Acetylcholine receptor gating is influenced by the polarity of amino acids at position 9' in the M2 domain.

Ligand-gated ion channels contain a conserved leucine at position 9' (L9') in the M2 transmembrane domain. We used multiple substitutions at this position in the gamma subunit of the mouse acetylcholine receptor (AChR) (gammaL9') to examine the role of residue polarity at this position in the gating process at both the macroscopic and single-channel levels. The midpoint of the macroscopic dose-response relationship (EC(50)) and the channel closing rate constant, alpha, decreased as the polarity of the residue at that position increased, suggesting a stabilization of the open state of the channel. Both parameters showed similar dependencies on the polarity of the substituted residue. These data support the notion that during AChR gating, the amino acid at the 9' position moves into a polar environment, and that interactions between this residue and the polar environment determine the stability of the open state. Since this residue is conserved in all other members of the ligand-gated ion channel family, we suggest that a similar mechanism applies to the other members of the family.

Inactivation and secondary structure in the D4/S4-5 region of the SkM1 sodium channel.

The D4/S4-5 interhelical region plays a role in sodium channel fast inactivation. Examination of S4-5 primary structure in all domains suggests a possible amphipathic helical conformation in which a conserved group of small hydrophobic residues occupies one contiguous surface with a more variable complement of nonpolar and polar residues on the opposite face. We evaluated this potential structure by replacing each residue in D4/S4-5 of the rat SkM1 skeletal muscle sodium channel with substitutions having different side chain properties. Of the 63 mutations analyzed, 44 produced functional channels. P1473 was intolerant of substitutions. Nonpolar substitutions in the conserved hydrophobic region were functionally similar to wild type, while charged mutations in this region before P1473 were nonfunctional. Charged mutations at F1466, M1469, M1470, and A1474, located on the opposite surface of the predicted helix, produced functional channels with pronounced slowing of inactivation, shifted voltage dependence of steady-state inactivation, and increased rate of recovery from inactivation. The substituted-cysteine-accessibility method was used to probe accessibility at each position. Residues L1465, F1466, A1467, M1469, M1470, L1472, A1474, and F1476C were easily accessible for modification by sulfhydryl reagents; L1464, L1468, S1471, and L1475 were not accessible within the time frame of our measurements. Molecular dynamics simulations of residues A1458 to N1477 were then used to explore energetically favorable local structures. Based on mutagenesis, substituted-cysteine-accessibility method, and modeling results, we suggest a secondary structure for the D4/S4-5 region in which the peptide chain is alpha-helical proximal to P1473, bends at this residue, and may continue beyond this point as a random coil. In this configuration, the entire resultant loop is amphipathic; four residues on one surface could form part of the binding site for the inactivation particle.

Analysis of local structure in the D2/S1-S2 region of the rat skeletal muscle type 1 sodium channel using insertional mutagenesis.

A reporter epitope was inserted at 11 positions in a region encompassing proposed transmembrane segments S1 and S2 in the second repeat domain (D2) of the rat skeletal muscle type 1 sodium channel. All mutations produced full-length membrane-associated protein following transfection into cultured cells, although the level of expression varied with insertion position. Characterization of cognate cRNAs for each mutation in Xenopus oocytes by two-electrode voltage clamp defined a permissive region between the proposed transmembrane regions in which these large insertions did not interfere with channel function. Two of the mutations, in which the point of insertion was within the proposed S1-S2 loop, demonstrated extracellular membrane labeling when studied either by antibody binding in oocytes or by confocal analysis following transfection into primary muscle cells. Our results define the likely boundaries of an extramembrane region linking the S1 and S2 transmembrane segments in D2 and confirm the extracellular location of this S1-S2 loop predicted by current models of channel tertiary structure.

The role of conserved leucines in the M2 domain of the acetylcholine receptor in channel gating.

A highly conserved leucine is found in the middle of the porelining (M2) domain of the members of the ligand-gated ion channel family. Two very different roles have been proposed for this leucine. In one model, this residue swings into the lumen of the channel during desensitization to form the nonconducting desensitized state, whereas in the other model, the leucines from each subunit interact with each other to form a constriction in the channel that constitutes the actual gate of the channel. We examined the role of this leucine in the muscle-type acetylcholine receptor by replacing it with the polar amino acid threonine. Replacement of the leucine in any one subunit slows desensitization and shifts the dose-response relationship toward lower concentrations. Replacement of leucines in additional subunits leads to progressively larger shifts in the dose-response curves. The shift depends only on the number of leucines replaced, not on which particular subunits contain the mutation; in other words, the mutations act independently. At the single-channel level, the mutation greatly increases the channel mean open time. We conclude that the role of the conserved leucine is to set the mean open time of the channel through interactions with other regions of the receptor rather than to serve as the gate per se of the ion channel.

Characterization of d-tubocurarine binding site of Torpedo acetylcholine receptor.

d-Tubocurarine (curare) is a well-characterized competitive antagonist of nicotinic acetylcholine receptors (AChRs), and it is usually assumed that curare and agonists share a common binding site. We have examined the role of several highly conserved residues of the alpha-, gamma-, and delta-subunits in the interaction of curare with the Torpedo acetylcholine receptor (AChR). Curare inhibition of wild-type receptors is consistent with curare binding to a single high-affinity binding site [inhibitor constant (Ki) = 20 nM]. Phenylalanine substitutions for two tyrosine residues implicated as being in the ligand binding site (alpha Y93F, alpha Y190F) reduce curare affinity, indicating that these residues are also important for high-affinity curare binding. Phenylalanine substitution for alpha Y198 [alpha Y198F (notation used here: subunit/amino acid in wild-type/residue number/substitution)] causes a 10-fold increase in curare affinity (Ki = 3.1 nM), and measurement of the recovery from curare inhibition indicates that this increase in affinity is due to a reduction in the rate of curare dissociation from the receptor. In addition to the alpha-subunits, portions of the ligand binding sites also reside on the gamma- and delta-subunits, and photoaffinity studies have implicated two residues (gamma W55 and delta W57) as forming part of the curare sites. The gamma W55L mutation results in an eightfold decrease in curare affinity (Ki = 170 nM), whereas the delta W57L mutation has no effect. These data support the notion that the high-affinity curare binding site is formed by segments of the alpha- and gamma-subunits.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective enhancement of the interaction of curare with the nicotinic acetylcholine receptor.

Alteration of the ligand-binding domain of the nicotinic acetylcholine receptor through site-directed mutagenesis offers a powerful approach to the elucidation of structure-function relations in the receptor. Several conserved tyrosine residues in the large extracellular amino terminus of the alpha subunit of the receptor have been implicated by both chemical labeling and mutagenesis studies as playing an important role in the interaction of acetylcholine with the receptor. We and others have previously shown that substitution of phenylalanine for tyrosine at position 198 of the alpha subunit (alpha Y198F) leads to a rightward shift in the dose-response curve for acetylcholine-elicited currents. We have further investigated this particular mutation by examining the interaction of the competitive antagonist d-tubocurarine (curare) with the receptor. In contrast to the effect on the interaction of agonists with the receptor, this mutation leads to a marked increase in the affinity of the receptor for curare. Furthermore, this enhancement in affinity is selective for curare and is not seen with other competitive antagonists (pancuronium, beta-erythroidine, and gallamine). Examination of the structures of these competitive antagonists leads to the proposal that this enhancement is due to the formation of an aromatic-aromatic interaction between the phenylalanine ring at position alpha 198 in the mutant and one of the aromatic rings of curare and that this can provide information about the spatial arrangement of this residue in the binding site.

On the activation of phosphodiesterase by a 26 kDa protein.

The effects of a 26 kDa protein isolated from vertebrate retina rod outer segments (ROS) and its reconstituted analog on the phosphodiesterase (PDE) activity and cGMP-dependent conductance have been studied [Nature 313 (1985) 310-313]. Using the patch-clamp technique it was shown that the 26 kDa protein in concentrations up to 1 microM accelerates hydrolysis of cGMP by near-membrane PDE by 1-2 orders of magnitude. This process is suggested to be mediated by some intracellular agent. At the same concentrations the 26 kDa protein was shown to inhibit cGMP-dependent conductance of the photoreceptor membrane. A possible role of these effects in the processes of phototransduction and adaptation is discussed.

Antiidiotypic antibodies against anti-cGMP polyclonal antibodies.

Affinity-purified polyclonal anti-cGMP antibodies were obtained from rabbit serum after immunization by succinyl derivative of cGMP coupled to bovine serum albumin. These antibodies were used to raise antiidiotypic antibodies in rats. Putative antiidiotypic serum inhibited the binding of [3H]cGMP to affinity-purified anti-cGMP antibodies. The influence of immunoglobulins isolated from antiidiotypic serum on the ion conductance of rod outer segment plasma membrane fragments from frog retina was studied in patch-clamp experiments. These immunoglobulins increased the conductance of ion channels acting like a natural agonist (cGMP). Preimmune immunoglobulins did not act. The data obtained suggest that antiidiotypic antibodies interact with regulatory cGMP-binding sites of the plasma membrane channels.

Antiidiotypic antibodies interacting with cGMP-dependent channels of frog retinal rod outer segments.

Antiidiotypic approach was used to obtain antibodies interacting with cGMP-binding site of the cGMP-activated channel of the photoreceptor cell. Monoclonal anti-BrcGMP antibodies having characteristics of binding of agonist and its analogs close to those for a natural receptor have been obtained. These antibodies were used to raise polyclonal antiidiotypic antibodies capable of interacting with a natural cGMP-receptor. Application of immunoglobulins, isolated from antiidiotypic serum, to inside-out fragments of the rod plasma membrane led to an irreversible increase of the conductance of cGMP-dependent channels.

Regulation of cGMP-dependent conductance in cytoplasmic membrane of rod outer segments by transducin.

A preparation of the photoreceptor G-protein, transducin, containing mainly the T alpha-subunit in a GTP-gamma-S-bound form, has been used for perfusion of the intracellular surface of excised patches of rod outer segment cytoplasmic membrane from frog retina. The preparation has been shown to result in the complete suppression of the cGMP-activated ionic conductance of the cytoplasmic membrane patch. The effect is entirely reversible after the protein has been washed out and is not observed in the absence of cGMP. The degree of conductance inhibition depends on the protein concentration, half-maximal inhibition occurring at 1 microM T alpha-GTP-gamma-S.

The effect of ATP, GTP and cAMP on the cGMP-dependent conductance of the fragments from frog rod plasma membrane.

Using a 'patch-clamp' method in the 'inside-out' configuration, ATP, ADP, AMP-PCP and AMP-PNP have been shown to increase the cGMP-dependent component of the rod plasma membrane conductance 2-4-fold and GTP, GDP but not GMP or nonhydrolyzable GTP analogs GMP-PNP and GTP-gamma-S to abolish the ATP action. The ATP and GTP effects were observed at [EDTA] = 1 mM when magnesium and calcium ions were absent. In about half of the experiments the cGMP-dependent conductance was shown to be increased by cAMP in the micromolar concentration range by 10-50%, the cAMP action did not depend on the presence of nucleoside triphosphates. In vivo ATP, GTP and cAMP are assumed to modulate the sensitivity of the photoreceptor plasma membrane to cGMP.