Bacterial Glycolipid Acting on Protein Transport Across Membranes.

The process of protein transport across membranes involves a variety of factors and has been extensively investigated. Traditionally, proteinaceous translocons and chaperones have been recognized as crucial factors in this process. However, recent studies have highlighted the significant roles played by lipids and a glycolipid present in biological membranes in membrane protein transport. Membrane lipids can influence transport efficiency by altering the physicochemical properties of membranes. Notably, our studies have revealed that diacylglycerol (DAG) attenuates mobility in the membrane core region, leading to a dramatic suppression of membrane protein integration. Conversely, a glycolipid in Escherichia coli inner membranes, named membrane protein integrase (MPIase), enhances integration not only through the alteration of membrane properties but also via direct interactions with membrane proteins. This review explores the mechanisms of membrane protein integration mediated by membrane lipids, specifically DAG, and MPIase. Our results, along with the employed physicochemical analysis methods such as fluorescence measurements, nuclear magnetic resonance, surface plasmon resonance, and docking simulation, are presented to elucidate these mechanisms.

Key contributions of a glycolipid to membrane protein integration.

Regulation of membrane protein integration involves molecular devices such as Sec-translocons or the insertase YidC. We have identified an integration-promoting factor in the inner membrane of Escherichia coli called membrane protein integrase (MPIase). Structural analysis revealed that, despite its enzyme-like name, MPIase is a glycolipid with a long glycan comprising N-acetyl amino sugars, a pyrophosphate linker, and a diacylglycerol (DAG) anchor. Additionally, we found that DAG, a minor membrane component, blocks spontaneous integration. In this review, we demonstrate how they contribute to Sec-independent membrane protein integration in bacteria using a comprehensive approach including synthetic chemistry and biophysical analyses. DAG blocks unfavorable spontaneous integrations by suppressing mobility in the membrane core, whereas MPIase compensates for this. Moreover, MPIase plays critical roles in capturing a substrate protein to prevent its aggregation, attracting it to the membrane surface, facilitating its insertion into the membrane, and delivering it to other factors. The combination of DAG and MPIase efficiently regulates the integration of membrane proteins.

Structural characteristics of patients with superior vena cava foci initiating atrial fibrillation: Analysis with electrocardiogram-triggered computed tomography.

INTRODUCTION: The superior vena cava (SVC) is the most common source of non-pulmonary vein foci in atrial fibrillation (AF); therefore, predicting the existence of non-pulmonary vein foci before the catheter ablation procedure helps construct a proper ablation strategy in preparation for SVC isolation. This study aimed to clarify the structural characteristics of patients with SVC foci initiating AF. METHODS: We enrolled 331 consecutive patients with AF who underwent cardiac computed tomography imaging before radiofrequency catheter ablation treatment, and they were divided into SVC (+) and (-) groups based on the presence or absence of SVC foci initiating AF. RESULTS: The SVC (+) group (n = 27) exhibited SVC crescent signs-defined as a curve-shaped SVC with two narrow pointed ends-more frequently (37% vs. 9%, p < .001), and larger right atrial volume (95.6 ± 20.8 vs. 80.5 ± 26.1 mL, p = .004) than the SVC (-) group (n = 304). Multivariate logistic regression analysis revealed that the SVC crescent sign (odds ratio, 8.88; 95% confidence interval [CI], 3.21-24.60) and right atrial volume (odds ratio, 1.03; 95% CI, 1.01-1.04) were independent predictors of SVC foci. CONCLUSION: Patients with SVC foci exhibited more frequent SVC crescent signs and larger right atrial volumes, and these characteristics may help clinicians choose the appropriate ablation technology.

Structural Requirements of a Glycolipid MPIase for Membrane Protein Integration.

MPIase is a glycolipid involved in membrane protein integration in the inner membrane of Escherichia coli. To overcome the trace amounts and heterogeneity of natural MPIase, we systematically synthesized MPIase analogs. Structure-activity relationship studies revealed the contribution of distinctive functional groups and the effect of the MPIase glycan length on membrane protein integration activity. In addition, both the synergistic effects of these analogs with the membrane chaperone/insertase YidC, and the chaperone-like activity of the phosphorylated glycan were observed. These results verified the translocon-independent membrane integration mechanism in the inner membrane of E. coli, in which MPIase captures the highly hydrophobic nascent proteins via its characteristic functional groups, prevents protein aggregation, attracts the proteins to the membrane surface, and delivers them to YidC in order to regenerate its own integration activity.

Transvenous lead extraction versus surgical lead extraction or conservative treatment for cardiac implantable electronic device infections: Propensity score-weighted analyses of a nationwide claim-based database.

INTRODUCTION: Infection is one of the most important complications associated with cardiac implantable electronic device (CIED) therapy. The number of reports comparing the outcomes of transvenous lead extraction (TLE), surgical lead extraction, and conservative treatment for CIED infections using a real-world database is limited. This study investigated the association between the treatment strategies for CIED infections and their outcomes. METHODS: We performed a retrospective analysis of 3605 patients with CIED infections admitted to 681 hospitals using a nationwide claim-based database collected between April 2012 and March 2018. RESULTS: We divided the 3605 patients into TLE (n = 938 [26%]), surgical lead extraction (n = 182 [5.0%]), and conservative treatment (n = 2485 [69%]) groups. TLE was performed more frequently in younger patients and at larger hospitals (p for trend < .001 for both). The rate of TLE increased during the study period, whereas that of surgical lead extraction decreased (p for trend < .001 for both). TLE was associated with lower in-hospital mortality (vs. surgical lead extraction: odds ratio [OR], 0.20; 95% CI, 0.06-0.70; vs. conservative treatment: OR, 0.45; 95% CI: 0.22-0.94) and lower 30-day readmission rates (vs. surgical lead extraction: OR, 0.18; 95% CI: 0.06-0.56; vs. conservative treatment: OR, 0.06; 95% CI, 0.03-0.13) in propensity score-weighted analyses. CONCLUSIONS: Only 26% of patients hospitalized for CIED infections received TLE. TLE was associated with significantly lower in-hospital mortality and 30-day recurrence rates than surgical lead extraction and conservative treatment, suggesting that TLE should be more widely recommended as a first-line treatment for CIED infections.

Alcohol consumption and the risk of heart failure: the Suita Study and meta-analysis of prospective cohort studies.

BACKGROUND: Alcohol consumption is a modifiable lifestyle, but its role in heart failure (HF) development is controversial. Herein, we investigated the prospective association between alcohol consumption and HF risk. METHODS: A total of 2,712 participants (1,149 men and 1,563 women) from the Suita Study were followed up every two years. Cox regression was applied to calculate the hazard ratios (HRs) and 95% confidence intervals (95% CIs) of HF risk for heavy drinking (≥46 g/day in men or ≥23 g/day in women) and never drinking compared to light drinking (<23 g/day in men or <11.5 g/day in women). Then, we combined the results of the Suita Study with those from other eligible prospective cohort studies in a meta-analysis using the random-effects model. RESULTS: In the Suita Study, within a median follow-up period of 8 years, 319 HF cases (162 in men and 157 in women) were detected. In men, but not women, never and heavy drinking carried a higher risk of HF than light drinking: HRs (95% CIs) = 1.65 (1.00, 2.73) and 2.14 (1.26, 3.66), respectively. Alike, the meta-analysis showed a higher risk of HF among heavy drinkers: HR (95% CI) = 1.37 (1.15, 1.62) and abstainers: HR (95% CI) = 1.18 (1.02, 1.37). CONCLUSION: We indicated a J-shaped association between alcohol consumption and HF risk among Japanese men. The results of the meta-analysis came in line with the Suita Study. Heavy-drinking men should be targeted for lifestyle modification interventions.

Zero-fluoroscopy ablation in patients with cardiac electronic implantable devices.

INTRODUCTION: Utilizing a three-dimensional (3-D) mapping system and intracardiac echocardiography (ICE) has allowed ablation procedures with less or without fluoroscopy; however, there is limited data for patients with cardiac electronic implantable device (CIED) leads regarding the suspected risk of lead injury. Therefore, we sought to explore technics to perform safe trans-septal approach and catheter manipulation technique in patients with CIED leads. METHODS AND RESULTS: This study comprised 49 consecutive patients (59% males, median 73 years old) with CIED who underwent catheter ablation for supraventricular tachycardia requiring the trans-septal approach, 15 without fluoroscopy (zero-fluoro group), and 34 with fluoroscopy (conventional-fluoro group), between July 2019 and April 2021. All procedures were performed under a 3-D mapping system and ICE guidance. We compared the differences in treatment and development of complications between the two groups. The procedures were for atrial fibrillation (82%) and atrial tachycardia (76%). Coronary sinus catheter insertion and the trans-septal procedure were successfully performed in all patients. The median time from venipuncture to trans-septal procedure (zero-fluoro vs. conventional-fluoro group: 28 [18-37] min vs. 24 [21-31] min, p = .70), total procedure time (231 [142-274] min vs. 175 [163-225] min, p = .63), and the acute procedural success rate (100% vs. 97%, p = 1.00) did not differ between both groups. No patient showed lead-related complications in both groups. CONCLUSION: This is the first study to show zero-fluoro ablation for supraventricular arrhythmia using 3-D mapping and ICE in patients with CIED leads was feasible under careful catheter manipulation.

Association Between White Blood Cell Count and Atrial Fibrillation Risk　- A Population-Based Prospective Cohort Study.

BACKGROUND: The incidence and prevalence of atrial fibrillation (AF) are increasing. The white blood cell (WBC) count is an indicator of systemic inflammation and is related to increased cardiovascular disease risk. Using data from the Suita Study, we investigated the association between WBC count and AF risk in the general Japanese population.Methods and Results: This prospective cohort study included 6,884 people, aged 30-84 years, with no baseline AF. Cox regression was used to calculate hazard ratios (HRs) and 95% confidence intervals (CIs) for AF incidence by WBC count quintile. Within a median follow-up period of 14.6 years, 312 AF cases were diagnosed. Compared with the lowest WBC count quintile, the highest quintile was associated with an increased AF risk (HR 1.57; 95% CI 1.07-2.29). The association was more pronounced among women than men (HR 2.16 [95% CI 1.10-4.26] and 1.55 [95% CI 0.99-2.44], respectively; P interaction=0.07), and among current than non-smokers (HR 4.66 [95% CI 1.89-11.50] and 1.61 [95% CI 1.01-2.57], respectively; P interaction=0.20). For each 1.0×109-cells/L increment in WBC count, AF risk increased by 9% in men (9% in non-smokers, 10% in current smokers) and 20% in women (13% in non-smokers, 32% in current smokers). CONCLUSIONS: A higher WBC count was positively associated with an elevated AF risk in the general Japanese population, especially in women who smoked.

Stair climbing and incident atrial fibrillation: a prospective cohort study.

BACKGROUND: A protective role for physical activity against the development of atrial fibrillation (AF) has been suggested. Stair climbing is a readily available form of physical activity that many people practice. Herein, we investigated the association between stair climbing and the risk of AF in a Japanese population. METHODS: In this prospective cohort study, we used data of 6,575 people registered in the Suita Study, aged 30-84 years, and had no history of AF. The frequency of stair climbing was assessed by a baseline questionnaire, while AF was diagnosed during the follow-up using a 12-lead ECG, health records, check-ups, and death certificates. We used the Cox regression to calculate the hazard ratios and 95% confidence intervals of AF incidence for climbing stairs in 20-39%, 40-59%, and ≥60% compared with <20% of the time. RESULTS: Within 91,389 person-years of follow-up, 295 participants developed AF. The incidence of AF was distributed across the stair climbing groups <20%, 20-39%, 40-59%, and ≥60% as follows: 3.57, 3.27, 3.46, and 2.63/1,000 person-years, respectively. Stair climbing ≥60% of the time was associated with a reduced risk of AF after adjustment for age and sex 0.69 (0.49, 0.96). Further adjustment for lifestyle and medical history did not affect the results 0.69 (0.49, 0.98). CONCLUSION: Frequent stair climbing could protect from AF. From a preventive point of view, stair climbing could be a simple way to reduce AF risk at the population level.

Transethnic Genome-Wide Association Study Provides Insights in the Genetic Architecture and Heritability of Long QT Syndrome.

BACKGROUND: Long QT syndrome (LQTS) is a rare genetic disorder and a major preventable cause of sudden cardiac death in the young. A causal rare genetic variant with large effect size is identified in up to 80% of probands (genotype positive) and cascade family screening shows incomplete penetrance of genetic variants. Furthermore, a proportion of cases meeting diagnostic criteria for LQTS remain genetically elusive despite genetic testing of established genes (genotype negative). These observations raise the possibility that common genetic variants with small effect size contribute to the clinical picture of LQTS. This study aimed to characterize and quantify the contribution of common genetic variation to LQTS disease susceptibility. METHODS: We conducted genome-wide association studies followed by transethnic meta-analysis in 1656 unrelated patients with LQTS of European or Japanese ancestry and 9890 controls to identify susceptibility single nucleotide polymorphisms. We estimated the common variant heritability of LQTS and tested the genetic correlation between LQTS susceptibility and other cardiac traits. Furthermore, we tested the aggregate effect of the 68 single nucleotide polymorphisms previously associated with the QT-interval in the general population using a polygenic risk score. RESULTS: Genome-wide association analysis identified 3 loci associated with LQTS at genome-wide statistical significance (P<5×10-8) near NOS1AP, KCNQ1, and KLF12, and 1 missense variant in KCNE1(p.Asp85Asn) at the suggestive threshold (P<10-6). Heritability analyses showed that ≈15% of variance in overall LQTS susceptibility was attributable to common genetic variation (h2SNP 0.148; standard error 0.019). LQTS susceptibility showed a strong genome-wide genetic correlation with the QT-interval in the general population (rg=0.40; P=3.2×10-3). The polygenic risk score comprising common variants previously associated with the QT-interval in the general population was greater in LQTS cases compared with controls (P<10-13), and it is notable that, among patients with LQTS, this polygenic risk score was greater in patients who were genotype negative compared with those who were genotype positive (P<0.005). CONCLUSIONS: This work establishes an important role for common genetic variation in susceptibility to LQTS. We demonstrate overlap between genetic control of the QT-interval in the general population and genetic factors contributing to LQTS susceptibility. Using polygenic risk score analyses aggregating common genetic variants that modulate the QT-interval in the general population, we provide evidence for a polygenic architecture in genotype negative LQTS.

An International Multicenter Evaluation of Type 5 Long QT Syndrome: A Low Penetrant Primary Arrhythmic Condition.

BACKGROUND: Insight into type 5 long QT syndrome (LQT5) has been limited to case reports and small family series. Improved understanding of the clinical phenotype and genetic features associated with rare KCNE1 variants implicated in LQT5 was sought through an international multicenter collaboration. METHODS: Patients with either presumed autosomal dominant LQT5 (N = 229) or the recessive Type 2 Jervell and Lange-Nielsen syndrome (N = 19) were enrolled from 22 genetic arrhythmia clinics and 4 registries from 9 countries. KCNE1 variants were evaluated for ECG penetrance (defined as QTc >460 ms on presenting ECG) and genotype-phenotype segregation. Multivariable Cox regression was used to compare the associations between clinical and genetic variables with a composite primary outcome of definite arrhythmic events, including appropriate implantable cardioverter-defibrillator shocks, aborted cardiac arrest, and sudden cardiac death. RESULTS: A total of 32 distinct KCNE1 rare variants were identified in 89 probands and 140 genotype positive family members with presumed LQT5 and an additional 19 Type 2 Jervell and Lange-Nielsen syndrome patients. Among presumed LQT5 patients, the mean QTc on presenting ECG was significantly longer in probands (476.9±38.6 ms) compared with genotype positive family members (441.8±30.9 ms, P<0.001). ECG penetrance for heterozygous genotype positive family members was 20.7% (29/140). A definite arrhythmic event was experienced in 16.9% (15/89) of heterozygous probands in comparison with 1.4% (2/140) of family members (adjusted hazard ratio [HR] 11.6 [95% CI, 2.6-52.2]; P=0.001). Event incidence did not differ significantly for Type 2 Jervell and Lange-Nielsen syndrome patients relative to the overall heterozygous cohort (10.5% [2/19]; HR 1.7 [95% CI, 0.3-10.8], P=0.590). The cumulative prevalence of the 32 KCNE1 variants in the Genome Aggregation Database, which is a human database of exome and genome sequencing data from now over 140 000 individuals, was 238-fold greater than the anticipated prevalence of all LQT5 combined (0.238% vs 0.001%). CONCLUSIONS: The present study suggests that putative/confirmed loss-of-function KCNE1 variants predispose to QT prolongation, however, the low ECG penetrance observed suggests they do not manifest clinically in the majority of individuals, aligning with the mild phenotype observed for Type 2 Jervell and Lange-Nielsen syndrome patients.

An anatomical approach to determine the location of the sinoatrial node during catheter ablation.

INTRODUCTION: The sinoatrial node (SAN) should be identified before superior vena cava (SVC) isolation to avoid SAN injury. However, its location cannot be identified without restoring sinus rhythm. This study evaluated the usefulness of the anatomically defined SAN by comparing it with the electrically confirmed SAN (e-SAN) to predict the top-most position of e-SAN and thus establish a safe and more efficient anatomical reference for SVC isolation than the previously reported reference of the right superior pulmonary vein (RSPV) roof. METHODS AND RESULTS: The e-SAN was identified as the earliest activation site in the electroanatomical map obtained during sinus rhythm. The anatomically defined SAN, the cranial edge of the crista terminalis (CT) visualized with intracardiac echocardiography (CT top), and the RSPV roof, which was obtained from the overlaid electroanatomical image of SVC and RSPV, were tagged on one map. The distance from the e-SAN to each reference was measured. Among 77 patients, the height of the e-SAN from the CT top was a median (interquartile range) of -2.0 (-8.0 to 4.0) mm. The e-SAN existed from 10 mm above the CT top or lower in 74 (96%) patients and from the RSPV roof or below in 73 (95%) patients. The reference of 10 mm above the CT top is more proximal to the right atrium than the RSPV roof and can provide longer isolatable SVC sleeves (30.0 [20.0-35.0] vs. 24.0 [18.0-30.0] mm, p < .001). The e-SAN tended to be found above the CT top when the heart rate during mapping was faster (adjusted odds ratio [95% confidence interval] per 10-bpm increase: 1.71 [1.20-2.43], p < .01). CONCLUSION: The CT top is useful for predicting the upper limit of the e-SAN and can provide a better reference for SVC isolation than the RSPV roof.

A New Secretory Peptide of Natriuretic Peptide Family, Osteocrin, Suppresses the Progression of Congestive Heart Failure After Myocardial Infarction.

RATIONALE: An increase of severe ischemic heart diseases results in an increase of the patients with congestive heart failure (CHF). Therefore, new therapies are expected in addition to recanalization of coronary arteries. Previous clinical trials using natriuretic peptides (NPs) prove the improvement of CHF by NPs. OBJECTIVE: We aimed at investigating whether OSTN (osteocrin) peptide potentially functioning as an NPR (NP clearance receptor) 3-blocking peptide can be used as a new therapeutic peptide for treating CHF after myocardial infarction (MI) using animal models. METHODS AND RESULTS: We examined the effect of OSTN on circulation using 2 mouse models; the continuous intravenous infusion of OSTN after MI and the OSTN-transgenic (Tg) mice with MI. In vitro studies revealed that OSTN competitively bound to NPR3 with atrial NP. In both OSTN-continuous intravenous infusion model and OSTN-Tg model, acute inflammation within the first week after MI was reduced. Moreover, both models showed the improvement of prognosis at 28 days after MI by OSTN. Consistent with the in vitro study binding of OSTN to NPR3, the OSTN-Tg exhibited an increased plasma atrial NP and C-type NP, which might result in the improvement of CHF after MI as indicated by the reduced weight of hearts and lungs and by the reduced fibrosis. CONCLUSIONS: OSTN might suppress the worsening of CHF after MI by inhibiting clearance of NP family peptides.

Glutamate transporter GLAST controls synaptic wrapping by Bergmann glia and ensures proper wiring of Purkinje cells.

Astrocytes regulate synaptic transmission through controlling neurotransmitter concentrations around synapses. Little is known, however, about their roles in neural circuit development. Here we report that Bergmann glia (BG), specialized cerebellar astrocytes that thoroughly enwrap Purkinje cells (PCs), are essential for synaptic organization in PCs through the action of the l-glutamate/l-aspartate transporter (GLAST). In GLAST-knockout mice, dendritic innervation by the main ascending climbing fiber (CF) branch was significantly weakened, whereas the transverse branch, which is thin and nonsynaptogenic in control mice, was transformed into thick and synaptogenic branches. Both types of CF branches frequently produced aberrant wiring to proximal and distal dendrites, causing multiple CF-PC innervation. Our electrophysiological analysis revealed that slow and small CF-evoked excitatory postsynaptic currents (EPSCs) were recorded from almost all PCs in GLAST-knockout mice. These atypical CF-EPSCs were far more numerous and had significantly faster 10-90% rise time than those elicited by glutamate spillover under pharmacological blockade of glial glutamate transporters. Innervation by parallel fibers (PFs) was also affected. PF synapses were robustly increased in the entire dendritic trees, leading to impaired segregation of CF and PF territories. Furthermore, lamellate BG processes were retracted from PC dendrites and synapses, leading to the exposure of these neuronal elements to the extracellular milieus. These synaptic and glial phenotypes were reproduced in wild-type mice after functional blockade of glial glutamate transporters. These findings highlight that glutamate transporter function by GLAST on BG plays important roles in development and maintenance of proper synaptic wiring and wrapping in PCs.

Alteration of Membrane Physicochemical Properties by Two Factors for Membrane Protein Integration.

After a nascent chain of a membrane protein emerges from the ribosomal tunnel, the protein is integrated into the cell membrane. This process is controlled by a series of proteinaceous molecular devices, such as signal recognition particles and Sec translocons. In addition to these proteins, we discovered two endogenous components regulating membrane protein integration in the inner membrane of Escherichia coli. The integration is blocked by diacylglycerol (DAG), whereas the blocking is relieved by a glycolipid named membrane protein integrase (MPIase). Here, we investigated the influence of these integration-blocking and integration-promoting factors on the physicochemical properties of membrane lipids via solid-state NMR and fluorescence measurements. These factors did not have destructive effects on membrane morphology because the membrane maintained its lamellar structure and did not fuse in the presence of DAG and/or MPIase at their effective concentrations. We next focused on membrane flexibility. DAG did not affect the mobility of the membrane surface, whereas the sugar chain in MPIase was highly mobile and enhanced the flexibility of membrane lipid headgroups. Comparison with a synthetic MPIase analog revealed the effects of the long sugar chain on membrane properties. The acyl chain order inside the membrane was increased by DAG, whereas the increase was cancelled by the addition of MPIase. MPIase also loosened the membrane lipid packing. Focusing on the transbilayer movement, MPIase reduced the rapid flip-flop motion of DAG. On the other hand, MPIase could not compensate for the diminished lateral diffusion by DAG. These results suggest that by manipulating the membrane lipids dynamics, DAG inhibits the protein from contacting the inner membrane, whereas the flexible long sugar chain of MPIase increases the opportunity for interaction between the membrane and the protein, leading to membrane integration of the newly formed protein.

Catalytic Asymmetric Hydrogenation of Dehydroamino Acid Esters with Biscarbamate Protection and Its Application to the Synthesis of xCT Inhibitors.

Catalytic asymmetric hydrogenation of dehydroamino acid esters with biscarbamate protection was examined for the first time to prepare optically active amino acids. The new method was successfully applied to the synthesis of new cystine-glutamate exchanger inhibitors.

The Role of the Prod1 Membrane Anchor in Newt Limb Regeneration.

Prod1 is a protein that regulates limb regeneration in salamanders by determining the direction of limb growth. Prod1 is attached to the membrane by a glycosylphosphatidylinositol (GPI) anchor, but the role of membrane anchoring in the limb regeneration process is poorly understood. In this study, we investigated the functional role of the anchoring of Prod1 to the membrane by using its synthetic mimics in combination with solid-state NMR spectroscopy and fluorescent microscopy techniques. Anchoring did not affect the three-dimensional structure of Prod1 but did induce aggregation by aligning the molecules and drastically reducing the molecular motion on the two-dimensional membrane surface. Interestingly, aggregated Prod1 interacted with Prod1 molecules tethered on the surface of opposing membranes, inducing membrane adhesion. Our results strongly suggest that anchoring of the salamander-specific protein Prod1 assists cell adhesion in the limb regeneration process.

Structure-activity relationship study at C9 position of kaitocephalin.

Kaitocephalin (KCP) isolated from Eupenicillium shearii PF1191 is an unusual amino acid natural product in which serine, proline, and alanine moieties are liked with carbon-carbon bonds. KCP exhibits potent and selective binding affinity for one of the ionotropic glutamate receptor subtypes, NMDA receptors (Ki=7.8nM). In this study, new structure-activity relationship studies at C9 of KCP were implemented. Eleven new KCP analogs with different substituents at C9 were prepared and employed for binding affinity tests using native ionotropic glutamate receptors. Replacement of the 3,5-dichloro-4-hydroxybenzoyl group of KCP with a 3-phenylpropionyl group resulted in significant loss of binding affinity for NMDARs (Ki=1300nM), indicating an indispensable role of the aromatic ring of KCP in the potent and selective binding to NMDARs. Other analogs showed potent binding affinity in a range of 11-270nM. These findings would directly link to develop useful chemical tools toward imaging and labeling of NMDARs.