s2fieldsofstudy: Biology Engineering

text:
Passive Muscle-Tendon Unit Gearing Is Joint Dependent in Human Medial Gastrocnemius

Skeletal muscles change length and develop force both passively and actively. Gearing allows muscle fiber length changes to be uncoupled from those of the whole muscle-tendon unit. During active contractions this process allows muscles to operate at mechanically favorable conditions for power or economical force production. Here we ask whether gearing is constant in passive muscle; determining the relationship between fascicle and muscle-tendon unit length change in the bi-articular medial gastrocnemius and investigating the influence of whether motion occurs at the knee or ankle joint. Specifically, the same muscle-tendon unit length changes were elicited by rotating either the ankle or knee joint whilst simultaneously measuring fascicle lengths in proximal and distal muscle regions using B-mode ultrasound. In both the proximal and distal muscle region, passive gearing values differed depending on whether ankle or knee motion occurred. Fascicle length changes were greater with ankle motion, likely reflecting anatomical differences in proximal and distal passive tendinous tissues, as well as shape changes of the adjacent mono-articular soleus. This suggests that there is joint-dependent dissociation between the mechanical behavior of muscle fibers and the muscle-tendon unit during passive joint motions that may be important to consider when developing accurate models of bi-articular muscles.

INTRODUCTION
Skeletal muscles are an organ whose primary function is to produce force and hence movement. They are a structurally organized, composite material containing multiple contractile elements, internal and external connective tissues as well as nerve and vascular tissues. They often span two or more joints and many reports have evidenced different geometric arrangements of fibers both between and within individual muscles (for a review see: English et al., 1993); differences in connective tissue properties at sites of origin and insertion (Dalmau-Pastor et al., 2014) and complex myofascial connections between groups of muscles (Ahn et al., 2003;Higham and Biewener, 2011).
The forces developed within a muscle-tendon unit (MTU) during contraction are in part dependent upon the activation state, length, velocity, and orientation of the contracting fibers (Hill, 1938;Katz, 1939). The force-velocity relationship of muscle fibers shows that slower shortening velocities result in higher force and mechanical power production is reduced at higher velocities (Hill, 1938), suggesting a mechanical/energetic advantage may be gained if mechanisms to prevent fibers operating at very high shortening velocities during completion of a motor task are present.
In muscles with pennate fascicle architecture, the fascicles (i.e., fiber bundles) are arranged obliquely to the mid-line of the muscle ( Figure 1A). As the muscle belly shortens, the fascicles both shorten and rotate to greater pennation angles. Such fiber rotation means that the fiber velocity (V f ) can be uncoupled from that of the muscle tendon unit (V MTU ): a process termed MTU gearing .
In active contractions variable MTU gearing (V MTU /V f ) is a well-documented phenomenon, and suggests the presence of underlying mechanisms that enable fibers to shorten at optimal velocities for power production across a range of MTU velocities (Azizi et al., 2008;Wakeling et al., 2011). Variations in gearing are likely to be driven by the pattern of muscle activation which influences the forces developed within the muscle and underpins an ability for an individual muscle to complete tasks with different mechanical demands (Higham et al., 2008).
Muscles, however, also change length and generate forces passively. During many cyclical motor tasks (e.g., stepping, stair negotiation, cycling), there are alternating phases of muscle activation and phases of quiescence. During quiescence, MTU and fiber length changes still occur as joint angles are modified by the action of antagonistic and/or synergistic muscles or external loads. Without active forces being developed within the muscle, passive MTU gearing could be influenced by the kinematics of the movement with implications for the force generating ability of the muscle during the initial period of activation (i.e., influence fiber operating length at the onset of activation).
In humans the medial gastrocnemius muscle (MG) is a biarticular plantar flexor of the foot which spans both the knee and ankle joints. It has a simple uni-pennate fascicle architecture (Wolf and Kim, 1997), with fascicles tending to be shorter and more pennate in the more proximal region of the muscle (Narici et al., 1996). It originates from a short tendon attached to the medial epicondyle of the femur and inserts via the long Achilles tendon onto the calcaneus. Dynamic changes in MG fascicle geometric properties can also be studied relatively easily using B-mode ultrasound imaging (Namburete et al., 2011;Rana and Wakeling, 2011;Darby et al., 2013). Previous work, exploring the relationship between fascicle and MTU length changes in MG, has shown that during passive ankle joint rotation only a small proportion (∼27-30%) of the total MTU length change is reflected in the muscle fascicles (Herbert et al., 2002(Herbert et al., , 2011(Herbert et al., , 2014, which would lead to MTU gearing values greater than one. MG is however a bi-articular muscle (spanning both knee and ankle joint) and, as such, does undergo passive length changes as a result of movement of either the joints spanned (Tian et al., 2012).
In addition to the influence of joint kinematics, the tendons of origin and insertion of MG have very different characteristics. Specifically, the MG tendon of origin is much shorter and thicker than the long Achilles tendon, which is also shared with the synergistic soleus and lateral gastrocnemius muscles (Dalmau-Pastor et al., 2014). MTU gearing may therefore differ between passive ankle and knee joint rotation due to the influence of these tendinous structures. Further to this, given the material properties of MG and potential for epimuscular force transmission (Huijing, 2009), fascicle length changes in proximal and distal regions are likely to be affected differently by ankle or knee joint rotation. This could lead to regional variation in MTU gearing. To our knowledge no-one has previously reported passive MTU gearing in a bi-articular muscle resulting from independent rotation of each of the joints spanned. The purpose of this study was therefore to: (i) test whether fascicle and MTU velocity were uncoupled during passive movements of human MG; (ii) determine if MTU gearing varied in MG, depending on the joint at which motion occurred.

Overview of Experimental Approach
To test whether MTU gearing in MG was joint dependent, a combination of simulation and experimental work was completed with methods selected to provide: (i) estimation of MTU lengths that occurred within a range of both knee and ankle joint angles; and (ii) measures of fascicle geometric properties (e.g., fascicle length and pennation angle) that occurred for equivalent MTU lengths within the defined joint angle ranges of motion. The objective was to determine proximal and distal fascicle geometric property changes for the same MTU length change in both ankle and knee joint rotation conditions and hence calculate MTU gearing in each muscle region.

Participants
Eight healthy volunteers (six male, mean ± S.D. Age: 27.5 ± 1.9 years; height: 175.48 ± 7.16 cm; mass: 70.44 ± 7.44 kg) gave written informed consent to take part in the study, which was approved by Manchester Metropolitan University Local Ethics Committee and complied with principles laid down by the Declaration of Helsinki.

Muscle-Tendon Unit Length Estimation
For bi-articular muscles, the MTU length change is determined by the angle of the two joints spanned and their respective moment arms. Participant specific knee and ankle moment arm and shank length measures were therefore used to scale a kinematics model in computer simulation software (Delp et al., 2007). The model was used to estimate the relationship between MTU length and joint angle and enable identification of the ranges of knee and ankle joint angles that resulted in the same range of MTU lengths, which were subsequently used in the experimental protocol. Specifically, MTU length as a function of knee joint angle was calculated with ankle joint considered to be fixed at 0 • (foot segment perpendicular to the tibia), while MTU length as a function of ankle joint angle was calculated with the knee joint considered fixed at 180 • (full extension).
To determine proximal and distal MG moment arms, participants lay supine on the bed of a MRI scanner (0.25 Tesla, Esaote, G-Scan, Genova, Italy), with their right leg inserted into a coil, positioned to image either the ankle or knee joint. Axial images (T1-weighted spin echo protocol: repetition time: 580 ms; echo time: 16 ms; number of excitations: 1·0, field of view: 150 × FIGURE 1 | (A) Schematic of collected data. (i) MR images of knee and ankle joints, which were used to provide participant-specific moment arm (yellow lines) used in computer simulation to predict MTU lengths; (ii) ultrasound images from proximal and distal MG regions, providing fascicle (blue, broken lines) length (L f ), and pennation; (iii) joint angles during knee (blue) and ankle (red) rotation conditions (Top of panel); (B) Representative fascicle length data from one participant. Individual points (black dots) indicate fascicle length at each time point across all recorded movement cycles; solid lines (blue/red) indicate the two-harmonic Fourier series fit to these data with associated R 2 values displayed. 200 mm; slice thickness: 5 mm; gap between slices: 5.5 mm) were collected ( Figure 1A). Once images had been collected from one joint, the participant was repositioned and the protocol repeated for the second joint, with the order of joint positions randomized between participants.
Three MR images of the ankle joint (15 • plantar flexion; 15 • dorsiflexion; neutral) were used to estimate the joint center of rotation using a geometric approach. The moment arm was calculated as the perpendicular distance from this joint center to the Achilles tendon, as described by Reuleaux (1875) and reported in Maganaris et al. (1998a). Knee moment arms were calculated, from a single image, as the perpendicular distance from the intersection of the anterior and posterior cruciate ligaments to the MG proximal tendon (O'connor et al., 1989; Figure 1A).

Measurement of Muscle Characteristics
Participants lay prone on an isokinetic dynamometer (HUMAC R /NORM TM 770, Computer Sports Medicine Inc., Stoughton, MA, USA), with their right leg aligned to enable angular changes of either the ankle or knee joint ( Figure 1A). Two linear ultrasound probes (LogicScan 128 and Echo Blaster 128, Telemed, Vilnius, Lithuania) were aligned to the fascicle plane in the proximal and distal regions of MG and held in place using elasticized bandage (Coban TM , 3M TM Bracknell, UK).
Passive joint rotations, cycling between the pre-defined range of joint angles (ankle: −15 • dorsiflexion to +30 • plantar flexion, with the knee fixed at 180 • ; knee: 180 • full extension to 90 • flexion, with the ankle fixed at 0 • , both at 60 • s −1 ), were completed following a short warm up period. Three trials were completed before, without moving the participant, the dynamometer was realigned to enable testing of the second joint. During ankle joint rotation the knee was fully extended along the length of the bed of the dynamometer. During knee joint rotation the ankle was fixed at 0 • using a customized boot-style brace device ( Figure 1A). The order in which the joints were investigated was randomized between participants.
Ultrasound images from each muscle region were collected (40 f.p.s., one PC laptop dedicated to each ultrasound device), while joint angle measures from the dynamometer were collected (2000 Hz) through a 16-bit data acquisition card (USB 6210, National Instruments Corp., Austin, TX, USA) using customwritten software (LabVIEW 2009, National Instruments Corp.). All data sources were synchronized by a common trigger signal.

Calculation of Muscle Geometric Properties
Ultrasound images from the proximal muscle region were analyzed using an automated Bayesian multiple hypothesis approach that provided the average shape of all MG fascicles visible in each image (Darby et al., 2013). Variation in fascicle orientations in the distal muscle portion meant this approach was not appropriate for use on these images (Figure 1A), so they were manually digitized. Pennation angle (β) in the proximal and distal regions was calculated as the mean of the angles made by the fascicle with the superficial and deep aponeuroses ( Figure 1A).
Estimated geometric properties were split into individual movement cycles (e.g., ankle joint rotation from −15 • through to −15 • ) and all cycles of movement from each condition grouped. To provide smoothly evolving data values (and simultaneously acting to filter these data), two-harmonic Fourier series were fit to each geometric property (fascicle length and pennation angle; Figure 1B).
For each participant, based on the combination of their moment arm data and musculoskeletal simulation, a 20 mm range of MTU lengths that occurred for both ankle and knee rotations was identified. Using the identified Fourier series coefficients, muscle geometric properties were calculated for these equivalent regions (i.e., across range of joint angles over which identified 20 mm range of MTU lengths occurred). Values were interpolated to 20 equally spaced values per muscle lengthening or shortening epoch and displayed as a function of MTU length (L MTU ). Predicted fascicle lengths were converted to strains (ε) using a reference length, determined in each muscle region, as the mean fascicle length across all trials. MTU gearing is given as the ratio of MTU velocity to fascicle velocity (V MTU /V f ). As fascicle and L MTU changes were determined across the same time period, this MTU gearing was equivalent to the ratio of their change in lengths ( L MTU / L f ) for each epoch.

Statistical Analyses
In each participant a least squares linear line of best fit was determined for the relationship between MTU length and each geometric property (L f , β) for the lengthening and shortening phase of the movement cycle. Paired T-tests were used to identify significant differences (α = 0.05) in the slope of the best fit line between knee and ankle joint rotations within each phase. Mann Whitney U-tests were performed for nonparametric data, which were identified using Shapiro-Wilk tests.

Moment Arm and MTU Lengths
The recorded moment arm data, and other anthropometric measures, for each participant are presented in Table 1. Greater moment arm values were recorded at the ankle (49.16 ± 8.77 mm) than at the knee (18.65 ± 1.40 mm). These values resulted in a mean ankle:knee moment arm ratio of: 2.62 ± 0.35. The 20 mm range of L MTU over which muscle geometric properties and MTU gearing was calculated started from between 401.34 and 477.03 mm across the group (Table 1).

Muscle Geometric Properties
Fascicle lengths and pennations as a function of MTU length change are shown in Figures 2A,B, respectively. Large variation in fascicle lengths across the grouped data is evident, reflecting individual variation in fascicle lengths which is removed if fascicle strains are considered ( Figure 2C). These data show that ankle joint rotations resulted in the greatest changes in fascicle length (and hence strain), during both lengthening and shortening phases of the movement cycle. The effect was particularly evident in the distal muscle region, where strains were higher and changed at a greater rate in ankle compared to knee joint conditions during both phases of the movement (Paired T-tests: Shortening p = 0.008; Lengthening p < 0.001). The greater fascicle strains in the distal region also coincided with lower pennation and significant differences in the rate of change in pennation also occurred between conditions during both movement phases (Shortening: Paired T-Test p = 0.006; Lengthening: Mann Whitney p = 0.041).
Although not as large an effect, similar differences were also found in the proximal muscle region, with greater fascicle strains and smaller pennation occurring during ankle joint rotation. Therefore, in both proximal and distal regions knee joint rotation resulted in very small changes in fascicle length (and strain) (Figure 2).

MTU Gearing
MTU gearing values were typically greater than 1, with the median ranging to about 6 (Figure 3). This represents changes in fascicle length of 20-3 mm respectively and reflects that fascicle length, generally, changed less than the 20 mm MTU length change investigated. As would be predicted from the differences in L f , the biggest difference in gearing values occurred in the distal muscle region with significance occurring in the statistical analysis (Mann Whitney U-Tests: shortening phase p = 0.003, lengthening phase p = 0.004). No significant differences in MTU gearing between rotations occurred in the proximal muscle region (Mann Whitney U-Tests: p ≥ 0.13 all cases). In all muscle regions MTU gearing values were similar between shortening and lengthening phases of the movement cycle.

DISCUSSION
The purpose of this study was to test whether the extent and velocity of fascicle length change, and hence MTU gearing, within proximal and distal regions of the passive MG muscle varied depending on the joint at which the motion occurred. The results revealed passive MTU gearing values greater than 1, with striking differences in the velocity of fascicle length change (Figure 2A) and MTU gearing (Figure 3) between ankle and knee joint conditions for the same change in MTU length.
The MTU gearing values greater than 1 (Figure 3) indicate that the muscle fascicles in the MG changed length to a lesser extent than the 20 mm change in MTU length studied within the experimental protocol. Active MG MTU gearing during cycling has been reported to range between 1 and 2 . The larger gearing values reported here indicate that the muscle fascicles underwent relatively small length changes during the passive uni-articular rotations studied. This is in broad agreement with Herbert et al. (2002) who showed that changes in MG fascicle length reflected approximately 25% of the total MTU length change, indicating a passive MTU gearing value of 4. Here, we have additionally shown that passive MG MTU gearing values can vary and are influenced by the joint at which MTU length change is imposed.
The gearing values greater than 1 indicates that fascicles operated at lower velocities, and hence underwent smaller length changes, than the MTU during the passive joint rotations (Figures 2A, 3). Two possible explanations for this phenomenon are: (i) rotation of the muscle fascicles and (ii) introduction of slack into the tendons. Based on trigonometry, fascicles would be required to rotate to pennation angles beyond 50 • to accommodate the small changes in fascicle length recorded. Maximal voluntary contractions can lead to high pennation (Maganaris et al., 1998b), however such values did not occur during the passive movements studied here, where changes in pennation were strikingly small ( Figure 2B). Fascicle rotation does not, therefore, fully explain the differences between fascicle and MTU length changes observed. When considering the potential contribution of slack in tendinous structures, it is important to consider that MTU gearing (V MTU /V f ) is the product of two factors: (i) the ratio of muscle belly to muscle fascicle velocity (V b /V f ) and (ii) the ratio between MTU and muscle belly velocity (V MTU /V b ) . This means that displacements of the muscle belly and the MTU can be uncoupled as a result of the contribution of the tendon. Although difficult to predict without measures of force, it could be expected that, due to its location and its relatively larger moment arm (Table 1), greater slack would be introduced into the Achilles tendon during rotation of the ankle joint. If this were true, higher MTU gearing values for this condition would be predicted. This however is not seen, with lower MTU gearing found (Figure 3) resulting from the fact that the largest changes in fascicle lengths occurred during ankle joint rotation (Figure 2).
It is possible that anatomical differences of tendinous structures at the MG origin and insertion sites may influence how much of the changes in MTU length are "seen" by the muscle fascicles within each joint rotation condition. It has been reported that the long, compliant Achilles tendon contributes more than muscle fascicles to changes in MTU length imposed by ankle rotation (Herbert et al., 2002(Herbert et al., , 2011(Herbert et al., , 2014Abellaneda et al., 2009), however little is currently reported about the potential contribution of the shorter and thicker proximal tendon at its origin (Dalmau-Pastor et al., 2014), making it hard to determine its influence. It is apparent however that neither fascicle rotation nor tendon slack can fully account for the differences in fascicle and MTU length changes seen. This suggests that additional factors, which may also explain the variation in passive MTU gearing values, should be considered. One potential factor is the influence of surrounding structures such as muscles, connective tissue, and bone.
Assuming constant muscle volume (Van Leeuwen and Spoor, 1992), it is apparent that changes in MTU length must be accompanied by changes in the cross-sectional area of the muscle belly if tendon length does not change. This means that increases and decreases in muscle cross-sectional area must correspond with shortening and lengthening of the MTU, respectively. Changes in muscle fascicle characteristics (e.g., length and pennation) will be coupled to changes in muscle belly shape that, in turn, will depend on external forces applied from surrounding structures (i.e., connective tissues, musculature, bone). The greater influence of ankle joint rotation on fascicle length and pennation could therefore reflect significant interactions with surrounding musculature and connective tissues. For example, MTU length changes imposed by ankle rotation are translated to the MG via the Achilles tendon, which is shared with the adjacent soleus muscle. The mono-articular soleus only spans the ankle joint, and should therefore not have been subject to length changes during the knee rotation condition (although see text below regarding epimuscular myofascial force transmission). Rotation of the ankle joint therefore results in shape changes in the soleus that influence the shape of the MG, an influence that does not occur (or differs) during knee joint rotation and may also be influenced by interactions that occur with the lateral head of the gastrocnemius. The potential for such interaction is supported by the fact that the largest differences in MTU gearing between rotation conditions occurred in the distal MG (Figure 3) where the greatest overlap between MG and soleus occurs (see Figure 13 in Dalmau-Pastor et al., 2014).
Interaction between adjacent muscles during passive loading at physiologically relevant muscle lengths has, to our knowledge, not been widely investigated. Passive knee joint rotation, with a fixed ankle angle, has been shown to result in displacement of soleus muscle (Bojsen-Møller et al., 2010). While comparison of two different knee/hip joint angles, achieved with a fixed ankle angle, has been shown to result in deformation of soleus muscle tissue (Yaman et al., 2013). These phenomena have been attributed to epimuscular myofascial force transmission, related to connections including collagen enforced neurovascular tracts, intermuscular septa, and compartment fascia (Bojsen-Møller et al., 2010;Yaman et al., 2013). These factors, alongside other features such as connective tissues or friction between muscle bellies, may have influenced current results, and suggests that both the active and passive dynamic interaction between adjacent muscles in physiologically intact systems needs further consideration for accurate models of muscle-tendon mechanics.
The results presented here should be considered within the context of at least one potential limitation: no measure of muscle activation was included. Given the angular velocity used within the protocol (60 • s −1 ) reflex activation of MG may have occurred and influenced muscle stiffness in the two conditions. A small pilot trial revealed that for both ankle and knee joint conditions such tonic activation was low (<5% MVC). Previous reports have suggested that for any resistive torques to be considered passive muscle activation should be below 1% MVC (McNair et al., 2002;Gajdosik et al., 2004). The effect of such low levels of activation on fascicle length changes is however unknown. As the clarity of the ultrasound images was a priority for the work presented and the space required to appropriately place and secure the two ultrasound probes to the leg limited the ability to appropriately position and securely place surface electromyography (EMG) electrodes, they were omitted from the main data collection. Fascicle characteristics were derived from fitting Fourier series to data points collected across all cycles (Figure 1B), if activity did occur in some of the trials this approach ensures that it would be likely to have minimum influence on the results, however it must be stated that, whilst we are certain the muscles were relaxed and not actively contracting, we cannot be sure that the muscles were investigated under totally passive conditions. One unexpected feature of the results was the differences in fascicle lengths and pennation that are apparent between shortening and lengthening phases in both the proximal and distal muscle regions (Figure 2). Fascicle lengths were longer when the MTU was being "released" (i.e., shortening phase) and shorter when being stretched (i.e., lengthening phase), suggesting a form of history dependence within these passive movements (with analysis based on series of cycles recorded across multiple trials). Hysteresis in passive length-tension curves has previously been reported in isolated canine internal abdominal muscles (Hwang et al., 2005) and rabbit masseter muscle (Van Eijden et al., 2002). Hysteresis is also reported to occur in human Achilles tendon, although discrepancies between in vitro and in vivo measures have led to questions regarding the potential of ultrasound imaging and tracking methodologies to introduce variability and bias into these measures (Finni et al., 2013). Whether the differences between shortening and lengthening phase values for fascicle length and pennation angle found here reflect physiological properties or aspects of the ultrasound FIGURE 3 | Box plots of MTU gearing for each condition/muscle region. Left panel shows shortening phase, right panel lengthening phase. 95% confidence intervals (whiskers, calculated excluding outliers); 25 and 75% quantiles (box edges); median (thick line). Participant number is indicated by outlier points. To provide a suitable scale, three outliers are not shown: one proximal ankle shortening phase, two distal knee lengthening phase.
imaging approach is therefore unclear and would require further investigation.
Dissociation between changes in MTU and fascicle length are well documented during active contractions Randhawa et al., 2013) as well as during locomotion (Fukunaga et al., 2001) and jumping performance (Kawakami et al., 2002) in humans, with proposed benefits for mechanical power output and economic force production. Here we show that a similar dissociation can occur during passive joint movements. This phenomenon may play an important functional role by altering the region of the force-length relationship over which a fascicle operates when first activated during a locomotor task. In addition we show that, within a biarticular muscle, changes in fascicle length are affected by the joint at which the motion occurs. These findings suggest that mechanical constraints may be imposed on a muscle by the kinematics of the motor task, with the ability of a muscle to generate force influenced by the site at which MTU length changes are imposed. This undermines current assumptions of a constant relationship between muscle force/function and MTU length, which are often made in conceptual models of muscle-tendon mechanics. These results indicate the potential for more anatomically realistic muscle models to be implemented within musculoskeletal simulations of movement. This is increasingly important for investigating the mechanical and neuromuscular consequences of abnormal gait patterns/changes in muscle properties (e.g., spasticity) as well as to inform development of effective treatment and rehabilitation protocols (e.g., including robotics) in those with compromised motor abilities.

AUTHOR CONTRIBUTIONS
EH, JW, and TD contributed to the conception and design of the work; EH and TD acquired and analyzed these data; EH, JW, and TD interpreted data for the manuscript. EH, JW, and TD drafted the work and/or provided critical revisions for important intellectual content and provided final approval of the version to be published. EH, JW, and TD agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

text:
Investigating the Role of Loop C Hydrophilic Residue ‘T244’ in the Binding Site of ρ1 GABAC Receptors via Site Mutation and Partial Agonism

The loop C hydrophilic residue, threonine 244 lines the orthosteric binding site of ρ1 GABAC receptors was studied by point mutation into serine, alanine and cysteine, and tested with GABA, some representative partial agonists and antagonists. Thr244 has a hydroxyl group essential for GABA activity that is constrained by the threonine methyl group, orienting it toward the binding site. Significant decreases in activation effects of the studied ligands at ρ1 T244S mutant receptors, suggests a critical role for this residue. Results of aliphatic and heteroaromatic partial agonists demonstrate different pharmacological effects at ρ1 T244S mutant receptors when co-applied with GABA EC50 responses. ρ1 T244A and ρ1 T244C mutant receptors have minimal sensitivity to GABA at high mM concentrations, whereas, the ρ1 WT partial agonists, β-alanine and MTSEA demonstrate more efficacy and potency, respectively, than GABA at these mutant receptors. This study explores the role of Thr244 in the binding of agonists as an initial step during channel gating by moving loop C towards the ligand.

Introduction
The γ-aminobutyric acid type C (GABA C ) receptors, also known as γ-aminobutyric acid type A rho (ρ GABA A ) receptors are ligand gated ion channel receptors that activated by GABA, the most inhibitory neurotransmitter substance in the vertebrate CNS (central nervous system) [1]. The rational discovery of selective agents initially revealed two distinct classes of GABA receptors, the ionotropic GABA A and metabotropic GABA B receptors. The two classes are different in their pharmacology, biochemical and electrophysiology properties [2]. A subclass of ligand-gated ion channels were later identified, insensitive to ligands that typically affected GABA A and GABA B receptors, these were initially classed as GABA C receptors [3]. Receptors of the ligand-gated ion channel superfamily are pentameric requiring five subunits to assemble a single ion channel. The ion channel may be homomeric, formed by five identical subunits as is the case for ρ GABA C receptors or heteromeric, formed by a combination of at least two different subunits, such as the GABA A receptors [4]. GABA C ion channels expressed in Xenopus oocytes have different properties to GABA A ion channels in terms of potency, channel opening time and receptor desensitization. In general, GABA is between ten-and one hundred-fold more potent at GABA C receptors than at GABA A receptors, with slow activation and deactivation, and less readily desensitized [5][6][7]. The pharmacological profile of GABA C receptors is distinguished by many ligands that are selective at this subfamily over GABA A receptors.
The novel ρ1 GABA C subunit from a human retina cDNA library was cloned in the early 1990s [8]. The second member of this subfamily, designed ρ2 was cloned from human retina a year later [9]. A third member of this subfamily designed ρ3 has also been detected in retina as well as higher brain regions with lower expression levels compared with the retinal level [10,11]. In the retina, ρ3 is found expressed in ganglion neurons of the retina while ρ1 and ρ2 are specifically expressed in bipolar and horizontal cells [12][13][14].
Many Studies have suggested the involvement of them in the sleep-waking behaviour of rats [15], learning and memory in chicks and rats [16], inhibition of ammonia-induced apoptosis in hippocampal neurons [17], and hormone release in the pituitary [18]. More recently, the GABA C antagonist TPMPA, was shown to improve the symptoms of retinitis pigmentosa in rats [19], and evidence that ρ1 and ρ2 may be important for different specific in vivo effects of ethanol, has been reported [20].
The structure of ρ1 receptors was first studied by identifying critical residues that have direct effects on ligand sensitivity using site-directed mutagenesis and substituted cysteine accessibility methods. The T244 residue in loop C has the hydroxyl side-chain oriented toward the GABA binding site. Mutation of this residue to various amino acids resulted mostly in non-functional receptors even at high concentrations of GABA, and only the T244S mutation resulted in a functional receptor, although GABA potency was decreased 35-fold [21]. Additionally, ρ1 GABA C T244S mutant receptors have recently been studied with various ligands. This mutation resulted in many folds decrease in potency of the studied agonists, whilst the studied antagonists remain unaffected by the mutation [22].
The current study aims to explore the role of this hydrophilic residue in the orthosteric binding site in terms of receptor functionality, and to determine the interactions it may form with various ligands. Furthermore, the shift in the potency and efficacy of the studied ligands (Fig 1), and additive (or co-operative)/inhibition effects are also considered and discussed in terms of how different interactions may affect the receptor stability in apo state or open conformation.

Molecular modeling
Sequence alignment and homology modelling. The protein sequence of human ρ1 GABA C was obtained from the universal protein resource (http://www.uniprot.org/) [23], and the models were built on templates of the GluCl in open conformation (PDB ID 3RIF) and apo state (PDB ID 4TNV) (http://www.rcsb.org/). [24]. Sequence alignment was performed using the CLUSTALW program [25]. The models of the ρ1 GABA C receptor were built using Prime 3.2 software [26]. The preparation, generation, validation and selection of the homology model of the ρ1 GABA C receptors was recently described [27]. [28]. The preparation of all compounds for docking was performed by LigPrep (LigPrep v3.1) [29].
Molecular docking of ligands into the orthosteric binding site. The docking studies of ligands with the generated models were carried out using the default settings of the Glide program in the extra-precision mode implemented in Schrödinger Suite 2013 [30].
Induced fit docking was also performed for the studied ligands in the ρ1 GABA C orthosteric binding site. The full methodology of the docking studies has been previously described in conjunction with the ρ1 GABA C homology model used in this work [27]. For the models of each studied mutant, the studied ligands also underwent induced fit docking into the new binding sites, however no significant changes to the binding site was identified. The orientation of the introduced amino acid side-chains after after induced fit docking of the various ligands were also considered, and other side-chain rotamers also checked for further confirmation of ligand binding and interactions with the introduced residues at Thr244 position.
Primer design and site-directed mutagenesis. Pairs of the complementary mutagenic oligonucleotide primers were first designed to introduce single point mutations at human ρ1  The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors GABA C subunit using UniProt [34]. Primers were made by life technologies (Life Technologies TM Australia Pty Ltd).
Site-directed mutagenesis was performed using QuikChange II site-directed mutagenesis kit as described by the manufacturer (Stratagene) [35,36].
Transformation and preparation of wild type and mutant plasmids. The desired mutant or wild type plasmid DNAs of ρ1 subunit were transferred into TOP10 Chemically Competent E. coli according to the protocol (Invitrogen™)). The E.Coli were grown in LB broth with ampicillin (100 μg/ml) then the cell harvesting and the plasmids DNA purification were performed using Qiagen Spin Miniprep Kit (Qiagen, VIC, Australia).
In vitro transcription. ρ1 GABA C WT and mutant plasmids DNA were linearized by incubation with Xba1. ρ1 WT and mutant cRNA were synthesized using the T7 Transcription mMESSAGE mMACHINE Kit (Ambion, Austin, TX, USA) following the procedure described by the manufacture. The quantity and quality of synthesized C RNA was determined by absorbance at 260-280 nm using Nanodrop Spectrophotometer (Thermo Scientific, Wilmington, DE, USA). The purity of synthesized cRNA was confirmed by agarose gel electrophoresis (0.9%) using the GelDoc 1000 (Bio-Rad Laboratories, Hercules, CA USA).

Electrophysiology
Oocyte preparation and RNA injection. Xenopus laevis are purchased from Nasco in Fort Atkinson, Wisconsin, USA and housed at the University of Sydney Xenopus laevis facility. Frogs are maintained in groups of 18-22°C water at a depth of 12 cm with a 12 hrs light dark cycle, and fed twice weekly. Mature female Xenopus laevis frogs were anaesthetised (0.17% tricaine, buffered with 0.06% sodium bicarbonate) until the loss of righting reflex was confirmed (15 minutes) before transferring them on to ice where surgeries were performed. A small (1-2 cm) incision was made using surgical knives through both the skin and muscle layer of the abdomen. Ovary lobes were removed with a pair of forceps and placed in oocyte releasing 2 (OR2) buffer (82.5 mM NaCl, 2 mM KCl, 1 mM MgCl2, 5 mM HEPES hemisodium; pH 7.4). The skin and muscle layer were sutured separately, and frogs allowed to recover for six months before being reselected for surgery. A total of five recoverable surgeries were allowed on each frog prior to a terminal surgery in which a lethal dose of tricaine (0.5%) was administered. All the procedures involved in the use of Xenopus laevis frogs were approved by the Animal Ethics Committee of the University of Sydney (Reference number: 2013/5915). The lobes of ovaries were then separated evenly and incubated with Collagenase A (Boehringer Manheim, Germany). The released oocytes were first washed with OR2 buffer then stored in frog Ringer buffer (ND96).
A microprocessor-controlled micropipette puller (PUL-100, World Precision Instruments, Inc. FL, USA) was used to make micropipettes then RNA pulled up into the micropipette by a positive-displacement using micro-injector (Nanoliter micro-injector, World Precision Instruments, Inc. FL, USA). Oocytes were sorted and healthy ones were injected with 35-50 ng of ρ cRNA unless otherwise mentioned.
Two-electrode voltage-clamp electrophysiology. The injected oocytes were incubated for between two and four days after injection with desired RNA. Two-electrode voltage clamp instrument; Geneclamp 500 amplifier (Axon Instrument, Sydney, NSW, Australia) and Chart version 5.5.6 was used to measure the various activities at the receptors expressed by these oocytes. The recording microelectrodes were prepared by pulling using PUL-100 micropipette puller (World precision Instruments, Inc. FL, USA) and filled with 3 M KCl.
Data analysis. Current responses were normalized to the maximum GABA-activated current (or other ligand which is stated as required) and expressed as a percentage, which was fitted by least squares to Hill equation (Eq 1). Dose response curves were generated using GraphPad PRISM 5.02 (GraphPad Software San Diego, CA). The responses of sub-maximal agonists tested at ρ1 GABA WT and mutant receptors were normalized by the EC max concentrations of the highest efficacious ligand in order to calculate concentration response curves of these ligands.
Where I is the current response to a known concentration of agonist, I max is the maximum current obtained, [A] is the agonist concentration, EC 50 is the concentration of agonist at which current response is half maximal and nH is the Hill coefficient.
The inhibitory concentration curves were generated using GraphPad PRISM 5.02 and IC 50 values were calculated using Eq 2.
I is the peak current at a given concentration of agonist, I max is the maximal current generated by the concentration of agonist, [A] is the concentration of GABA, IC 50 is the antagonist concentration which inhibits 50% of the maximum GABA response and nH is the Hill coefficient.

Statistical analysis
All statistical calculations are presented as mean ± standard error of the mean (SEM) or as mean (95% confidence intervals (CI)). Student's t-test was performed to determine the statistical significance of the change in EC 50 and IC 50 at ρ1 GABA C WT and mutant receptors, where 0.05 was treated as the point of significance.

Results and Discussion
Homology modeling and GABA docking studies with threonine 244 Our generated model predicts a hydrogen bond between the hydroxyl group of the Thr244 in loop C and the carboxylate group of GABA (Fig 2A). This interaction assists in agonist binding and may facilitate gating due to the movement of loop C inward, leading to the open conformation and activating the receptor [27].

Mutation of threonine 244 into serine, alanine and cysteine
GABA was first tested on all mutant receptors in order to determine the functionality of these receptors. The ρ1 GABA C T244S mutant receptors showed a 35-fold decrease in GABA potency with an EC 50 of 35 μM as previously reported [21,22]. ρ1 GABA C T244A and ρ1 GABA C T244C mutant receptors initially appeared to be non-functional or insensitive to GABA at a concentration of 30mM. However, increasing the concentration of mutant RNA injected into the oocytes from 50-100 ng/μl to 300 ng/μl resulted in the expressions of receptors that were weakly responsive to GABA at high concentrations ( Fig 2B).

ρ1 GABA C T244S mutant receptors
The moderate decrease in GABA potency that occurs at ρ1 T244S mutant receptors suggests the importance of the threonine methyl group in restricting the position of the hydroxyl group which is predicted by modeling to form a hydrogen bond with the carboxylate group of GABA [27]. When the partial agonists imidazole-4-acetic acid and muscimol were tested at ρ1 T244S mutant receptors, their agonist effects were eliminated by the mutation and they acted only as competitive antagonists. Although the actions of agonists and partial agonists were affected by the T244S mutation, the activity of the competitive antagonists in this study remained unchanged at these mutant receptors compared to ρ1 WT receptors [22].
The efficacy of the unsaturated ligands was also altered as a result of the ρ1 T244S mutation. Interestingly TACA, which shows 95% efficacy relative to the GABA maximal response at ρ1 WT receptors is slightly more efficacious (110%) than GABA at ρ1 T244S receptors (P < 0.05) (Fig 3C). CACA which shows only 60% efficacy relative to GABA maximal response at ρ1 wild type receptors has only 2% efficacy at ρ1 T244S receptors (Fig 4A).
TACA has a slightly longer distance between the carboxylate and ammonium poles than GABA (Fig 4B), and our model predicts the bound orientation of the carboxylate adopting a slightly different orientation to that of GABA and CACA (Fig 4A and 4B) and is predicted to be more able to form contacts with the side chain of the serine residue at position 244 (Fig 4C  and 4D). This interaction may further stabilize the open conformation and be responsible for the higher efficacy of TACA at these mutant receptors.
Effects of CACA co-applied with GABA EC 50 at ρ1 T244S receptors. The co-application of CACA has an additive effect on GABA EC 50 responses at ρ1 WT receptors, reaching up to 150% of GABA EC 50 efficacy at 100 μM CACA (Fig 5A and 5B). The additional effect of CACA is similar in magnitude to the effect of CACA applied alone at ρ1 WT receptors. There are five equivalent binding sites and it has been predicted that at least three molecules of GABA are required to bind at each pentameric GABA C receptor in order to elicit a response.
These results suggest that CACA is binding at vacant sites on the receptors activating the receptor so that more individual receptors are open, leading to a maximal response.
Interestingly, despite the fact that CACA applied alone is almost inactive at ρ1 T244S receptors (Fig 5B), CACA applied in the presence of GABA EC 50 shows an additive effect on GABA EC 50 responses at these mutant receptors, The magnitude of 300 μM CACA applied alone is less than 2% of GABA I MAX , however the GABA EC 50 effect is enhanced by approximate 20% at 100 μM CACA (Fig 5). This suggests that binding and/or activation by GABA changes the conformation of the vacant binding sites allowing CACA activate the receptor more effectively that when CACA is applied alone, leading to an additive effect.
Agonist effects of glycine, β-alanine and 5-aminovaleric acid. At ρ1 WT receptors, the analogues of GABA with varying numbers of carbon atoms in the backbone, glycine, β-alanine and 5-aminovaleric acid, respectively (Fig 1) are weak partial agonists ( Fig 6A) [37,38]. Compared to their effects on WT receptors, the potencies and efficacies of these partial agonists were decreased at ρ1 T244S mutant receptors. Both glycine and β-alanine showed significant decreases in efficacy, with a maximum of 8% and 5% relative to the GABA maximal response at 30 mM. The least efficacious compound at ρ1 WT receptors, 5-aminovaleric acid showed a large decrease in potency, shifting the curve to the right, however the ligand was still able to weakly activate the mutant receptors at very high concentrations (6000μM, 3% efficacy relative to the GABA maximal response at 30 mM) (Fig 6B).
Effects of glycine, β-alanine and 5-aminovaleric acid co-applied with GABA EC 50 at ρ1 T244S receptors. The partial agonist glycine displays an additive effect on the GABA EC 50 responses at ρ1 WT receptors, 180% efficacy (relative to the GABA EC 50 response set to 100%), and has a similar effect at ρ1 T244S mutant receptors but with less magnitude, 145% efficacy. At ρ1 T244S receptors this additive effect is reduced at concentrations higher than 1 mM ( Fig  7A). Despite only being able to directly activate these receptors by 25% (ρ1 WT) and 2% (ρ1 T244S) at mM concentrations (Fig 7A), the significant additive effects of glycine at both ρ1 WT and T244S receptors suggests that glycine is able to bind at vacant binding sites and is able to more effectively activate these receptors. Glycine molecules are not able to effectively compete with GABA at sites where GABA is bound at ρ1 WT receptors. This is most likely because GABA has a much higher binding affinity at ρ1 WT receptors. However, at ρ1 T244S mutant receptors the binding affinity of GABA is likely to be reduced and, therefore at very high concentrations of glycine, some competition between the two ligands for the same binding site may occur, but without significant displacement of GABA molecules, resulting in fewer channels being activated and, therefore the additive effect of glycine is diminished at 10mM (Fig 7A). At concentrations of up to 100 μM β-alanine in the presence of GABA EC 50 has weak additive effects at ρ1 WT receptors (125%) and ρ1 T244S mutant receptors (115%), respectively. At concentrations of greater than 100 μM β-alanine, acts as an antagonist at both ρ1 WT and ρ1 T244S receptors (β-alanine inhibits 60% of GABA EC 50 at both receptors). However the IC 50 of β-alanine is increased 8-fold at ρ1 T244S receptors compared to ρ1 WT (120μM and 1 mM of β-alanine antagonize GABA EC 50 responses at ρ1 WT and ρ1 T244S receptors, respectively) ( Fig 7B).
In the presence of GABA EC 50, 5-aminovaleric acid acts as an antagonist at both ρ1 WT and ρ1 T244S receptors at concentrations of greater than 1 μM (Fig 7C) . However, the potency of 5-aminovaleric acid is increased two-fold ρ1 T244S receptors with an IC 50 = 17 μM compared to 37 μM at ρ1 WT receptors (Fig 7C). The extended chain length of 5-aminovaleric acid  compared to GABA permits 5-aminovaleric acid to form required interactions with the receptors in the apo state and the protein is predominantly not required to undergo a conformational change activating either ρ1 WT nor ρ1 T244S receptors (Fig 8). The additional methylene group of 5-aminovaleric acid may result in further hydrophobic contacts in the binding site that further stabilize the apo state over open conformation. The increase in antagonist potency of 5-aminovaleric acid at ρ1 T244S receptors is possibly due to the extended chain length resulting in increased flexibility allowing the carboxylate group to form interactions with the side chain of serine at position 244 more effectively than GABA.

Isoguvacine and ρ1 T244S mutant receptors
Agonist effect of isoguvacine. The heterocyclic GABA analogue; isoguvacine (Fig 1) was tested at ρ1 WT and ρ1 T244S receptors. This ligand is weak partial agonist, activating WT receptors with an EC 50 = 200 μM [39] and 45% efficacy relative to the GABA maximal response. However, at ρ1 T244S mutant receptors the potency and efficacy of isoguvacine are significantly reduced with only a 30% response elicited at a concentration of 600 μM (Fig 9A).
Docking studies of this ligand in the ρ1 GABA C homology model predict H-bonding of the hydroxyl group of Thr244 with the carboxylate group of isoguvacine ( Fig 10A). There are also hydrophobic interactions between the side chains of Thr244 and isoguvacine (Fig 10B). These interactions may possibly further stabilize the receptor in open conformation. The significant reduction in potency and efficacy of isoguvacine when serine is introduced at the Thr244 site suggests the importance of the H-bond and other interactions between side chain of isoguvacine and Thr244 in stabilizing the receptor in open conformation.
Antagonist effects of isoguvacine. At ρ1 WT receptors isoguvacine has a moderate additive effect on the GABA EC 50 response of up to 135% (Fig 9B and 9C). However, at ρ1 T244S mutant receptors isoguvacine acts as a full antagonist (IC 50 = 25.3 μM) against GABA EC 50 (Fig 9B and 9D).
These results suggest that isoguvacine alone binds to ρ1 T244S mutant receptors but does not stabilize the open conformation. In the presence of GABA EC 50 the effect of isoguvacine is changed from a additive effect at ρ1 WT receptors to antagonist ρ1 T244S mutant receptors. Removal of the methyl group from the side chain allows serine to adopt an orientation that permits the formation of an H-bond with isoguvacine that does not require the movement of loop C in the open conformation.
Studying the activity of structurally different partial agonists at ρ1 GABA C T244S mutant receptors indicates that the overall effect of the mutation is determined by the structure of the ligand. The efficacy and potency of the aliphatic partial agonists (glycine, β-alanine and 5-aminovaleric acid) decreased significantly however they were still able to weakly activate ρ1 T244S receptors at very high concentrations. However, the heterocyclic partial agonist, isoguvacine, which lacks conformational flexibility, was converted to full antagonist.

Effect of the T244S mutation on the activity of the antagonists TPMPA and THIP
Compared to the effect of the GABA C antagonists, TPMPA and THIP (Fig 1) on WT receptors, the T244S mutation has no effect on the potencies of these ligands [40,41]. Modeling and docking studies with the GABA C model based on GluCl in the apo state predicts that loop C is sterically prevented from moving inward and forming a shield above the ligand as an essential The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors step in the open conformation. The bulkier structure of the antagonists may inhibit the movement of loop C closer to the ligand preventing the necessary initial step for channel activation [27].
ρ1 GABA C T244A mutant receptors Substitution of alanine for threonine at position 244 removes the hydroxyl group. At ρ1 T244A receptors, both the potency and efficacy of GABA (Fig 11A and 11B) was significantly reduced The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors (ρ1 WT = 1 μM, ρ1 T244A = 4960 μM). The ρ1 WT agonists, TACA and muscimol and partial agonists, glycine and 5-aminovaleric acid were found to be less efficacious than GABA. However, β-alanine with one carbon less than GABA, was found to be more potent (EC 50 = 1496 μM , Fig 11 A) and approximately two times more efficacious than GABA at a 30 mM concentration on ρ1 T244A mutant receptors (Fig 11B). Compared to its effects on ρ1 WT receptors (Fig 6), the potency of β-alanine was reduced only two-fold at T244A receptors.
Our model indicates that β-alanine does not form a salt bridge between its ammonium terminal and Glu196 in same manner as GABA. The ammonium group of β-alanine may be stabilized by the aromatic box in the binding site ( Fig 12A). This may also explain the mobility of βalanine in the binding site compared to GABA, allowing it to move slightly closer to and form more hydrophobic contacts between the alanine of T244A receptors, stabilizing the open conformation ( Fig 12B).
Threonine 244 was also mutated to alanine in silico in the ρ1 GABA C homology model that was used for docking studies with GABA and β-alanine. The docking studies predict more hydrophobic interactions of the carboxylate group of β-alanine shows with the alanine residue at position 244, than the carboxylate group of GABA. These hydrophobic interactions may provide additional stabilization to β-alanine in the binding site during gating, which may also enhance efficacy and potency of the ligand (Fig 12B). The greater number of interactions of βalanine with the alanine residue at position 244 may stabilize loop C closer to ligand, which is an essential for channel activation.
The antagonist effects of GABA and TPMPA against the β-alanine response (30 mM) at ρ1 T244A receptors were also investigated. When 30 mM of GABA was co-applied with 30 mM of β-alanine, GABA moderately inhibits the responses elicited by β-alanine (Fig 11C). These results suggest that GABA is acting as partial agonist at ρ1 T244A mutant receptors. On the other hand, the antagonist TPMPA has no effect when applied alone, but weakly antagonizes β-alanine responses at 1 mM (Fig 11C). However, the potency of TPMPA was significantly  while it forms a lower number of hydrophobic contacts with a serine at position 244, and the least number of hydrophobic contacts between TPMPA and the alanine at T244A receptors (Fig 12C).

ρ1 GABA C T244C mutant receptors
Initial experiments suggested that T244C mutant receptors did not express, however injection of five times the concentration of RNA normally injected resulted in very weak response to GABA (30 mM). Interestingly, the thio-reactive GABA analogue; 2-aminoethylmethane thiosulfonate (MTSEA) was found to elicit reversible responses at ρ1 T244C mutant receptors with greater potency and efficacy than GABA (Fig 13A and 13B).
MTSEA activates ρ1 WT receptors with very weak efficacy and potency ( Fig 13C). When this ligand is co-applied with GABA EC 50 , at concentrations below 100 μM it has weak additive effects to the GABA EC 50 response, but at concentrations of 100 μM and above it weakly antagonizes the GABA EC 50 responses ( Fig 13B).
Docking studies of MTSEA in the orthosteric binding site of ρ1 homology model predicts that a H-bond does not form between the sulfonyl oxygen of MTSEA and hydroxyl group of Thr244, Although a number of interactions occur between the side chains of MTSEA and Thr244 (Fig 14A). Moreover, replacing threonine with cysteine at position 244 in silico predicted that the introduced cysteine exists predominantly as a rotamer in which the thiol is pointed away from the binding site (Chi1 = -64°) which may explain why an irreversible S-S bond does not form between MTSEA and cysteine ( Fig 14B). However, MTSEA is still able to activate ρ1 T244C receptors with a similar magnitude to ρ1 WT receptors, although GABA sensitivity at the ρ1 T244C mutant receptors is significantly decreased.
Additionally, due to the methyl group of the methane thiosulfonate, MTSEA is predicted by docking studies to form slightly more interactions with the cysteine residue at the 244 position than the carboxylate group of GABA. Therefore, once the hydroxyl of threonine has been removed, MTSEA may confer better stability of the open channel conformation through hydrophobic interactions at the ρ1 T244C mutant receptors compared to GABA (Fig 14B).

Threonine 244 and channel activation
GABA in the orthosteric binding site of ρ1 GABA C forms a H-bond with the hydroxyl group of Thr244, and this interaction requires Loop C to move towards GABA. A competitive antagonist essentially stabilizes the closed conformation holding the receptor in the closed conformation and preventing the priming necessary for the channel opening [42], therefore preventing GABA from binding. Those ligands which inhibit agonist responses bind tightly in the site and stabilize the receptor in the inactive state. This is can be achieved in the orthosteric binding site of ρ1 GABA C receptors through antagonists forming additional hydrophobic contacts with aromatic residues that surround the binding site. In addition to blocking the site preventing GABA or other agonists from binding and activating the channel, these interactions stabilize the closed conformation by inhibiting the movement of loop C.
In the case of ρ1 GABA C ion channels, the bulkier and conformationally restricted heterocyclic ligand, TPMPA binds in the site and forms hydrophobic contacts with Tyr241 and Tyr247 The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors located in the loop C which prevent the loop from moving forward and thus leads to the channel stabilized in close conformation (Fig 15).

Conclusion
Threonine 244 is a hydrophilic residue with the side chain functional group oriented toward the binding side. GABA docking studies predicted that the hydroxyl group of Thr244 forms an H-bond with carboxylate group of GABA. This residue is also predicted to have various contacts with GABA that may contribute to stability during the gating process. The study of this hydrophilic residue by rational point mutations and testing against GABA, some representative ligands was able to identify the essential role of Thr244 in the closed/open conformations and during channel gating.
The predicted H-bond between the hydroxyl group of Thr244 and the side chain of GABA during the gating process is postulated to require the movement of loop C forward closer to the agonist in the priming step that initiates activation. The efficacy of GABA moderately decreases when serine is introduced at the site. Although GABA is able to activate ρ1 T244A and ρ1 T244C receptors, a greatly increased concentration of RNA was required for expression of The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors these mutant receptors. This suggests that the expression level may have significantly decreased with these mutant receptors. The partial agonists studied demonstrated significant decreases in their potencies and efficacies at ρ1 GABA C T244S mutant receptors. The additive/inhibition effects of these partial agonists were also shown to moderately decrease at the same mutant receptors. However the tested aliphatic partial agonists retained their agonist/antagonist effects while the tested heterocyclic partial agonists became inactive at these mutant receptors, fully antagonizing GABA EC 50 responses. These results suggest that structural differences of partial agonists may result in the formation of different interactions leading to the different efficacies despite similar structural features. The studied competitive antagonists did not show changes in the potency at T244S mutant receptors in comparison to wild type receptors. The bulkier structure of these antagonists may prevent the movement of loop C as the two aromatic residues (Tyr241 and Tyr247) in loop C could stabilize the ligand in the binding site without moving the loop forward which may confer what model predicted that antagonists aren't forming the H-bonding with Thr244 (Fig 15). Suggesting both the priming and flipping steps in the early stages of receptor activation appear to be key determinants in the efficacy of an agonist.
In the absence of the hydroxyl side chain at the Thr244 position through the T244A mutation, β-alanine demonstrated the highest efficacy of all agonists and partial agonists tested at ρ1 GABA C T244A mutant receptors, including GABA. Homology model studies predicted that βalanine forms more contacts with the alanine residue at position 244 than GABA. At ρ1 GABA C T244A receptors, β-alanine exhibited decreased responses when GABA or TPMPA was co-applied with it. These results suggest that TPMPA is binding at the orthosteric binding site of these mutant receptors however a significant decrease in its potency was noticed.
GABA elicited only very small responses while MTSEA with a thiosulfonate side chain demonstrated greater efficacy than GABA at ρ1 GABA C T244C mutant receptors. Docking studies predict that MTSEA is not forming S-S interactions with either cysteine at position 244, or with other residues, which suggests that MTSEA does not form a covalent bond. MTSEA The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors which shows only weak activation and additive/inhibition effects at ρ1 GABA C WT receptors, still has the same magnitude of activation at ρ1 T244C mutant receptors. However the responses to GABA are significantly decreased, and it appears that GABA is unable to stabilize the open conformation as at it does at ρ1 wild type receptors.
In summary, Thr244 is an important residue for channel activation as it forms a H-bond with agonists, initiating the conformational changes required for priming of the receptor and stabilizes the open conformation. The forward movement of loop C to form a shield-like cover over the agonist is the essential initial step for channel gating.  The Role of Loop C Hydrophilic Residue 'T244' in ρ1 GABAC Receptors


extfieldsofstudy: Medicine Biology

text:
Receptor for detection of a Type II sex pheromone in the winter moth Operophtera brumata

How signal diversity evolves under stabilizing selection in a pheromone-based mate recognition system is a conundrum. Female moths produce two major types of sex pheromones, i.e., long-chain acetates, alcohols and aldehydes (Type I) and polyenic hydrocarbons and epoxides (Type II), along different biosynthetic pathways. Little is known on how male pheromone receptor (PR) genes evolved to perceive the different pheromones. We report the identification of the first PR tuned to Type II pheromones, namely ObruOR1 from the winter moth, Operophtera brumata (Geometridae). ObruOR1 clusters together with previously ligand-unknown orthologues in the PR subfamily for the ancestral Type I pheromones, suggesting that O. brumata did not evolve a new type of PR to match the novel Type II signal but recruited receptors within an existing PR subfamily. AsegOR3, the ObruOR1 orthologue previously cloned from the noctuid Agrotis segetum that has Type I acetate pheromone components, responded significantly to another Type II hydrocarbon, suggesting that a common ancestor with Type I pheromones had receptors for both types of pheromones, a preadaptation for detection of Type II sex pheromone.

Scientific RepoRts | 6:18576 | DOI: 10.1038/srep18576 and references therein. The characterized lepidopteran PRs comprise a particular subfamily of insect olfactory receptors (ORs) 26 . To date, all the characterized PRs are receptors for Type I pheromones; no PRs responsive to Type II pheromones have been found. Thus, it is unknown how the transition from detection of ancestral Type I pheromones to Type II pheromones occurred. Did moths evolve a novel type of receptors to match the novel pheromone components or were existing receptors recruited and adapted to match the new signal?

Results
Morphology and sexual dimorphism of the antennal olfactory apparatus. We first examined the sexual dimorphism of the O. brumata antennae by using scanning electron microscopy (SEM). There are abundant sensilla trichodea distributed on the ventral and lateral sides across the whole male antennae, grouping into two clusters at both ends of each segment (Fig. 1a). This type of sensilla was proven to be involved in sex pheromone reception in various moth species 30 . In the female antennae, however, the sensilla are much less abundant and covered by scales, thus not as apparent as on the male antennae (Fig. 1a).

Responses of male O. brumata antennae to the pheromone component and structural analogues.
We tested the response of the male antennae to the tetraene pheromone and three polyene analogues by gas chromatography with electroantennographic detection (GC-EAD). The male antennae showed the strongest response to the C 19 pheromone 1,3Z,6Z,9Z-19:H, and the second largest response to the C 19 triene analogue, 3Z,6Z,9Z-19:H, a weak response to the bis-homolog tetraene 1,3Z,6Z,9Z-21:H, and no response to the bis-homolog triene 3Z,6Z,9Z-21:H (Fig. 1b).
Responses from single sensilla. We subsequently tested the response of the single sensilla trichodea using the same set of compounds. For all the tested sensilla trichodea we observed a large-spike neuron responding to the tetraene pheromone, 1,3Z,6Z,9Z-19:H, and a small-spike neuron responding to the triene analogue 3Z,6Z,9Z-19:H, whereas the other two analogues elicited no response (Fig. 1c). In addition, increased spike frequencies were observed when the two antennal-active stimuli were applied at a 10x higher dose (data not shown).
Candidate genes of PR and the co-receptor. We next aimed to identify the gene encoding the receptor tuned to the pheromonal tetraene. Based on the sequences of the previously functionally identified moth PRs, we designed degenerate primers and PCR amplified the candidate cDNA fragments from male antennae. We found three partial sequences and obtained one full-length sequence by 5′ and 3′-RACE PCR. We verified the gene by full-length amplification and sequencing, and termed it ObruOR1 (Genbank accession number KJ592711). ObruOR1 encodes a protein of 430 amino acids, sharing 58% identity with the candidate PR HvirOR11 in Heliothis virescens that belongs to the Noctuidae family 31 . The full-length of the other two partial sequences could not be obtained in the present study, probably because of the low abundance of the transcripts.
In addition, we cloned the olfactory co-receptor gene from O. brumata, which encodes a predicted protein of 472 amino acids and was termed Obru\Orco (Genbank accession number KJ592712) according to the unified nomenclature system for the insect olfactory co-receptor 32 . Obru\Orco shares 65%, 83%, and 88% amino acid identity with Orcos in Drosophila melanogaster, Bombyx mori, and H. virescens, respectively 33-35 . Male-biased expression of ObruOR1. Using qPCR, we compared the expression levels of ObruOR1 and Obru\Orco genes in male and female antennae as well as in abdomens (Fig. 2). The expression level of ObruOR1 was significantly higher in male antennae than in female antennae (19.9 ± 7.8-fold), suggesting its role in the detection of female produced sex pheromones by male individuals, whereas the gene showed very low expression in abdominal tissue. Transcripts of Obru\Orco were also more abundant in male antennae than in female antennae (4.4 ± 1.0-fold), probably due to the fact that the female antennae bear much fewer sensilla (Fig. 1a).
Wide distribution of ObruOR1 in the sensilla trichodea on male antennae. To visualize the expression pattern of Obru\Orco and ObruOR1 in male antennae, we performed whole mount fluorescence in situ hybridization (FISH) with the digoxigenin (DIG) and biotin labelled antisense RNA-probes for Obru\Orco and ObruOR1 respectively. ObruOR1 was extensively expressed across the whole antennae. The labelled cell bodies were observed in clusters at both ends of each segment, exactly corresponding to the positions where the clusters of long sensilla trichodea are localized (Fig. 3a). Compared to ObruOR1, Obru\Orco was more widely expressed and distributed more evenly in each segment, associated not only to the long sensilla trichodea, but also to other olfactory sensilla (Fig. 3b).
Furthermore, we observed the co-expression pattern of Obru\Orco and ObruOR1 on the longitudinal sections of male antennae via double in situ hybridization, where the red Obru\Orco labelling and the green ObruOR1 labelling overlapped and displayed as yellow signals (Fig. 3c). In addition, we found examples of the pairing of OSNs in the long sensilla trichodea, where one of the two adjacent cells displayed a merged signal in yellow, indicating the presence of both Obru\Orco and ObruOR1, whereas the other cell showed a red signal of Obru\Orco alone (Fig. 3d). The specific receptor for the Type II sex pheromone of O. brumata. We tested the response profile of ObruOR1 by two-electrode voltage clamp (TEVC) recording on Xenopus oocytes co-expressing ObruOR1 and Obru\Orco. When challenged by the four polyenes found in the pheromone gland, the injected oocytes responded most strongly to the pheromonal tetraene 1,3Z,6Z,9Z-19:H, and only slightly to the analogues 1,3Z,6Z,9Z-21:H, 3Z,6Z,9Z-19:H, and 3Z,6Z,9Z-21:H (Fig. 4a,b). This ligand selectivity was further confirmed by a dose-response experiment. At all doses the response to the tetraene pheromone was stronger than those to the analogues. The threshold dose for 1,3Z,6Z,9Z-19:H was at 10 nM, ten times lower than for the other three compounds (Fig. 4c).
Notably, we found that after stimulating the ObruOR1 expressing oocytes with the pheromone 1,3Z,6Z,9Z-19:H, the inactivation of the inward current was much slower compared to what had been observed with the previously identified receptors for Type I pheromones. For the receptors of Type I pheromones, the recovery typically takes a few minutes [36][37][38][39][40] , however, it took approximately 40 min for ObruOR1 to get the inward current back to the baseline, even if we reduced the stimulation time from 20 s to 10 s or 5 s.

Phylogenetic analysis of moth pheromone receptors.
We examined the phylogeny of lepidopteran PRs, including ObruOR1 and it´s ligand-unknown orthologues (Fig. 5). Following previous studies 25, 40 , we recognize four apparent orthologous clusters containing receptors from the most advanced lepidopteran families. ObruOR1 is not separated from previously reported PRs, but clusters with bootstrap support 100 within Cluster III. Except for ObruOR1, Cluster III only contains candidate PRs of noctuid species. It consists of a group of ligand-unknown PRs and is under strong purifying selective pressure, indicated by the previously reported ω value (or dN/dS, the ratio of nonsynonymous to synonymous substitutions) significantly less than 1 40 . We re-calculated the ω value of this cluster after the addition of ObruOR1. By comparing the log likelihoods (lnL) of model M0 (assuming one ω ratio for all branches) and model M1 (assuming one ω ratio for each branch), we found that the one ratio model (M0) could not be rejected (P> 0.01). Thus, all branches in Cluster III share a normalized ω ratio (ω = 0.04). The low ω ratio suggests that this cluster is under strong purifying selection (Fig. 5, Table S1), which may result in a conserved ligand profile of the orthologous receptors.
Functional assay of a noctuid orthologue in Cluster III. Active ligands for PRs in Cluster III have not been previously reported. To test the above hypothesis of a conserved ligand profile within this cluster, we co-expressed AsegOR3, a noctuid orthologue that showed very weak responses to Type I pheromone components in our previous study 40 , with the corresponding co-receptor Aseg\Orco from the turnip moth Agrotis segetum, and tested it against the four polyenes that we used for ObruOR1. Indeed, the orthologous receptor responded strongly to the triene 3Z,6Z,9Z-21:H, but less so to 3Z,6Z,9Z-19:H, 1,3Z,6Z,9Z-21:H, and the O. brumata pheromone tetraene 1,3Z,6Z,9Z-19:H (Fig. 6).

Discussion
Males and females of the winter moth O. brumata show clear sexual dimorphism in their antennal morphology. We showed that a receptor neuron in sensilla trichodea on the male antennae, responded strongly to the pheromone tetraene 1,3Z,6Z,9Z-19:H. These neurons expressed the pheromone receptor gene ObruOR1, which responded specifically to the tetraene when tested in the Xenopus oocyte assay. This is the first identification of a receptor for a lepidopteran Type II pheromone.
ObruOR1 resembles receptors for Type I pheromones in most aspects. It shows male-biased expression comparing antennae from males and females, as most of the receptors for Type I pheromones 25 , and references therein. The expression is confined to long sensilla trichodea, a type of sensilla relevant to the detection of sex pheromones in moths 30 . The single-sensillum recording (SSR) results demonstrate that in the long sensilla trichodea, the large-spike neuron is responsive to the tetraene pheromone, 1,3Z,6Z,9Z-19:H, and the neighbouring small-spike neuron is responsive to the triene analogue, 3Z,6Z,9Z-19:H (Fig. 1c). We, therefore, suggest that ObruOR1 is assigned to the large-spike neuron in the long sensilla trichodea and that another unidentified receptor is expressed in the adjacent small-spike neuron responding to 3Z,6Z,9Z-19:H.
Compared to previously identified PRs, the ObruOR1 showed a much slower inactivation of the inward current to the target ligand 1,3Z,6Z,9Z-19:H in the TEVC recording (Fig. 4a). The immediate strong response was followed by an unusual slower secondary response. Notably the same pattern was observed when stimulating AsegOR3 with 3Z,6Z,9Z-21:H, the most active ligand on this receptor. We can only speculate on the explanation for these patterns. The slow inactivation process might imply a much higher affinity between the polyene and the receptor in the heterologous system than that between Type I pheromones and corresponding receptors. Under natural conditions, other related proteins such as pheromone degrading enzymes or sensory neuron membrane proteins may be involved to facilitate the fast release of the bound polyenes from the receptors and thus accelerate the inactivation process and maintain an effective signal transduction, whereas in the oocytes such proteins are not present.
The winter moth pheromone receptor ObruOR1 shares sequence identity with the receptors for Type I pheromones and nests within the orthologous Cluster III together with noctuid receptors in the phylogenetic tree. Previously, no active ligands for the ObruOR1 orthologues in Cluster III have been reported. Although they have been assumed to be relevant to the reception of structurally similar pheromones, behavioural antagonists, or the degradation products of the major sex pheromone component 41,42 , these assumptions have not received any support by functional data. We noticed that Cluster III is under strong purifying selection (Fig. 5). This is an indication of conserved ligand profiles, which suggested to us that these orthologues might also be responsive to the Type II pheromone compounds. Indeed, we found that AsegOR3, one of the orthologues, did respond to the tested polyenes which lends support to this hypothesis. To our knowledge, the noctuid species hosting the PR orthologues in Cluster III do not use polyenes as sex pheromones. Thus the fact that one of these PRs is responsive to polyene compounds is of great interest, and the behavioural significance of this finding as well as the active ligands of the other orthologues await further studies.
Our results demonstrate that evolution of the novel Type II pheromones in Lepidoptera does not require the evolution of entirely novel receptors. Instead, members of the existing pheromone receptor subfamily can be recruited for detection of the new ligands. This is different to the production of the two types of pheromones in female moths, where distinct pathways and enzymes are involved 21,22 . Type II pheromones mainly appear in the advanced moth superfamilies Geometroidea and Noctuoidea 17 and we have not found any ObruOR1 orthologues outside Geometridae and Noctuidae among available sequences of PR candidate genes. We suggest that Cluster III might have evolved in a common ancestor of the geometroid and noctuoid lineages, but testing this hypothesis will require cloning and functional characterization of pheromone receptors in a number of additional phylogenetically relevant species. Interestingly, in the Pyraloidea superfamily some species use both Type I and Type II pheromone components [43][44][45] . The co-existence of the two types of pheromone components in the same species is intriguing and invites studies of the evolution of receptors for Type II pheromone components in the pyraloids.

Materials and Methods
Ethics statement. The care and use of Xenopus laevis frogs in this study were approved by the Swedish Board of Agriculture, and the methods were carried out in accordance with the approved guidelines. Chemicals. For the synthesis of 3Z,6Z,9Z-19:H and 3Z,6Z,9Z-21:H, the tosylate of (9Z,12Z,15Z)-octadeca-9,12,15-trienol 46 was chain elongated with methyl magnesium iodide resp. propyl magnesium iodide employing cuprate chemistry 47 and using the protocol of Molnár et al. 48 . The synthesis of 1,3Z,6Z,9Z-19:H and 1,3Z,6Z,9Z-21:H started from commercially available (Aldrich) 1-bromononane and 1-bromoundecane, following the iterative chain extension described by Zhu et al. 49 . Spectroscopic data of the four polyenes were in accord with those reported in the literature [50][51][52] . , one to two day-old, were used in these experiments. After cutting off the tips, the antennae together with the head were mounted to a PRG-2 EAG (10X gain) probe (Syntech, Kirchzarten, Germany) by conductive gel (Blågel, Cefar, Malmö, Sweden). The GC inlet was set at 230 °C, and a split of the carrier gas (hydrogen) at the end of the column allowed a 1:1 split of the GC effluent to the FID and the antennal preparation. Charcoal-filtered and humidified air passed over the antennal preparation from a glass tube outlet, which was connected to the GC transfer line heated at

SSR.
For the antennal preparation, a male O. brumata was placed in a 200 μ L pipette tip fastened by dental wax on a glass slide. The antennae were carefully pulled out from the tip cut opening and fixed on a cover slip using double sided sticky tape. The recording was performed using sharpened tungsten microelectrodes, of which the reference electrode was inserted through the eye, and the recording electrode was inserted in the base of the long sensillum trichodea. The antennae were flushed with a charcoal-filtered and humidified airflow at a rate of 0.5 mL/s through a glass tube (i.d. 5.9 mm). The outlet of the tube was approximately 25 mm away from the antennae. Each tested polyene was diluted in hexane to two working concentrations, 1 μ g/μ L and 10 μ g/μ L, respectively. Ten microliters of a one-component solution were loaded on a piece of filter paper (1 × 2 cm) and inserted into a Pasteur pipette. Four to ten replicates were recorded for different tested compounds. The stimulus was introduced into the airflow at a rate of 3 mL/s for 500 ms by a stimulus controller CS-02 (Syntech). The responses were recorded and analysed with Autospike software v3.9 (Syntech).

RNA preparation and first strand cDNA synthesis.
For each RNA sample, approximately 40 pairs of antennae were dissected from male or female adults. The dissected material was separately homogenized on ice in Trizol reagent (Invitrogen, Carlsbad, CA, USA) using a tissue tearor. Total RNA isolation was performed immediately after homogenization according to the manufacturer's instructions. In the qPCR experiment, the total RNA was treated with TURBO RNase free DNase (Life technologies), followed by acid phenol: chloroform (Life technologies) purification. Non-RT control PCR was performed to verify the absence of genomic DNA contamination. First-strand cDNAs were synthesized in a 20 μ L reaction mixture containing 150 ng total RNA and 1 μ L of oligo-dT primers by ThermoScript RT-PCR System (Invitrogen, Carlsbad, CA, USA).
Gene cloning. The cloning strategy was as previously described 40,53 . Briefly, degenerate primers were designed based on the lepidopteran pheromone receptor and Orco sequences (Table S3). To amplify the partial cDNA sequences, two rounds of PCR were performed. Several pairs of primers for RACE PCR were subsequently designed based on the cDNA fragment sequences (Table S3). According to the assembled full-length cDNA sequences, primers containing restriction sites were designed for amplifying the full-length cDNAs, which were subcloned into the pCS2+ vectors (Table S3). All the sequences were verified with a capillary 3130xL Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA) and analysed with Vector NTI Advance 10 software (Invitrogen, Carlsbad, CA, USA). Quantitative PCR. The relative expression levels of ObruOR1 and Obru\Orco transcripts in male and female antennae were compared by qPCR on a Stratagene Mx3005P Real-Time PCR System (Agilent technologies), with RPS3 as the reference gene 26,38,54 and the abdomen as the control tissue. Primers used for qPCR are shown in Table  S3. Prior to qPCR, we performed RT-PCR and confirmed that RPS3 is expressed at similar level in different tissues ( Figure S1). The primer efficiencies were validated by standard curves with 10X serial dilutions of the cDNA templates. Three biological replicates were performed in separate plates, with three technical replicates on each plate. The reaction was carried out in a 20 μ L system containing 1 μ L cDNA template, 10 μ L Power SYBR Green PCR 2X Master Mix (Life technologies), and 0.3 μ M of each primer. The thermal cycling parameters consisted of an initial denaturation step at 95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min. No-template controls were run in parallel for each primer pair. A subsequent melting curve analysis was performed to ensure the primer specificity. Gel electrophoresis analysis was carried out after amplification to confirm the amplicon size. The expression level of each gene relative to RPS3 was calculated by the comparative CT method 55 . Fluorescence in situ hybridization. Biotin-or digoxigenin-labelled antisense riboprobes for ObruOR1 and Obru\Orco were transcribed from linearized recombinant pCS2+ plasmids using the T7 RNA transcription system (Roche). In situ hybridization with either a single probe or double probes was performed following the previously described protocols 31,34,56 . Briefly, digoxigenin-labelled probes were detected by anti-digoxigenin AP-conjugated antibodies in combination with the substrate HNPP/Fast Red (Fluorescent detection Set; Roche), and biotin-labelled probes were detected by streptavidin horseradish peroxidase conjugates in combination with the substrate FITC-tyramides in a TSA kit (Perkin Elmer, Boston, MA, USA). A sense probe was included as control when in situ hybridization with single probe was performed ( Figure S2). In addition, the two probes with distinct sequences (ObruOR1 and Obru\Orco) worked as controls of each other. For cryosections specimen, antennae were first embedded in Tissue-Tek O.C.T. compound (Sakura Finetek Europe, Zoeterwoude, the Netherlands) and sliced by 10 μ m thickness at − 20 °C. The cryosections were then thaw mounted on slides for subsequent hybridization and washes. For whole mount specimen, antennae were cut into pieces of ca. 4-5 mm length and gently squeezed by fine forceps. After the hybridization and washes in 0.25 mL PCR tubes, the tissues were mounted in mowiol solution. The slides were visualized on a laser-scanning microscope (Zeiss LSM510 Meta, Oberkochen, Germany).
Functional assay of candidate PRs. The oocyte microinjection and two-electrode voltage clamp recording were performed as previously described 40 . The oocytes were collected from adult female X. laevis (purchased from Xenopus Express France, Vernassal, Haute-Loire, France) and pretreated by 1.5 mg/mL collagenase (Sigma-Aldrich Co., St. Louis, MO, USA). cRNAs of ObruOR1 and Obru\Orco were synthesized from the linearized recombinant pCS2+ plasmids with mMESSAGE mMACHINE Kit (Ambion), and co-injected into the oocytes in the amount of 50 ng of each cRNA per oocyte. The injected oocytes were incubated at 18 ± 1 °C for 3-5 days, then the whole-cell inward currents at the holding potential of − 80 mV were recorded by the two-electrode voltage clamp coupled with a TEC-03BF amplifier (npi electronic GmbH, Tamm, Germany). Oocytes without cRNA injection were set as negative control. Stock solutions of tested compounds were prepared using dimethyl sulfoxide (DMSO) (Sigma-Aldrich Co., St. Louis, MO, USA), which were diluted to the indicated concentrations by Ringer's buffer before use. A perfusion system was used to apply the tested compounds to the oocytes at a rate of 2 mL/min. After the stimulation, the cell chamber was extensively washed with Ringer's buffer at a flow rate of 4 mL/min to recover the inward current to baseline. Ringer's buffer containing 0.1% DMSO alone was used as solvent control. Data were collected and analysed by Cellworks software (npi electronic GmbH, Tamm, Germany).
Phylogenetic construction and selective pressure analysis. The evolutionary history was inferred with the software MEGA 6 by using the Maximum Likelihood method based on the LG model 57,58 . The so far functionally characterized lepidopteran PRs were included, as well as ObruOR1 and it´s ligand-unknown orthologues. Initial tree for the heuristic search was obtained by applying the Neighbor-Joining method to a matrix of pairwise distances estimated using a JTT model. A discrete Gamma distribution was used to model evolutionary rate differences among sites (5 categories (+ G, parameter ≈ 1.2)). The tree was rooted with the Orco lineage. Bootstrap analysis of 100 replicates was performed. The ω value was estimated by the maximum likelihood method 59 using the Codeml program in the PAML 4.6 package 60 . Genbank accession numbers of all the PR protein sequences in the tree are presented in Table S2.