Dimensions of the narrow portion of a recombinant NMDA receptor channel

Villarroel, Alfredo; Burnashev, Nail; Sakmann, Bert

doi:10.1016/s0006-3495(95)80263-8

Cited by 113 publications

(102 citation statements)

References 41 publications

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“…This suggests that the serotonergic agents also act within the ionic channel but at a different site, possibly in the vestibule of the channel. Similar results for cations of different sizes have been reported for NMDA receptors (Villarroel et al 1995). where C represents the non-conducting receptor, Aµ represents two molecules of the agonist, R* is the receptor in the open conformation, S is the serotonergic agent, D is the receptor in the desensitized state, á and â are the rate constants for channel closing and opening, G' is the effective blocking rate, which depends on serotonergic agent concentration, F is the backward rate constant for channel blocking, k1 and k−1 are the rate constants for receptor desensitized and non-desensitized states in the absence of the blocking agents (Feltz & Trautmann, 1982), and kµ and k−2, are the rate constants for channel desensitized and non-desensitized states in the presence of the blocking agents (Neher & Steinbach, 1978;Clapham & Neher, 1984).…”

Section: Effects Of Serotonergic Agents On Muscle Achrssupporting

confidence: 88%

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Garcı́a-Colunga¹,

Miledi²

1999

Exp. Physiol.

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summaryXenopus laevis oocytes were used to analyse the effects of serotonin (5-hydroxytryptamine, 5_HT) and serotonergic agents on ionic currents elicited by the activation of mammalian muscle nicotinic acetylcholine receptors (AChRs). 5_HT as well as other serotonergic agents, such as ketanserin, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), methysergide, spiperone, or fluoxetine alone (up to 1 mÒ), did not elicit membrane currents in Xenopus oocytes expressing AChRs, but they reversibly reduced the current elicited by acetylcholine (ACh-current). Serotonin was applied before, together with or after ACh application, and its effects were examined on desensitizing and non-desensitizing ACh-currents. 5_HT reduced the amplitude and accelerated the desensitization of the desensitizing currents. In contrast, non-desensitizing currents were reduced in amplitude but their time course was not significantly affected. With the same concentration of 5_HT the inhibition was stronger on desensitizing than on non-desensitizing ACh-currents. For example, 100 ìÒ 5_HT reduced the peak of a desensitizing ACh-current to 0·48 ± 0·06 (peak current ratio) and after 40 s the current was reduced to a ratio of 0·25 ± 0·04, whereas a non-desensitizing ACh-current was reduced to a ratio of 0·66 ± 0·01. All the serotonergic agents tested inhibited the ACh-currents rapidly and reversibly, suggesting that they are acting directly on the AChRs. The half-inhibitory concentration, IC50, of 5_HT acting on nondesensitizing currents elicited by 250 nÒ ACh was 247 ± 26 ìÒ and the Hill coefficient was •0·88, suggesting a single site for the interaction of 5_HT with the receptor. It appears that 5_HT inhibits AChRs non-competitively because neither the half-effective concentration of ACh, EC50, for ACh-current nor the Hill coefficient were affected by 5_HT. Furthermore, the extent of inhibition of 5_HT on AChRs did not depend on the nicotinic agonist (suberyldicholine, ACh or nicotine). The inhibition of AChRs by serotonergic agents was voltage-dependent. The electrical distance of the binding site for 5_HT was •0·75, whereas for the other serotonergic agents tested it was •0·22, suggesting that ketanserin, 8-OH-DPAT, methysergide, spiperone and fluoxetine act within the ion channel, but at a site more external than that for 5_HT. These substances inhibited the AChcurrent more potently than 5_HT. We conclude that 5_HT and serotonergic agents inhibit, in a noncompetitive manner, the ACh-current in muscle AChRs by blocking the open receptor-channel complex. Moreover, 5_HT appears to promote the desensitized state of the receptor when the current is elicited by high ACh concentrations. introduction The muscle nicotinic acetylcholine receptor (AChR), a neurotransmitter-gated ion channel, is a member of a gene superfamily that includes GABAA, glycine and 5_HT× receptors (Cockcroft et al. 1995). Structurally, the AChR is a pentamer composed of four different subunits (á, â, ã or å, and ä) with a stoichiometry of áµâãä (Karlin & Akabas, 1995; Unwin, ...

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Section: Effects Of Serotonergic Agents On Muscle Achrssupporting

confidence: 88%

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Garcı́a-Colunga¹,

Miledi²

1999

Exp. Physiol.

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“…Ascher & Nowak, 1988;Jahr & Stevens, 1990a) as well as in the present study places the apparent blocking site almost entirely across the transmembrane electric field. Our results demonstrate that the narrow constriction represents a critical blocking site for extracellular Mg¥, yet recent studies examining the block by impermeant organic cations suggest that it is positioned about 0·5-0·6 of the way across the electric field (Villarroel et al 1995;Zarei & Dani, 1995). Since the adjacent NR2A-subunit asparagines form an apparent barrier for Mg¥ influx, it is unlikely that in terms of simple block Mg¥ penetrates any deeper into the channel than to the narrow constriction.…”

Section: Asparagines Forming the Narrow Constriction Contribute Diffementioning

confidence: 58%

“…The second goal was to determine whether the size of the narrow constriction contributes to the block. The size of hydrated Mg¥, at least 0·7 nm, is larger than the estimated 0·55 nm pore size of NMDA receptor channels (Villarroel, Burnashev & Sakmann, 1995;Zarei & Dani, 1995;Wollmuth et al 1996), and Mg¥ block at the narrow constriction could arise by steric occlusion. Since the substitutions change the pore size to a known degree ; see Methods), the contribution of the size of this region to the block can be determined.…”

mentioning

confidence: 84%

“…Since Mg¥ is a small ion (diameter, •0·13 nm; Frausto da Silva & Williams, 1991), giving it a large charge to surface area ratio, the removal of its inner hydration shell would require considerable free energy and its effective size would be considerably larger, at least 0·7 nm. Since NMDA receptor channels have a pore size of about 0·55 nm (Villarroel et al 1995;Zarei & Dani, 1995;Wollmuth et al 1996), hydrated Mg¥ could block NMDA receptor channels at the narrow constriction by steric occlusion. However, the relationship between how substitutions change pore size and block by Mg¥ is poor (Fig.…”

Section: Asparagines Forming the Narrow Constriction Contribute Diffementioning

confidence: 99%

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Adjacent asparagines in the NR2‐subunit of the NMDA receptor channel control the voltage‐dependent block by extracellular Mg²⁺

Wollmuth

Kuner

Sakmann

1998

The Journal of Physiology

138

159

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1. The voltage-dependent block of N-methyl-ª_aspartate (NMDA) receptor channels by extracellular Mg¥ is a critical determinant of its contribution to CNS synaptic physiology. The function of the narrow constriction of the channel in determining the block was investigated by analysing the effects of a set of different amino acid substitutions at exposed residues positioned at or near this region. NMDA receptor channels, composed of wild-type and mutant NR1-and NR2A-subunits, were expressed in Xenopus oocytes or human embryonic kidney (HEK) 293 cells. 2. In wild-type channels, the voltage dependence (ä) of the block by Mg¥ was concentration dependent with values of ä of •0·58 in 0·01 mÒ and •0·82 in 0·07 mÒ and higher concentrations. Under biionic conditions with high extracellular Mg¥ and K¤ as the reference ion, Mg¥ weakly permeated the channel. Over intermediate potentials (•−60 to −10 mV), this weak permeability had no apparent effect on the block but at potentials negative to •−60 mV, it attenuated the extent and voltage dependence of the block. 3. Substitutions of glycine, serine, glutamine or aspartate for the N-site asparagine in the NR1-subunit enhanced the extent of block over intermediate potentials but left the voltage dependence of the block unchanged indicating that structural determinants of the block remained. These same substitutions either attenuated or left unchanged the apparent Mg¥ permeability. 4. In channels containing substitutions of glycine, serine or glutamine for the N-site asparagine in the NR2A-subunit, the block by Mg¥ was reduced at negative potentials. Over intermediate potentials, the block was not strongly attenuated except for the glutamine substitution which reduced the voltage dependence of the block to •0·57 in 0·7 mÒ Mg¥. 5. Equivalent substitutions for the N + 1 site asparagine in the NR2A-subunit strongly attenuated the block over the entire voltage range. In 0·7 mÒ Mg¥, the voltage dependence of the block was reduced to 0·50 (glycine), 0·53 (serine) and 0·46 (glutamine). 6. Channels containing substitutions of the N-site or N + 1 site asparagines in the NR2A-subunit showed an increased Mg¥ permeability suggesting that these adjacent asparagines form a barrier for inward Mg¥ flux. Changes in this barrier contribute, at least in part, to the mechanism underlying disruption of the block following substitution of these residues. 7. The adjacent NR2A-subunit asparagines are positioned at or near the narrow constriction of the channel. Pore size, however, did not determine how effectively Mg¥ blocks mutant channels. 8. It is concluded that, at the narrow constriction in the NMDA receptor channel, the adjacent NR2A-subunit asparagines, the N-site and N + 1 site, but not the N-site asparagine of the NR1-subunit, form a critical blocking site for extracellular Mg¥. The contribution to the blocking site, in contrast to the prevailing view, is stronger for the N + 1 site than for the N_site asparagine. The block may involve binding of Mg¥ to these residues.

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“…After agonist activation, MTSEA can reach N-site residues at the narrowest point of constriction (Ͻ5.5 Å) (Villarroel et al, 1995) in either the NR1 or NR2 subunit. In contrast, none of the cysteine substitutions in the M2 segment of either NR1 or NR3A is accessible to external MTSEA.…”

Section: Discussionmentioning

confidence: 99%

NR3A Modulates the Outer Vestibule of the “NMDA” Receptor Channel

Wada¹,

Takahashi²,

Lipton³

et al. 2006

J. Neurosci.

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Classical NMDA receptors (NMDARs), activated by glycine and glutamate, are heteromultimers comprised of NR1 and NR2 subunits. Coexpression of the novel NR3 family of NMDAR subunits decreases the magnitude of NR1/NR2 receptor-mediated currents or forms glycine-activated channels with the NR1 subunit alone. The second (M2) and third (M3) membrane segments of NR1 and NR2 subunits of classical NMDARs form the core of the channel permeation pathway. Structural information regarding NR1/NR3 channels remains unknown. Using the Xenopus oocyte expression system and the SCAM (substituted cysteine accessibility method), we found that M3 segments of both NR1 and NR3A form a narrow constriction in the outer vestibule of the channel, which prevents passage of externally applied sulfhydryl-specific agents. The most internal reactive residue in each M3 segment is the threonine in the conserved SYTANLAAF motif. These threonines appear to be symmetrically aligned. Several NR3A M3 mutations change the behavior of NR1/NR3A channels. Unlike NR1, however, the M3 segment of NR3A does not undergo extensive molecular rearrangement during channel gating by added glycine. Additionally, in the M2 segment, our data suggest that the amino acid at the asparagine (N) site of NR1, but not NR3A, contributes to the selectivity filter of NR1/3A channels. We therefore conclude that NR3A modulates the NR1/NR3A permeation pathway via a novel mechanism of forming a narrow constriction at the outer channel vestibule. This modified channel vestibule may also explain the dominant-negative effect of the NR3 subunit on channel behavior when coexpressed with NR1 and NR2 subunits.

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Dimensions of the narrow portion of a recombinant NMDA receptor channel

Cited by 113 publications

References 41 publications

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Adjacent asparagines in the NR2‐subunit of the NMDA receptor channel control the voltage‐dependent block by extracellular Mg²⁺

NR3A Modulates the Outer Vestibule of the “NMDA” Receptor Channel

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Dimensions of the narrow portion of a recombinant NMDA receptor channel

Cited by 113 publications

References 41 publications

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Blockage of Mouse Muscle Nicotinic Receptors by Serotonergic Compounds

Adjacent asparagines in the NR2‐subunit of the NMDA receptor channel control the voltage‐dependent block by extracellular Mg2+

NR3A Modulates the Outer Vestibule of the “NMDA” Receptor Channel

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Adjacent asparagines in the NR2‐subunit of the NMDA receptor channel control the voltage‐dependent block by extracellular Mg²⁺