scholarly journals Kinetic Aspects of Verapamil Binding (On-Rate) on Wild-Type and Six hKv1.3 Mutant Channels

2017 ◽  
Vol 44 (1) ◽  
pp. 172-184
Author(s):  
Ann-Kathrin Diesch ◽  
Stephan Grissmer
Keyword(s):  
Binding Site ◽  
Time Course ◽  
Open Channel ◽  
Binding Pocket ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Patch Clamp Technique ◽  
Open Channel Block ◽  
Mutant Channel ◽  
Whole Cell Patch Clamp

Background/Aims: The human-voltage gated Kv1.3 channel (hKv1.3) is expressed in T- and B lymphocytes. Verapamil is able to block hKv1.3 channels. We characterized the effect of verapamil on currents through hKv1.3 channels paying special attention to the on-rate (kon) of verapamil. By comparing on-rates obtained in wild-type (wt) and mutant channels a binding pocket for verapamil and impacts of different amino acid residues should be investigated. Methods: Using the whole-cell patch clamp technique the action of verapamil on currents through wild-type and six hKv1.3 mutant channels in the open state was investigated by measuring the time course of the open channel block in order to calculate kon of verapamil. Results: The on-rate of verapamil to block current through hKv1.3_T419C mutant channels is similar to that obtained for hKv1.3_wt channels whereas the on-rate of verapamil to block currents through hKv1.3_L417C and hKv1.3_L418C mutant channels was ∼ 3 times slower compared to in wt channels. The on-rate of verapamil to block currents through hKv1.3_L346C and the double mutant hKv1.3_L346C_L418C channel was ∼ 2 times slower compared to that obtained in the wt channel. The hKv1.3_I420C mutant channel reduced the on-rate of verapamil to block currents ∼ 6 fold. Conclusions: We conclude that position 420 in hKv1.3 channels maximally interferes with verapamil reaching its binding site to block the channel. Positions 417 and 418 in hKv1.3 channels partially hinder verapamil reaching its binding site to block the channel whereas position 419 may not interfere with verapamil at all. Mutant hKv1.3_L346C and hKv1.3_L346C_L418C mutant channels might indirectly influence the ability of verapamil reaching its binding site to block current.

Acute effects of repetitive depolarization on sodium current in chick myocytes

1991 ◽  
Vol 260 (6) ◽  
pp. H1810-H1818
Author(s):  
M. R. Gold ◽  
G. R. Strichartz
Keyword(s):  
Time Course ◽  
Sodium Current ◽  
Complete Recovery ◽  
Acute Effects ◽  
Na Current ◽  
Patch Clamp Technique ◽  
Membrane Hyperpolarization ◽  
Atrial Cells ◽  
Whole Cell Patch Clamp ◽  
Recovery From Inactivation

Acute effects of repetitive depolarization on the inward Na+ current (INa) of cultured embryonic chick atrial cells were studied using the whole cell patch-clamp technique. Stimulation rates of 1 Hz or greater produced a progressive decrement of peak INa. With depolarizations to 0 mV of 150-ms duration, applied at 2 Hz from a holding potential of -100 mV, the steady-state decrement was approximately 20%. The magnitude of this effect increased with stimulation frequency and with test potential depolarization and decreased with membrane hyperpolarization. Analysis of INa kinetics revealed that reactivation was sufficiently slow to preclude complete recovery from inactivation with interpulse intervals less than 1,000 ms. Moreover, reactivation accelerated markedly with membrane hyperpolarization, in parallel with the response to repetitive stimulation. The multiexponential time course of recovery of peak INa from repetitive depolarization was similar to that observed after single stimuli; however, there was a shift toward a greater proportion of current recovering with the slower of two time constants. It is concluded that incomplete recovery from inactivation is responsible for the decrement in INa observed with short interpulse intervals.

scholarly journals State-dependent Block of Wild-type and Inactivation-deficient Na+ Channels by Flecainide

2003 ◽  
Vol 122 (3) ◽  
pp. 365-374 ◽  
Author(s):  
Ging Kuo Wang ◽  
Corinna Russell ◽  
Sho-Ya Wang
Keyword(s):  
Time Constant ◽  
Open Channel ◽  
Inhibitory Concentration ◽  
Antiarrhythmic Agent ◽  
Na Channels ◽  
Fast Inactivation ◽  
Wild Type ◽  
Open Channel Block ◽  
State Dependent ◽  
Na Currents

The antiarrhythmic agent flecainide appears beneficial for painful congenital myotonia and LQT-3/ΔKPQ syndrome. Both diseases manifest small but persistent late Na+ currents in skeletal or cardiac myocytes. Flecainide may therefore block late Na+ currents for its efficacy. To investigate this possibility, we characterized state-dependent block of flecainide in wild-type and inactivation-deficient rNav1.4 muscle Na+ channels (L435W/L437C/A438W) expressed with β1 subunits in Hek293t cells. The flecainide-resting block at −140 mV was weak for wild-type Na+ channels, with an estimated 50% inhibitory concentration (IC50) of 365 μM when the cell was not stimulated for 1,000 s. At 100 μM flecainide, brief monitoring pulses of +30 mV applied at frequencies as low as 1 per 60 s, however, produced an ∼70% use-dependent block of peak Na+ currents. Recovery from this use-dependent block followed an exponential function, with a time constant over 225 s at −140 mV. Inactivated wild-type Na+ channels interacted with flecainide also slowly at −50 mV, with a time constant of 7.9 s. In contrast, flecainide blocked the open state of inactivation-deficient Na+ channels potently as revealed by its rapid time-dependent block of late Na+ currents. The IC50 for flecainide open-channel block at +30 mV was 0.61 μM, right within the therapeutic plasma concentration range; on-rate and off-rate constants were 14.9 μM−1s−1 and 12.2 s−1, respectively. Upon repolarization to −140 mV, flecainide block of inactivation-deficient Na+ channels recovered, with a time constant of 11.2 s, which was ∼20-fold faster than that of wild-type counterparts. We conclude that flecainide directly blocks persistent late Na+ currents with a high affinity. The fast-inactivation gate, probably via its S6 docking site, may further stabilize the flecainide-receptor complex in wild-type Na+ channels.

Regulation of calcium current by voltage and cytoplasmic calcium in canine gastric smooth muscle

1992 ◽  
Vol 262 (3) ◽  
pp. C691-C700 ◽  
Author(s):  
F. Vogalis ◽  
N. G. Publicover ◽  
K. M. Sanders
Keyword(s):  
Time Course ◽  
Fast Component ◽  
Patch Clamp Technique ◽  
Similar Time ◽  
Whole Cell Patch Clamp ◽  
Gastric Smooth Muscle ◽  
Double Exponential ◽  
Circular Layer ◽  
Double Exponential Function ◽  
Ca2 Channels

The regulation of Ca2+ current by intracellular Ca2+ was studied in isolated myocytes from the circular layer of canine gastric antrum. Ca2+ current was measured with the whole cell patch-clamp technique, and changes in cytoplasmic Ca2+ ([Ca2+]i) were simultaneously measured with indo-1 fluorescence. Ca2+ currents were activated by depolarization and inactivated despite maintained depolarization. Ca2+ current inactivation was fit with a double exponential function. Using Ba2+ or Na+ as charge carriers removed the fast component of inactivation, whereas enhanced intracellular buffering of Ca2+ did not remove the fast component. Ca2+ currents were associated with a rise in [Ca2+]i. The decrease in [Ca2+]i following repolarization was exponential, and during the relaxation of [Ca2+]i, Ca2+ current was inactivated. The inward current recovered with a similar time course as the decrease in [Ca2+]i, suggesting that [Ca2+]i regulates the basal availability of Ca2+ channels. These data support the hypothesis that, although [Ca2+]i may influence the resting level of inactivation, it is the "submembrane" compartment of [Ca2+]i that regulates the development of inactivation.

Glycine regulation of synaptic NMDA receptors in hippocampal neurons

1996 ◽  
Vol 76 (5) ◽  
pp. 3415-3424 ◽  
Author(s):  
K. S. Wilcox ◽  
R. M. Fitzsimonds ◽  
B. Johnson ◽  
M. A. Dichter
Keyword(s):  
Nmda Receptor ◽  
Nmda Receptors ◽  
Hippocampal Neurons ◽  
Time Course ◽  
Hippocampal Slices ◽  
Maximal Effect ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Dissociated Cell Culture ◽  
Cultured Hippocampal Neurons

1. Although glycine has been identified as a required coagonist with glutamate at N-methyl-D-aspartate (NMDA) receptors, the understanding of glycine's role in excitatory synaptic neurotransmission is quite limited. In the present study, we used the whole cell patch-clamp technique to examine the ability of glycine to regulate current flow through synaptic NMDA receptors at excitatory synapses between cultured hippocampal neurons and in acutely isolated hippocampal slices. 2. These studies demonstrate that the glycine modulatory site on the synaptic NMDA receptor is not saturated under baseline conditions and that increased glycine concentrations can markedly increased NMDA-receptor-mediated excitatory postsynaptic currents (EPSCs) in hippocampal neurons in both dissociated cell culture and in slice. Saturation of the maximal effect of glycine takes place at different concentrations for different cells in culture, suggesting the presence of heterogenous NMDA receptor subunit compositions. 3. Bath-applied glycine had no effect on the time course of EPSCs in either brain slice or culture, indicating that desensitization of the NMDA receptor is not prevented by glycine over the time course of an EPSC. 4. When extracellular glycine concentration is high, all miniature EPSCs recorded in the cultured hippocampal neurons contained NMDA components, indicating that segregation of non-NMDA receptors at individual synaptic boutons does not occur.

Force measurements from voltage-clamped guinea pig ventricular myocytes

1990 ◽  
Vol 258 (2) ◽  
pp. H452-H459 ◽  
Author(s):  
N. Shepherd ◽  
M. Vornanen ◽  
G. Isenberg
Keyword(s):  
Guinea Pig ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Isometric Force ◽  
Contractile Force ◽  
Patch Clamp Technique ◽  
Thin Glass ◽  
Tonic Component ◽  
Time To Peak ◽  
Whole Cell Patch Clamp

We describe the first observations of isolated mammalian guinea pig ventricular myocytes that combine measurements of contractile force with the voltage-clamp method. The myocytes were attached by poly-L-lysine to the beveled ends of a pair of thin glass rods having a compliance of 0.76 m/N. The contractile force of a cell caused a 1- to 3-microm displacement of the rods; the motion of which was converted to an output voltage by phototransistors. By the use of the whole cell patch-clamp technique, the cells were depolarized at 1 Hz with 200-ms-long clamp pulses from -45 to +5 mV (35 degrees C, 3.6 mM CaCl2). Isometric force began after a latency of 7 +/- 2 ms, peaked at 93 +/- 21 ms, and relaxed (90%) at 235 +/- 63 ms. The time course of force was always faster than that of isotonic shortening (time to peak 154 +/- 18 ms). With 400-ms-long depolarizations, a tonic component was recorded as either sustained force or sustained shortening that decayed on repolarization. Substitution of Ca by Sr in the bath increased the inward current through Ca channels but slowed down the time course of force development. The results are consistent with the hypothesis that activator calcium derives mainly from internal stores and that Ca release needs Ca entry through channels.

scholarly journals Cx46 hemichannels contribute to the sodium leak conductance in lens fiber cells

2014 ◽  
Vol 306 (5) ◽  
pp. C506-C513 ◽  
Author(s):  
Lisa Ebihara ◽  
Yegor Korzyukov ◽  
Sorabh Kothari ◽  
Jun-Jie Tong
Keyword(s):  
Open Channel ◽  
Basal Membrane ◽  
Resting Potential ◽  
Channel Noise ◽  
Lens Fiber ◽  
Patch Clamp Technique ◽  
Fiber Cells ◽  
Whole Cell Patch Clamp ◽  
Ionic Fluxes ◽  
Persistent Inward Current

The lens is proposed to have an internal microcirculation system consisting of continuously circulating ionic fluxes that play an essential role in maintaining lens transparency. One of the key components of this system is the sodium leak conductance. Here we investigate the contribution of Cx46 hemichannels to the basal membrane permeability of peripheral fiber cells isolated from transgenic mouse lenses lacking Cx50 or both Cx50 and Cx46 (dKO) using the whole cell patch-clamp technique. Our results show that Cx46 hemichannels were largely closed at a resting voltage of −60 mV in the presence of millimolar divalent cation concentrations. However, even though the vast majority of these channels were closed at −60 mV, a small, persistent, inward current could still be detected. This current could be mostly blocked by exposure to 1 mM La3+ and was not observed in fiber cells isolated from dKO mouse lenses suggesting that it was due to Cx46 hemichannels. In addition, Cx50−/− fiber cells showed increased open channel noise and a depolarized resting potential compared with dKO fiber cells. Exposure of Cx50−/− fiber cells to La3+ hyperpolarized the resting potential to −58 mV, which is similar to the value of resting potential measured in dKO fiber and significantly reduced the open channel noise. In conclusion, these results suggest that Cx46 hemichannels may contribute to the sodium leak conductance in lens fiber cells.

Identification of Amino Acid Residues in the C1 Domain of Human Factor Va2 That Affect Phosphatidylserine-Triggered Cofactor Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1711-1711
Author(s):  
Rinku Majumder ◽  
Mary Ann Quinn-Allen ◽  
Barry R. Lentz ◽  
William H. Kane
Keyword(s):  
Amino Acid ◽  
Binding Site ◽  
Factor Xa ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Hydrophobic Residues ◽  
Factor Va ◽  
C1 Domain ◽  
Cofactor Activity ◽  
Prothrombin Activation

Abstract Tightly associated factors Va and Xa serve as the essential prothrombin-activating complex whose assembly is triggered by occupancy of phosphatidylserine (PS) regulatory sites on both proteins. Factor Va C2 domain contains a binding site for soluble, short chain PS (C6PS) that includes the indole moieties of Trp2063/Trp2064 at the apex of a loop (“spike-1”) (Srivastava A, Quinn-Allen MA, Kim SW, Kane WH, Lentz BR. Biochemistry, 2001, 40(28): 8246–55). Our recent data show that there is a C6PS site in the factor Va2 C1 domain that serves as a regulatory site for assembly and/or activity of the FVa2-FXa complex (Majumder R, Quinn-Allen MA, Kane WH & Lentz BR. Manuscript in Preparation). This C6PS-binding site also involves aromatic and hydrophobic residues (Tyr1956/Tyr1957) located in a homologous loop whose apex is termed “spike 3”. In order to identify the amino acid residues in the C1 domain that contribute to the PS-mediated cofactor activity of factor Va2, charged and hydrophobic residues predicted to be exposed in FVa2-C1 domain were mutated to alanine in clusters of 1–3 mutations per construct. The resultant 20 mutants (R1880A, D1892A, (K1896,E1899)A, (F1900,L1901,Y1903)A, (E1905,R1907)A, Y1917A, (E1923,K1924)A, (K1941,E1942)A, (K1954,H1955)A, (Y1956,L1957)A, Y1956A, L1957A, K1958A, E1964A, K1980A, D1995A, R2019A, (R2023,R2027)A, R2023A, R2027A,) and factor V wild type were expressed in Cos-7 cells followed by activation with thrombin, partial purification and concentration using HiTrap SP HP columns. The specific activities of all factor Va mutants were greater than 70% of wild type, with concentrations in the 1.5-7μM range. Recently it has been shown that two mutants (Y1956, L1957)A and (R2023,R2027)A showed decreased binding to immobilized PS and a selective decrease in prothrombinase activity on membranes containing 5% PS (Saleh M, Peng W, Quinn-Allen MA, Macedo-Ribeiro S, Fuentes-Prior P, Bode W & Kane WH. Thromb. Haemost.2004, 91:16–27). Here we report the rate of prothrombin activation in the presence of 1 nM factor Xa, 5 nM factorVa2 (mutants and wild type) and 400 mM C6PS. Enhancement of cofactor activity (E) of factor Va-C1 wild type and mutants by C6PS was measured using the following equation ( Zhai X, Srivastava A, Drummond DC, Daleke D and Lentz BR. Biochemistry. 2002, 41: 5675–84): \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \[E=\ (r_{Xa.Va.PL}/r_{Xa.PL})/(r_{Xa.Va}/r_{Xa})\] \end{document} Here, rXa·Va·PL is the rate of prothrombin activation measured as the initial slope of the rate of change of normalized DAPA fluorescence with time by enzyme in the presence of factor Va and lipid, and other terms are defined analogously. The cofactor activities of (Y1956, L1957) A, Y1956A and L1957A were drastically reduced (values are 1.1, 4.2 and 5.1 respectively) relative to the cofactor activity of the wild type factor Va2 (15). The cofactor activities of (R2023, R2027) A, E1964A and (K1954, H1955) A were also reduced but to a lesser extent (values are 8, 10.6 and 12 respectively). We plan to monitor the binding of these mutants to C6PS and to factor Xa in the presence of C6PS in order to determine the role of these mutations on the assembly and activity of prothrombinase. Supported by grants from the NHLBI (HL43106 to W. Kane and HL 072827 to BRL).

The Novel BCR-ABL1 V304D Mutation Induces Pan-Tyrosine Kinase Inhibitor Resistance by a Unique Kinase Lateral Escape Mechanism and Is Associated with Very Poor Prognosis In Patients (PTS) with Chronic Myeloid Leukemia (CML).

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3402-3402
Author(s):  
Sabrina Pricl ◽  
Don L Gibbons ◽  
Paola Posocco ◽  
Erik Laurini ◽  
Maurizio Fermeglia ◽  
...  
Keyword(s):  
Binding Site ◽  
Research Funding ◽  
Poor Prognosis ◽  
Binding Pocket ◽  
Activation Loop ◽  
Wild Type ◽  
Tki Resistance ◽  
Ic50 Values ◽  
Lateral Escape ◽  
Free Energy Of Binding

Abstract Abstract 3402 We discovered a novel BCR-ABL1 mutation (V304D) in pts with CML failing imatinib by DNA expansion of specific clones followed by DNA sequencing of ≥10 clones. BCR-ABL1V304D was detected in a median of 37% (range, 20% to 80%) resistant clones from 13 (18%) of 70 imatinib-resistant pts with CML in chronic phase (CP). Pts received imatinib for a median of 35 months (range 2 to 66) at doses ≥600 mg/d. No pt achieved a cytogenetic response. Four received nilotinib: 3 had hematologic resistance and 1 progressed to blast phase (BP). All pts died with negligible response to second-line TKIs: 8/12 pts on dasatinib had disease progression and 4 responded (2 hematologic and 2 transient minor cytogenetic responses). BCR-ABL1V304D failed to induce cytokine-independence or activate Stat5 in Ba/F3 cells. Phosphorylation of CrkL and specific BCR-ABL1 substrates were detectable but diminished compared to unmutated BCR-ABL1-transduced cells. BCR-ABL1V304D failed to catalyze autophosphorylation and the catalytic domain of ABL1V304D demonstrated deficient kinase activity. Enforced expression of BCR-ABL1V304Din CML cells induced quiescence and protection from imatinib-induced apoptosis. In vitro analyses of cells from a pt in CP expressing BCR-ABL1V304D in 50% of clones failed to detect CrKL phosphorylation in the presence of normal BCR-ABL1 protein levels, suggesting that BCR-ABL1V304D encodes a kinase-deficient protein and is associated with remarkable TKI resistance and extremely poor prognosis. To determine the mechanism of resistance imposed by BCR-ABL1V304D, we modeled this mutation in water and counterions and compared it to unmutated and mutant BCR-ABL1 isoforms. We first correlated the free energy of binding (DGbind) to the corresponding IC50 (DGbind = -RT lnIC50) and calculated the difference in free energy of binding between wild-type and mutant kinases (DDGbind = DGbind(WT) – DGbind(MUT)). DGbind <0 indicates a tighter binding to a TKI of the unmutated kinase relative to the mutant kinase. A negative increase of 1.4 kcal/mol in DGbind corresponds to a decrease by a factor of 10 in the IC50 value. The DGbind (IC50) values of imatinib for wild-type, Y253H, and T315I kinases were -10.47kcal/mol (21nM), -7.45 kcal/mol (3.4mM), and -6.38 kcal/mol (21mM), similar to published experimental data (25nM, 1.8–3.9mM, and >10mM, respectively), thus validating our modeling. DGbind and IC50 values for imatinib and dasatinib against V304D are -9.86kcal/mol (59nM) and -12.27 kcal/mol (1.02nM), respectively. 3D images generated from an equilibrated frame of 10 ns molecular dynamics (MD) simulations demonstrated that the 304 position is not in direct contact with imatinib, nor does it directly alter imatinib binding. Rather, V304D disturbs the position of the regulatory αC helix (Figure1). Longer standard molecular dynamics simulations coupled with steered MD recipes indicate that V304D induces a rearrangement of the ATP/drug binding pocket and water-mediated disruption of some fundamental hydrogen bonds regulating the transition of the activation loop to a “semi-open” conformation and the apt overall conformation of the SH3-binding segment of the TK (residues K294-F311). Furthermore, a decrease in the number of total interactions causes unidirectional drug translation toward the binding site exit. Iterative simulations revealed significant ATP/inhibitor diversion with subsequent complete imatinib expulsion. Thus, the V304D-induced semi-opened conformation of the activation loop favors 1) the lateral escape of imatinib, thus increasing the rate of TKI dissociating from the kinase and 2) does not allow the passage of ATP to reach deep into the binding pocket, thus hampering tyrosine phosphorylation. A similar phenomenon is observed in the activation loop in the active conformation of the V304D kinase bound to dasatinib, which results in greater exposure to water solvent of a part of the binding site and almost complete loss of hydrophobic contacts in the opposite end of the binding site. Fig. 1 MD snapshots of Imatinib (colored sticks) bound to (top) and “escaped” from (bottom) SCTABLIV304D. The mutant residue D304 is highlighted in yellow. Note the rearrangement of the activation loop, the SH3 binding region, and the helix C, colored blue, spring green, and orange in the lower panel. Some waters and counterions are shown as colored spheres. Fig. 1. MD snapshots of Imatinib (colored sticks) bound to (top) and “escaped” from (bottom) SCTABLIV304D. The mutant residue D304 is highlighted in yellow. Note the rearrangement of the activation loop, the SH3 binding region, and the helix C, colored blue, spring green, and orange in the lower panel. Some waters and counterions are shown as colored spheres. In summary, BCR-ABL1V304D results in kinase inactivation, pan-TKI resistance mediated by a novel mechanism of lateral escape at the kinase domain, less control of protein autoinhibition via perturbation of the SH3 binding domain and very poor prognosis. Complete modeling data against a panel of novel TKIs and potential modes of overcoming this novel mechanism of resistance will be presented. Disclosures: Kantarjian: Bristol Myers Squibb: Research Funding; ARIAD: Research Funding; Nerviano: Research Funding. Cortes:Bristol Myers Squibb: Research Funding; ARIAD: Research Funding; Nerviano: Research Funding.

scholarly journals Mutations in the cyclic AMP binding site of the cyclic AMP receptor protein of Escherichia coli

1988 ◽  
Vol 253 (3) ◽  
pp. 801-807 ◽  
Author(s):  
A M Gronenborn ◽  
R Sandulache ◽  
S Gärtner ◽  
G M Clore
Keyword(s):  
Escherichia Coli ◽  
Cyclic Amp ◽  
Binding Site ◽  
Cyclic Nucleotides ◽  
Binding Pocket ◽  
Receptor Protein ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Cyclic Amp Receptor Protein ◽  
Better Than

Mutants in the cyclic AMP binding site of the cyclic AMP receptor protein (CRP) of Escherichia coli have been constructed by oligonucleotide-directed mutagenesis. They have been phenotypically characterized and their ability to enhance the expression of catabolite-repressible operons has been tested. In addition, the binding of cyclic nucleotides to the mutants has been investigated. It is shown that the six mutants made fall into one of three classes: (i) those that bind cyclic AMP better than the wild type protein (Ser-62→Ala) and result in greater transcription enhancement; (ii) those that bind cyclic AMP similarly to wild type (Ser-83→Ala, Ser-83→Lys, Thr-127→Ala, Ser-129→Ala); and (iii) those that do not bind cyclic AMP at all (Arg-82→Leu). Implications of these findings with respect to present models of the cyclic nucleotide binding pocket of CRP are discussed.

scholarly journals OR24-01 Mutational Study of the GPR119 Receptor Binding Site

2020 ◽  
Vol 4 (Supplement_1) ◽  
Author(s):  
Matthew D Rosales ◽  
Frank Dean ◽  
Evangelia Kotsikorou
Keyword(s):  
Hydrogen Bonding ◽  
Binding Site ◽  
Specific Activity ◽  
Md Simulations ◽  
Binding Pocket ◽  
Receptor Activation ◽  
Activation Mechanism ◽  
Receptor Model ◽  
Wild Type

Abstract The GPR119 receptor, a class A G-protein coupled receptor located in the pancreatic β cells, induces insulin production when activated. Due to its specific activity, the pharmaceutical industry has identified GPR119 as a target for the treatment for type 2 diabetes. The lack of a GRP119 crystal structure has hindered the study of the receptor so our laboratory developed GPR119 active and inactive homology models. Docking studies with the inactive receptor model indicated that two leucine residues facing the binding pocket, L5.43(169) and L6.52(242), may be involved in ligand activation. Additionally, a serine at the extracellular end of the pocket, S1.32(4), may help orient of the ligand in the binding pocket via hydrogen bonding. To gain further insight into the role of these residues and the receptor activation mechanism, molecular dynamics (MD) simulations and in vitro cAMP assays of the wild type and mutant receptors were employed. The software NAMD employing the CHARMM force field was used to carry out MD simulations of the active receptor model bound with the agonist AR231453 embedded in a hydrated lipid bilayer. Preliminary results indicate that L6.52(242), located on transmembrane helix (TMH) 6, does not face directly into the binding site and does not interact with the ligand, while L5.43(169), located on TMH5, does face into the binding site, potentially interacting directly with the ligand. Also, S1.32(4), because of its extracellular location, is solvated instead of interacting with the ligand. The in vitro studies overall support the MD simulations. The mutations L6.52(242)M and L6.52(242)A appear to have minimal to no effect on agonist-induced cAMP production, compared to the wild type. In contrast, the L5.43(169)M and L5.43(169)A mutations decrease the potency of activation by AR231453, indicating that L5.43(169) changes the shape of the binding pocket, affecting ligand binding and activation. Finally, the cAMP assays show that the S1.32(4)A mutant also shows decreased activity compared to the wild type, implying that the ligand may be losing a hydrogen bonding interaction when S1.32(4) is mutated to alanine.

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