Mutant Cycle Analysis Research Articles

A computational design approach for virtual screening of peptide interactions across K+ channel families

Ion channels represent a large family of membrane proteins with many being well established targets in pharmacotherapy. The ‘druggability’ of heteromeric channels comprised of different subunits remains obscure, due largely to a lack of channel-specific probes necessary to delineate their therapeutic potential in vivo. Our initial studies reported here, investigated the family of inwardly rectifying potassium (Kir) channels given the availability of high resolution crystal structures for the eukaryotic constitutively active Kir2.2 channel. We describe a ‘limited’ homology modeling approach that can yield chimeric Kir channels having an outer vestibule structure representing nearly any known vertebrate or invertebrate channel. These computationally-derived channel structures were tested in silico for ‘docking’ to NMR structures of tertiapin (TPN), a 21 amino acid peptide found in bee venom. TPN is a highly selective and potent blocker for the epithelial rat Kir1.1 channel, but does not block human or zebrafish Kir1.1 channel isoforms. Our Kir1.1 channel-TPN docking experiments recapitulated published in vitro findings for TPN-sensitive and TPN-insensitive channels. Additionally, in silico site-directed mutagenesis identified ‘hot spots’ within the channel outer vestibule that mediate energetically favorable docking scores and correlate with sites previously identified with in vitro thermodynamic mutant-cycle analysis. These ‘proof-of-principle’ results establish a framework for virtual screening of re-engineered peptide toxins for interactions with computationally derived Kir channels that currently lack channel-specific blockers. When coupled with electrophysiological validation, this virtual screening approach may accelerate the drug discovery process, and can be readily applied to other ion channels families where high resolution structures are available.

A self-consistent approach for determining pairwise interactions that underlie channel activation.

Signaling proteins such as ion channels largely exist in two functional forms, corresponding to the active and resting states, connected by multiple intermediates. Multiparametric kinetic models based on sophisticated electrophysiological experiments have been devised to identify molecular interactions of these conformational transitions. However, this approach is arduous and is not suitable for large-scale perturbation analysis of interaction pathways. Recently, we described a model-free method to obtain the net free energy of activation in voltage- and ligand-activated ion channels. Here we extend this approach to estimate pairwise interaction energies of side chains that contribute to gating transitions. Our approach, which we call generalized interaction-energy analysis (GIA), combines median voltage estimates obtained from charge-voltage curves with mutant cycle analysis to ascertain the strengths of pairwise interactions. We show that, for a system with an arbitrary gating scheme, the nonadditive contributions of amino acid pairs to the net free energy of activation can be computed in a self-consistent manner. Numerical analyses of sequential and allosteric models of channel activation also show that this approach can measure energetic nonadditivities even when perturbations affect multiple transitions. To demonstrate the experimental application of this method, we reevaluated the interaction energies of six previously described long-range interactors in the Shaker potassium channel. Our approach offers the ability to generate detailed interaction energy maps in voltage- and ligand-activated ion channels and can be extended to any force-driven system as long as associated "displacement" can be measured.

Multiple Interactions between Cytoplasmic Domains Regulate Slow Deactivation of Kv11.1 Channels

The intracellular domains of many ion channels are important for fine-tuning their gating kinetics. In Kv11.1 channels, the slow kinetics of channel deactivation, which are critical for their function in the heart, are largely regulated by the N-terminal N-Cap and Per-Arnt-Sim (PAS) domains, as well as the C-terminal cyclic nucleotide-binding homology (cNBH) domain. Here, we use mutant cycle analysis to probe for functional interactions between the N-Cap/PAS domains and the cNBH domain. We identified a specific and stable charge-charge interaction between Arg(56) of the PAS domain and Asp(803) of the cNBH domain, as well an additional interaction between the cNBH domain and the N-Cap, both of which are critical for maintaining slow deactivation kinetics. Furthermore, we found that positively charged arginine residues within the disordered region of the N-Cap interact with negatively charged residues of the C-linker domain. Although this interaction is likely more transient than the PAS-cNBD interaction, it is strong enough to stabilize the open conformation of the channel and thus slow deactivation. These findings provide novel insights into the slow deactivation mechanism of Kv11.1 channels.

Role of the Cytoplasmic N-terminal Cap and Per-Arnt-Sim (PAS) Domain in Trafficking and Stabilization of Kv11.1 Channels

The N-terminal cytoplasmic region of the Kv11.1a potassium channel contains a Per-Arnt-Sim (PAS) domain that is essential for the unique slow deactivation gating kinetics of the channel. The PAS domain has also been implicated in the assembly and stabilization of the assembled tetrameric channel, with many clinical mutants in the PAS domain resulting in reduced stability of the domain and reduced trafficking. Here, we use quantitative Western blotting to show that the PAS domain is not required for normal channel trafficking nor for subunit-subunit interactions, and it is not necessary for stabilizing assembled channels. However, when the PAS domain is present, the N-Cap amphipathic helix must also be present for channels to traffic to the cell membrane. Serine scan mutagenesis of the N-Cap amphipathic helix identified Leu-15, Ile-18, and Ile-19 as residues critical for the stabilization of full-length proteins when the PAS domain is present. Furthermore, mutant cycle analysis experiments support recent crystallography studies, indicating that the hydrophobic face of the N-Cap amphipathic helix interacts with a surface-exposed hydrophobic patch on the core of the PAS domain to stabilize the structure of this critical gating domain. Our data demonstrate that the N-Cap amphipathic helix is critical for channel stability and trafficking.

Evaluation of multivalency as an organization principle for the efficient synthesis of doubly and triply threaded amide rotaxanes

Mono-, di- and trivalent pseudorotaxanes with tetralactam macrocycle hosts and axles containing diamide binding stations as the guests have been synthesised. Their threading behaviour was analyzed in detail by NMR experiments and isothermal titration calorimetry. An X-ray crystal structure of the monovalent pseudorotaxane confirms the binding motif. Double mutant cycle analysis provides the effective molarities and insight into the chelate cooperativity of multivalent binding. While the second binding event in a trivalent pseudorotaxane exhibits a slightly positive cooperativity, the third binding is nearly non-cooperative. Nevertheless, the enhanced binding affinities resulting from the multivalent interaction are the basis for a highly efficient synthesis of di- and trivalent rotaxanes through stoppering the axle termini by “click” chemistry. Evidence for the multiply threaded geometry comes from NMR spectroscopy as well as tandem mass-spectrometric fragmentation experiments of mass-selected rotaxane ions in the gas phase. Furthermore, the trivalent rotaxane can be controlled by external stimuli (chloride addition and removal) which lead to an elevator-type movement of the wheel along the axle.

Electrostatic Interactions in the Closed and Open States of Voltage-Gated Proton Channels

Voltage-gated proton channels (Hv1) play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane segment, S4, functions as the voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by counter charges in the other transmembrane segments both in the closed and open states. We construct models of both the closed and open states of Hv1 that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between transmembrane segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward movement of S4 removes the hydrophobic plug, as if this hydrophobic plug works as the gate that prevents protons to permeate the Hv1 channel in the closed state.

Inhibition of Voltage-Gated Hv1 Channel by Guanidine Derivatives

The voltage-gated proton channel Hv1 plays a key role in the regulation of ROS production by NOX enzymes in phagocytic cells. Excessive channel activity enhances proliferation and invasiveness in cancer cells, and worsens brain damage after ischemic stroke. The Hv1 channel is composed of two subunits, each containing a proton-permeable voltage-sensing domain (VSD) and lacking the pore domain typical of other voltage-gated ion channels. We have previously shown that the compound 2-guanidinobenzimidazole (2GBI) inhibits the Hv1 channel and that its binding site in the VSD is accessible from the intracellular side of the membrane only when the channel is open. Here, we examine the apparent binding affinities of a series of 2GBI derivatives on Hv1 channels mutated at positions located in the core of the VSD and apply mutant cycle analysis to determine the most likely interactions between channel and inhibitor. We identify four residues involved in 2GBI binding and determine the orientation of the blocker inside the VSD. Our data provide a simple explanation for the very large difference in binding affinity between 2GBI and the related compound 2-guanidinobenzoxazole (2GBOZ), and suggest ways to design more effective Hv1 inhibitors. We also investigate the molecular characteristics that allow guanidine derivatives to reach the binding site when added from the extracellular side of the membrane so that they can be used to block endogenous proton channels under physiological conditions. This work is supported by NIH -National Institute of General Medical Sciences, grant R01GM098973.

Transducer Residues are Thermodynamically Coupled in the Kinesin-5 Motor Domain

Motor proteins coordinate the activities of nucleotide hydrolysis and polymer binding to move along cellular tracks. In kinesin and myosin motor domains, the sites responsible for these two activities are located on opposite faces of a core beta-sheet. Three beta-strands and a mobile loop transduce information between the active and polymer-binding sites and, thus, are collectively termed the transducer. Herein we demonstrate residues in the core beta-sheet and the L5 loop are thermodynamically coupled. Two methods were employed in this study. Probabilistic methods to analyze evolutionarily correlations between residues in protein families identified coupling between transducer residues. Second, thermodynamic linkage between M115 in loop-5 and L263 in beta-7 in the human kinesin-5 motor domain was established using double mutant cycle analysis for wildtype, two single mutants, and the corresponding double mutant protein. The resulting changes in free energy were calculated from experimentally measured kinetic parameters and determined to be non-additive. While conformational changes of the beta strands and mobile loop have been observed in structural investigations of kinesins and myosins, these experiments establish energetic and evolutionary coupling of these distinct motifs. We conclude that loop-5 and the beta-strands are important kinesin building blocks that act in concert during mechanotransduction. This work was funded by NIH (GM097350 to SK).

Effect of Charged Amino Acid Side Chain Length on Lateral Cross-Strand Interactions between Carboxylate-Containing Residues and Lysine Analogues in a β-Hairpin

β-Sheets are one of the fundamental three-dimensional building blocks for protein structures. Oppositely charged amino acids are frequently observed directly across one another in antiparallel sheet structures, suggesting the importance of cross-strand ion pairing interactions. Despite the apparent electrostatic nature of ion pairing interactions, the charged amino acids Asp, Glu, Arg, Lys have different numbers of hydrophobic methylenes linking the charged functionality to the backbone. Accordingly, the effect of charged amino acid side chain length on cross-strand ion pairing interactions at lateral non-hydrogen bonded positions was investigated in a β-hairpin motif. The negatively charged residues with a carboxylate (Asp, Glu, Aad in increasing length) were incorporated at position 4, and the positively charged residues with an ammonium (Dap, Dab, Orn, Lys in increasing length) were incorporated at position 9. The fraction folded population and folding free energy were derived from the chemical shift deviation data. Double mutant cycle analysis was used to determine the interaction energy for the potential lateral ion pairs. Only the Asp/Glu-Dap interactions with shorter side chains and the Aad-Orn/Lys interactions with longer side chains exhibited stabilizing energetics, mostly relying on electrostatics and hydrophobics, respectively. This suggested the need for length matching of the interacting residues to stabilize the β-hairpin motif. A survey of a nonredundant protein structure database revealed that the statistical sheet pair propensity followed the trend Asp-Lys < Glu-Lys, also implying the need for length matching of the oppositely charged residues.

Hot‐Spot Residues in the Cytochrome P450cam–Putidaredoxin Binding Interface

Cytochrome P450cam (P450cam) is a heme-containing monooxygenase that catalyzes the hydroxylation of D-camphor to produce 5-exo-hydroxycamphor. The catalytic cycle of P450cam requires two electrons, both of which are donated by putidaredoxin (Pdx), a ferredoxin containing a [2 Fe-2 S] cluster. Atomic-resolution structures of the Pdx-P450cam complex have recently been solved by X-ray crystallography and paramagnetic NMR spectroscopy. The binding interface showed the potential electron transfer pathways and interactions between Pdx Asp38 and P450cam Arg112, as well as hydrophobic contacts between the Pdx Trp106 and P450cam residues. Several polar residues not previously recognized as relevant for binding were found in the interface. In this study, site-directed mutagenesis, kinetic measurements, and NMR studies were employed to probe the energetic importance and role of the polar residues in the Pdx-P450cam interaction. A double mutant cycle (DMC) analysis of kinetic data shows that favorable interactions exist between Pdx Tyr33 and P450cam Asp125, as well as between Pdx Ser42 and P450cam His352. The results show that alanine substitutions of these residues and several others do not influence the rates of electron transfer. It is concluded that these polar interactions contribute to partner recognition rather than to electronic coupling of the redox centers.

Analysis of Hyperekplexia Mutations Identifies Transmembrane Domain Rearrangements That Mediate Glycine Receptor Activation

Pentameric ligand-gated ion channels (pLGICs) mediate numerous physiological processes and are therapeutic targets for a wide range of clinical indications. Elucidating the structural differences between their closed and open states may help in designing improved drugs that bias receptors toward the desired conformational state. We recently showed that two new hyperekplexia mutations, Q226E and V280M, induced spontaneous activity in α1 glycine receptors. Gln-226, located near the top of transmembrane (TM) 1, is closely apposed to Arg-271 at the top of TM2 in the neighboring subunit. Using mutant cycle analysis, we inferred that Q226E induces activation via an enhanced electrostatic attraction to Arg-271. This would tilt the top of TM2 toward TM1 and hence away from the pore axis to open the channel. We also concluded that the increased side chain volume of V280M, in the TM2-TM3 loop, exerts a steric repulsion against Ile-225 at the top of TM1 in the neighboring subunit. We infer that this steric repulsion would tilt the top of TM3 radially outwards against the stationary TM1 and thus provide space for TM2 to relax away from the pore axis to create an open channel. Because the transmembrane domain movements inferred from this functional analysis are consistent with the structural differences evident in the x-ray atomic structures of closed and open state bacterial pLGICs, we propyose that the model of pLGIC activation as outlined here may be broadly applicable across the eukaryotic pLGIC receptor family.

Cooperative Activation of the T-type CaV3.2 Channel: INTERACTION BETWEEN DOMAINS II AND III

T-type CaV3 channels are important mediators of Ca(2+) entry near the resting membrane potential. Little is known about the molecular mechanisms responsible for channel activation. Homology models based upon the high-resolution structure of bacterial NaV channels predict interaction between the S4-S5 helix of Domain II (IIS4-S5) and the distal S6 pore region of Domain II (IIS6) and Domain III (IIIS6). Functional intra- and inter-domain interactions were investigated with a double mutant cycle analysis. Activation gating and channel kinetics were measured for 47 single mutants and 20 pairs of mutants. Significant coupling energies (ΔΔG(interact) ≥ 1.5 kcal mol(-1)) were measured for 4 specific pairs of mutants introduced between IIS4-S5 and IIS6 and between IIS4-S5 and IIIS6. In agreement with the computer based models, Thr-911 in IIS4-S5 was functionally coupled with Ile-1013 in IIS6 during channel activation. The interaction energy was, however, found to be stronger between Val-907 in IIS4-S5 and Ile-1013 in IIS6. In addition Val-907 was significantly coupled with Asn-1548 in IIIS6 but not with Asn-1853 in IVS6. Altogether, our results demonstrate that the S4-S5 and S6 helices from adjacent domains are energetically coupled during the activation of a low voltage-gated T-type CaV3 channel.

Molecular determinants of common gating of a ClC chloride channel

Uniquely, the ClC family harbours dissipative channels and anion/H(+) transporters that share unprecedented functional characteristics. ClC-1 channels are homodimers in which each monomer supports an identical pore carrying three anion-binding sites. Transient occupancy of the extracellular binding site by a conserved glutamate residue, E232, independently gates each pore. A common gate, the molecular basis of which is unknown, closes both pores simultaneously. Mutations affecting common gating underlie myotonia congenita in humans. Here we show that the common gate likely occludes the channel pore via interaction of E232 with a highly conserved tyrosine, Y578, at the central anion-binding site. We also identify structural linkages important for coordination of common gating between subunits and modulation by intracellular molecules. Our data reveal important molecular determinants of common gating of ClC channels and suggest that the molecular mechanism is an evolutionary vestige of coupled anion/H(+) transport.

P.12.5 Subunit specific effects of the invariant AChR Cys-loop Aspartate residues on gating and AChR expression

The signature Cys-loop at the interface between binding and gating domains of AChR is crucial for coupling agonist binding to gating but the mechanism is not fully understood. An invariant Cys-loop Asp is present at equivalent positions in all subunits of Cys-loop superfamily receptors of all species, but its functional significance is not known. After discovering that a phenotype-determining Asp140Asn in the AChR δ subunit causes a severe congenital myasthenic syndrome (CMS), we introduced the same mutation into the AChR α, β, and e subunits and examined the expression, burst openings, and gating efficiency of each mutant in HEK cells. We employed mutation analysis, mutagenesis, single-channel recordings, and mutant cycle analysis using wild-type and mutant receptors expressed in HEK cells. Compared to wild-type, the respective protein expressions of the α, β, δ and e mutants were 54%, 79%, 21%, and 100%; the corresponding burst durations were 7%, 18%, 55%, and 62%; and the corresponding gating efficiencies were 1%, 23%, 75%, and 108%. To understand why α Asp138Asn has the greatest effects on gating, we examined its thermodynamic interactions with nearby residues by mutant cycle analysis. This revealed that corresponding αAsp138 is functionally linked to nearby αGln208 which is conserved in mammalian α-subunits and is adjacent to the invariant αArg209 which was previously shown to be essential for rapid and efficient gating. (1) The invariant Cys-loop Asp residues contribute to receptor expression, burst duration, and gating efficiency in a subunit-specific manner. (2) The α-subunit Asp has profound effects on burst length and gating efficiency whereas δ-Asp predominantly affects AChR expression. (3) Mutant cycle analysis indicates that αAsp138 is an essential link in the chain of conformational changes that couple agonist binding to gating. (4) The δAsp140Asn causes CMS mainly by reducing AChR expression.

Functional interactions of voltage sensor charges with an S2 hydrophobic plug in hERG channels

Human ether-à-go-go–related gene (hERG, Kv11.1) potassium channels have unusually slow activation and deactivation kinetics. It has been suggested that, in fast-activating Shaker channels, a highly conserved Phe residue (F290) in the S2 segment forms a putative gating charge transfer center that interacts with S4 gating charges, i.e., R362 (R1) and K374 (K5), and catalyzes their movement across the focused electric field. F290 is conserved in hERG (F463), but the relevant residues in the hERG S4 are reversed, i.e., K525 (K1) and R537 (R5), and there is an extra positive charge adjacent to R537 (i.e., K538). We have examined whether hERG channels possess a transfer center similar to that described in Shaker and if these S4 charge differences contribute to slow gating in hERG channels. Of five hERG F463 hydrophobic substitutions tested, F463W and F463Y shifted the conductance–voltage (G-V) relationship to more depolarized potentials and dramatically slowed channel activation. With the S4 residue reversals (i.e., K525, R537) taken into account, the closed state stabilization by F463W is consistent with a role for F463 that is similar to that described for F290 in Shaker. As predicted from results with Shaker, the hERG K525R mutation destabilized the closed state. However, hERG R537K did not stabilize the open state as predicted. Instead, we found the neighboring K538 residue to be critical for open state stabilization, as K538R dramatically slowed and right-shifted the voltage dependence of activation. Finally, double mutant cycle analysis on the G-V curves of F463W/K525R and F463W/K538R double mutations suggests that F463 forms functional interactions with K525 and K538 in the S4 segment. Collectively, these data suggest a role for F463 in mediating closed–open equilibria, similar to that proposed for F290 in Shaker channels.

Hydrophobic interactions between the voltage sensor and pore mediate inactivation in Kv11.1 channels

Kv11.1 channels are critical for the maintenance of a normal heart rhythm. The flow of potassium ions through these channels is controlled by two voltage-regulated gates, termed “activation” and “inactivation,” located at opposite ends of the pore. Crucially in Kv11.1 channels, inactivation gating occurs much more rapidly, and over a distinct range of voltages, compared with activation gating. Although it is clear that the fourth transmembrane segments (S4), within each subunit of the tetrameric channel, are important for controlling the opening and closing of the activation gate, their role during inactivation gating is much less clear. Here, we use rate equilibrium free energy relationship (REFER) analysis to probe the contribution of the S4 “voltage-sensor” helix during inactivation of Kv11.1 channels. Contrary to the important role that charged residues play during activation gating, it is the hydrophobic residues (Leu529, Leu530, Leu532, and Val535) that are the key molecular determinants of inactivation gating. Within the context of an interconnected multi-domain model of Kv11.1 inactivation gating, our REFER analysis indicates that the S4 helix and the S4–S5 linker undergo a conformational rearrangement shortly after that of the S5 helix and S5P linker, but before the S6 helix. Combining REFER analysis with double mutant cycle analysis, we provide evidence for a hydrophobic interaction between residues on the S4 and S5 helices. Based on a Kv11.1 channel homology model, we propose that this hydrophobic interaction forms the basis of an intersubunit coupling between the voltage sensor and pore domain that is an important mediator of inactivation gating.

Q/R site interactions with the M3 helix in GluK2 kainate receptor channels revealed by thermodynamic mutant cycles

RNA editing at the Q/R site near the apex of the pore loop of AMPA and kainate receptors controls a diverse array of channel properties, including ion selectivity and unitary conductance and susceptibility to inhibition by polyamines and cis-unsaturated fatty acids, as well as subunit assembly into tetramers and regulation by auxiliary subunits. How these different aspects of channel function are all determined by a single amino acid substitution remains poorly understood; however, several lines of evidence suggest that interaction between the pore helix (M2) and adjacent segments of the transmembrane inner (M3) and outer (M1) helices may be involved. In the present study, we have used double mutant cycle analysis to test for energetic coupling between the Q/R site residue and amino acid side chains along the M3 helix. Our results demonstrate interaction with several M3 locations and particularly strong coupling to substitution for L614 at the level of the central cavity. In this location, replacement with smaller side chains completely and selectively reverses the effect of fatty acids on gating of edited channels, converting strong inhibition of wild-type GluK2(R) to nearly 10-fold potentiation of GluK2(R) L614A.

Human PIEZO1: Removing Inactivation

PIEZO1 is an inactivating eukaryotic cation-selective mechanosensitive ion channel. Two sites have been located in the channel that when individually mutated lead to xerocytotic anemia by slowing inactivation. By introducing mutations at two sites, one associated with xerocytosis and the other artificial, we were able to remove inactivation. The double mutant (DhPIEZO1) has a substitution of arginine for methionine (M2225R) and lysine for arginine (R2456K). The loss of inactivation was accompanied by ∼30-mmHg shift of the activation curve to lower pressures and slower rates of deactivation. The slope sensitivity of gating was the same for wild-type and mutants, indicating that the dimensional changes between the closed and open state are unaffected by the mutations. The unitary channel conductance was unchanged by mutations, so these sites are not associated with pore. DhPIEZO1 was reversibly inhibited by the peptide GsMTx4 that acted as a gating modifier. The channel kinetics were solved using complex stimulus waveforms and the data fit to a three-state loop in detailed balance. The reaction had two pressure-dependent rates, closed to open and inactivated to closed. Pressure sensitivity of the opening rate with no sensitivity of the closing rate means that the energy barrier between them is located near the open state. Mutant cycle analysis of inactivation showed that the two sites interacted strongly, even though they are postulated to be on opposite sides of the membrane.

Insights on the mechanisms of Ca2+ regulation of connexin26 hemichannels revealed by human pathogenic mutations (D50N/Y)

Because of the large size and modest selectivity of the connexin hemichannel aqueous pore, hemichannel opening must be highly regulated to maintain cell viability. At normal resting potentials, this regulation is achieved predominantly by the physiological extracellular Ca2+ concentration, which drastically reduces hemichannel activity. Here, we characterize the Ca2+ regulation of channels formed by wild-type human connexin26 (hCx26) and its human mutations, D50N/Y, that cause aberrant hemichannel opening and result in deafness and skin disorders. We found that in hCx26 wild-type channels, deactivation kinetics are accelerated as a function of Ca2+ concentration, indicating that Ca2+ facilitates transition to, and stabilizes, the closed state of the hemichannels. The D50N/Y mutant hemichannels show lower apparent affinities for Ca2+-induced closing than wild-type channels and have more rapid deactivation kinetics, which are Ca2+ insensitive. These results suggest that D50 plays a role in (a) stabilizing the open state in the absence of Ca2+, and (b) facilitating closing and stabilization of the closed state in the presence of Ca2+. To explore the role of a negatively charged residue at position 50 in regulation by Ca2+, this position was substituted with a cysteine residue, which was then modified with a negatively charged methanethiosulfonate reagent, sodium (2-sulfanoethyl) methanethiosulfonate (MTSES)−. D50C mutant hemichannels display properties similar to those of D50N/Y mutants. Recovery of the negative charge with chemical modification by MTSES− restores the wild-type Ca2+ regulation of the channels. These results confirm the essential role of a negative charge at position 50 for Ca2+ regulation. Additionally, charge-swapping mutagenesis studies suggest involvement of a salt bridge interaction between D50 and K61 in the adjacent connexin subunit in stabilizing the open state in low extracellular Ca2+. Mutant cycle analysis supports a Ca2+-sensitive interaction between these two residues in the open state of the channel. We propose that disruption of this interaction by extracellular Ca2+ destabilizes the open state and facilitates hemichannel closing. Our data provide a mechanistic understanding of how mutations at position 50 that cause human diseases are linked to dysfunction of hemichannel gating by external Ca2+.

Function of Interfacial Prolines at the Transmitter-binding Sites of the Neuromuscular Acetylcholine Receptor

The neuromuscular acetylcholine (ACh) receptor has two conserved prolines in loop D of the complementary subunit at each of its two transmitter-binding sites (α-ε and α-δ). We used single-channel electrophysiology to estimate the energy changes caused by mutations of these prolines with regard to unliganded gating (ΔG0) and the affinity change for ACh that increases the open channel probability (ΔGB). The effects of mutations of ProD2 (εPro-121/δPro-123) were greater than those of its neighbor (εPro-120/δPro-122) and were greater at α-ε versus α-δ. The main consequence of the congenital myasthenic syndrome mutation εProD2-L was to impair the establishment of a high affinity for ACh and thus make ΔGB less favorable. At both binding sites, most ProD2 mutations decreased constitutive activity (increased ΔG0). LRYHQG and RL substitutions reduced substantially the net binding energy (made ΔGB(ACh) less favorable) by ≥2 kcal/mol at α-ε and α-δ, respectively. Mutant cycle analyses were used to estimate energy coupling between the two ProD2 residues and between each ProD2 and glycine residues (αGly-147 and αGly-153) on the primary (α subunit) side of each binding pocket. The distant binding site prolines interact weakly. ProD2 interacts strongly with αGly-147 but only at α-ε and only when ACh is present. The results suggest that in the low to-high affinity change there is a concerted inter-subunit strain in the backbones at εProD2 and αGly-147. It is possible to engineer receptors having a single functional binding site by using a α-ε or α-δ ProD2-R knock-out mutation. In adult-type ACh receptors, the energy from the affinity change for ACh is approximately the same at the two binding sites (approximately -5 kcal/mol).