-binding sites that are largely protonated at pH 6.0 (3,4). Moreover, our results led us to propose that the difference between the muscle-AChR glutamates that catalyze cation permeation and those that do not is the conformation adopted by their side chains in the open channel (2).…”

contrasting

confidence: 42%

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Harpole

Grosman

2014

Proc. Natl. Acad. Sci. U.S.A.

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On the basis of single-channel currents recorded from the muscle nicotinic acetylcholine receptor (AChR), we have recently hypothesized that the conformation adopted by the glutamate side chains at the first turn of the pore-lining α-helices is a key determinant of the rate of ion permeation. In this paper, we set out to test these ideas within a framework of atomic detail and stereochemical rigor by conducting all-atom molecular dynamics and Brownian dynamics simulations on an extensively validated model of the open-channel muscle AChR. Our simulations provided ample support to the notion that the different rotamers of these glutamates partition into two classes that differ markedly in their ability to catalyze ion conduction, and that the conformations of the four wild-type glutamates are such that two of them "fall" in each rotamer class. Moreover, the simulations allowed us to identify the mm (χ 1 ≅ -60°; χ 2 ≅ -60°) and tp (χ 1 ≅ 180°; χ 2 ≅ +60°) rotamers as the likely conduction-catalyzing conformations of the AChR's selectivity-filter glutamates. More generally, our work shows an example of how experimental benchmarks can guide molecular simulations into providing a type of structural and mechanistic insight that seems otherwise unattainable.nicotinic receptor | glutamate rotamers T he role an ion channel can play in the physiology of a cell is dictated by the rate at which ions permeate, the type of ions that permeate, the stimulus that gates the channel open, the rates of interconversion among all conductive and nonconductive conformations, and the channel's level of expression in the membrane. In this paper, we are concerned with the chemical determinants of the rate at which cations permeate through the muscle nicotinic acetylcholine receptor (AChR), an archetypal neurotransmitter-gated ion channel.Several "rings" of negatively charged residues decorate the walls of the ion-permeation pathway of the muscle nicotinic AChR. Of these, it is the ring of four glutamates and one glutamine in the first (N-terminal) turn of the pore-lining M2 transmembrane α-helices (the "intermediate ring of charge" at position -1′; Fig. 1A) that lowers the energetic barrier to cation permeation the most (1). Recently, on the basis of single-channel currents recorded from mutant AChRs, we proposed that only two of the four glutamates in the ring contribute to set the size of the unitary currents, and that these glutamates are deprotonated even at pH 6.0 (2). This is in stark contrast with the situation in voltage-dependent Ca 2+ channels (Ca V channels) and cyclicnucleotide-gated channels (CNG channels), for example, where all four selectivity-filter glutamates have been suggested to contribute (directly or indirectly) to the formation of one (in Ca V channels) or two (in CNG channels) proton-binding sites that are largely protonated at pH 6.0 (3, 4). Moreover, our results led us to propose that the difference between the muscle-AChR glutamates that catalyze cation permeation and those that do not is the conformation adopte...

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contrasting

confidence: 42%

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Harpole

Grosman

2014

Proc. Natl. Acad. Sci. U.S.A.

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“…The pKa of this group is proposed to be influenced by other positively charged residues that are forced into proximity by tertiary structure of the channel complex (Schulte et al, 1999). L-type Ca 2ϩ and cyclic nucleotide-gated channels are similarly controlled by protons, and similar mechanisms have been proposed (Root and MacKinnon, 1994;Chen et al, 1996). In each of these channels, the working hypothesis is that the pKa of a critical residue is shifted to physiological range by the repulsive forces of either ionized carboxylic acids or amino/guanidino groups located at some distance within the polypeptide chain but forced close together by tertiary structure, illustrating how the proton sensor might be a multipart entity.…”

Section: Structural Implicationsmentioning

confidence: 96%

Protons Trap NR1/NR2B NMDA Receptors in a Nonconducting State

Banke¹,

Dravid²,

Traynelis³

2005

J. Neurosci.

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NMDA receptors are highly expressed in the CNS and are involved in excitatory synaptic transmission, as well as synaptic plasticity. Given that overstimulation of NMDA receptors can cause cell death, it is not surprising that these channels are under tight control by a series of inhibitory extracellular ions, including zinc, magnesium, and H ϩ . We studied the inhibition by extracellular protons of recombinant NMDA receptor NR1/NR2B single-channel and macroscopic responses in transiently transfected human embryonic kidney HEK 293 cells using patch-clamp techniques. We report that proton inhibition proceeds identically in the absence or presence of agonist, which rules out the possibility that protonation inhibits receptors by altering coagonist binding. The response of macroscopic currents in excised patches to rapid jumps in pH was used to estimate the microscopic association and dissociation rates for protons, which were 1.4 ϫ 10 9 M Ϫ1 sec Ϫ1 and 110 -196 sec Ϫ1 , respectively (K d corresponds to pH 7.2). Protons reduce the open probability without altering the time course of desensitization or deactivation. Protons appear to slow at least one time constant describing the intra-activation shut-time histogram and modestly reduce channel open time, which we interpret to reflect a reduction in the overall channel activation rate and possible proton-induced termination of openings. This is consistent with a modest proton-dependent slowing of the macroscopic response rise time. From these data, we propose a physical model of proton inhibition that can describe macroscopic and single-channel properties of NMDA receptor function over a range of pH values.

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“…This mechanism fundamentally differs from that found for cyclic nucleotide-gated and inwardrectifying K ϩ channels in animals. In the latter protons block the open channel and thereby reduce the single-channel conductance (15,23). Consequently, we would expect a different molecular basis for pH dependence in plant K ϩ channels.…”

Section: Discussionmentioning

confidence: 99%

Molecular basis of plant-specific acid activation of K ⁺ uptake channels

Hoth¹,

Drèyer²,

Dietrich³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

123

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During stomatal opening potassium uptake into guard cells and K ؉ channel activation is tightly coupled to proton extrusion. The pH sensor of the K ؉ uptake channel in these motor cells has, however, not yet been identified. Electrophysiological investigations on the voltage-gated, inward rectifying K ؉ channel in guard cell protoplasts from Solanum tuberosum (KST1), and the kst1 gene product expressed in Xenopus oocytes revealed that pH dependence is an intrinsic property of the channel protein. Whereas extracellular acidification resulted in a shift of the voltage-dependence toward less negative voltages, the single-channel conductance was pH-insensitive. Mutational analysis allowed us to relate this acid activation to both extracellular histidines in KST1. One histidine is located within the linker between the transmembrane helices S3 and S4 (H160), and the other within the putative pore-forming region P between S5 and S6 (H271). When both histidines were substituted by alanines the double mutant completely lost its pH sensitivity. Among the single mutants, replacement of the pore histidine, which is highly conserved in plant K ؉ channels, increased or even inverted the pH sensitivity of KST1. From our molecular and biophysical analyses we conclude that both extracellular sites are part of the pH sensor in plant K ؉ uptake channels.Plant growth, differentiation, cell and tissue polarity, as well as movements strongly depend on the formation of pH gradients across individual cell types (1-3). Stomata that are formed by two guard cells represent a unique model for plant movement. Regulation of stomatal movement in higher plants is essential for efficient uptake of CO 2 at minimal water loss. Stomatal opening requires accumulation of potassium ions into guard cells. K ϩ uptake is mediated by K ϩ uptake channels, a process accompanied by the acidification of the apoplast (4-7). Due to differential pumping activity of the plasma membrane H ϩ -pump the apoplastic pH around closed and open stomata varies between 7 and 5 (8, 9). Within this range changes in the extracellular proton concentration affect the activity of guard cell inward rectifying K ϩ channels (10-13). In contrast to their functional and structural animal counterparts, which are either not activated or even inhibited by protons (14, 15), the guard cell K ϩ channel is activated upon extracellular acidification (10-13).The molecular cloning of plant K ϩ inward rectifiers revealed a structural homology to animal outward rectifying K ϩ channels of the Shaker gene family (12,16,17). Hydrophobicity analyses of their primary structure predicted six transmembrane domains (S1-S6) (12,16,17). Whereas S4 is likely to represent the voltage sensor of these voltage-dependent channels, the amphiphilic linker between S5 and S6 (P) forms the conductive pore (18). Based on the current molecular model of the Shaker channel we were able to relate structural elements of guard cell K ϩ inward rectifiers to stomatal physiology. In this context we have previously sho...

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Two Identical Noninteracting Sites in an Ion Channel Revealed by Proton Transfer

Cited by 150 publications

References 30 publications

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Protons Trap NR1/NR2B NMDA Receptors in a Nonconducting State

Molecular basis of plant-specific acid activation of K ⁺ uptake channels

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Two Identical Noninteracting Sites in an Ion Channel Revealed by Proton Transfer

Cited by 150 publications

References 30 publications

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Side-chain conformation at the selectivity filter shapes the permeation free-energy landscape of an ion channel

Protons Trap NR1/NR2B NMDA Receptors in a Nonconducting State

Molecular basis of plant-specific acid activation of K + uptake channels

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Product

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Molecular basis of plant-specific acid activation of K ⁺ uptake channels