Activation by dopamine, norepinephrine and serotonin of human dopamine D4 receptor polymorphic variants coupled to GIRK channels in Xenopus oocytes.

WEDEMEYER, C.; GOUTMAN, JD; AVALE, M. E.; RUBINSTEIN, M.; DANIEL JUAN CALVO

Atlanta

Congreso; 36th Meeting of the Society for Neuroscience; 2005

ACTIVATION BY DOPAMINE, NOREPINEPHRINE AND SEROTONIN OF HUMAN D4 RECEPTOR POLYMORPHIC VARIANTS COUPLED TO GIRK CHANNELS IN XENOPUSXENOPUS OOCYTES C. Wedemeyer, J.D. Goutman, M.E. Avale, M. Rubinstein* & D.J. Calvo*. INGEBI DFBMC FCEyN UBA Buenos Aires, Argentina. Human dopamine D4 receptors (hD4R) are 7 transmembrane domain G protein–coupled receptors. They present polymorphic variation in the third intracellular loop due to the presence of a variable number of imperfect tandem repeats of 16 amino acids. This loop may be involved in coupling to G-proteins, so the occurrence of receptor isoforms might cause functional diversities. Alleles containing 2, 4 or 7 repeats (D4.2, D4.4 and D4.7) were identified in the 10%, 60% and 14% of the human population respectively, being other variants less frequent. The properties of diverse hD4R, as well as their possible role in brain physiology and disease, remain to be established. In order to characterize hD4R pharmacological properties, we co-expressed D4.2, D4.4 and D4.7 receptors with G protein–regulated inward rectifying potassium channels (GIRK1) in4.2, D4.4 and D4.7) were identified in the 10%, 60% and 14% of the human population respectively, being other variants less frequent. The properties of diverse hD4R, as well as their possible role in brain physiology and disease, remain to be established. In order to characterize hD4R pharmacological properties, we co-expressed D4.2, D4.4 and D4.7 receptors with G protein–regulated inward rectifying potassium channels (GIRK1) in4.2, D4.4 and D4.7 receptors with G protein–regulated inward rectifying potassium channels (GIRK1) in4.4 and D4.7 receptors with G protein–regulated inward rectifying potassium channels (GIRK1) in Xenopus laevis oocytes, and carry out functional analysis of the variants by using two-electrode voltage-clamp. Dopamine (DA) induced a dose-dependent increase in GIRK1 currents amplitude recorded at -100 mV only in cells injected with hD4R. D4.2 and D4.7 showed similar sensitivity (EC50= 1.02 ± 0.05 and 1.07 ± 0.03 nM), whereas D4.4 was five times less sensitive (EC50=4.89 ± 0.23 nM). Other monoamines, such as norepinephrine (NE) and serotonin (5HT), also activated D4R. NE EC50´s were: D4.2= 42.7 ± 0.8; D4.4= 51.8 ± 1.8 and D4.7= 60.1 ± 2.8 nM. The maximum response was evoked by 10 µM NE, and was similar to the maximum response elicited by DA. 5HT EC50´s were: D4.2= 1.1 ± 0.1, D4.4=1.4 ± 0.1 and D4.7= 1.7 ± 0.1 µM. 5HT elicited maximum responses at 100 µM and behaved as partial agonist (65% of the maximum DA response). The selective D4R antagonist PNU10138 reversibly blocked DA, NE and 5HT responses mediated by all variants. Our results suggest functional differences among different hD4R subtypes. In addition, we demonstrated that NE and 5HT, besides DA, activate hD4R polymorphic variants.oocytes, and carry out functional analysis of the variants by using two-electrode voltage-clamp. Dopamine (DA) induced a dose-dependent increase in GIRK1 currents amplitude recorded at -100 mV only in cells injected with hD4R. D4.2 and D4.7 showed similar sensitivity (EC50= 1.02 ± 0.05 and 1.07 ± 0.03 nM), whereas D4.4 was five times less sensitive (EC50=4.89 ± 0.23 nM). Other monoamines, such as norepinephrine (NE) and serotonin (5HT), also activated D4R. NE EC50´s were: D4.2= 42.7 ± 0.8; D4.4= 51.8 ± 1.8 and D4.7= 60.1 ± 2.8 nM. The maximum response was evoked by 10 µM NE, and was similar to the maximum response elicited by DA. 5HT EC50´s were: D4.2= 1.1 ± 0.1, D4.4=1.4 ± 0.1 and D4.7= 1.7 ± 0.1 µM. 5HT elicited maximum responses at 100 µM and behaved as partial agonist (65% of the maximum DA response). The selective D4R antagonist PNU10138 reversibly blocked DA, NE and 5HT responses mediated by all variants. Our results suggest functional differences among different hD4R subtypes. In addition, we demonstrated that NE and 5HT, besides DA, activate hD4R polymorphic variants.4.2 and D4.7 showed similar sensitivity (EC50= 1.02 ± 0.05 and 1.07 ± 0.03 nM), whereas D4.4 was five times less sensitive (EC50=4.89 ± 0.23 nM). Other monoamines, such as norepinephrine (NE) and serotonin (5HT), also activated D4R. NE EC50´s were: D4.2= 42.7 ± 0.8; D4.4= 51.8 ± 1.8 and D4.7= 60.1 ± 2.8 nM. The maximum response was evoked by 10 µM NE, and was similar to the maximum response elicited by DA. 5HT EC50´s were: D4.2= 1.1 ± 0.1, D4.4=1.4 ± 0.1 and D4.7= 1.7 ± 0.1 µM. 5HT elicited maximum responses at 100 µM and behaved as partial agonist (65% of the maximum DA response). The selective D4R antagonist PNU10138 reversibly blocked DA, NE and 5HT responses mediated by all variants. Our results suggest functional differences among different hD4R subtypes. In addition, we demonstrated that NE and 5HT, besides DA, activate hD4R polymorphic variants.50= 1.02 ± 0.05 and 1.07 ± 0.03 nM), whereas D4.4 was five times less sensitive (EC50=4.89 ± 0.23 nM). Other monoamines, such as norepinephrine (NE) and serotonin (5HT), also activated D4R. NE EC50´s were: D4.2= 42.7 ± 0.8; D4.4= 51.8 ± 1.8 and D4.7= 60.1 ± 2.8 nM. The maximum response was evoked by 10 µM NE, and was similar to the maximum response elicited by DA. 5HT EC50´s were: D4.2= 1.1 ± 0.1, D4.4=1.4 ± 0.1 and D4.7= 1.7 ± 0.1 µM. 5HT elicited maximum responses at 100 µM and behaved as partial agonist (65% of the maximum DA response). The selective D4R antagonist PNU10138 reversibly blocked DA, NE and 5HT responses mediated by all variants. Our results suggest functional differences among different hD4R subtypes. In addition, we demonstrated that NE and 5HT, besides DA, activate hD4R polymorphic variants.50´s were: D4.2= 42.7 ± 0.8; D4.4= 51.8 ± 1.8 and D4.7= 60.1 ± 2.8 nM. The maximum response was evoked by 10 µM NE, and was similar to the maximum response elicited by DA. 5HT EC50´s were: D4.2= 1.1 ± 0.1, D4.4=1.4 ± 0.1 and D4.7= 1.7 ± 0.1 µM. 5HT elicited maximum responses at 100 µM and behaved as partial agonist (65% of the maximum DA response). The selective D4R antagonist PNU10138 reversibly blocked DA, NE and 5HT responses mediated by all variants. Our results suggest functional differences among different hD4R subtypes. In addition, we demonstrated that NE and 5HT, besides DA, activate hD4R polymorphic variants.50´s were: D4.2= 1.1 ± 0.1, D4.4=1.4 ± 0.1 and D4.7= 1.7 ± 0.1 µM. 5HT elicited maximum responses at 100 µM and behaved as partial agonist (65% of the maximum DA response). The selective D4R antagonist PNU10138 reversibly blocked DA, NE and 5HT responses mediated by all variants. Our results suggest functional differences among different hD4R subtypes. In addition, we demonstrated that NE and 5HT, besides DA, activate hD4R polymorphic variants.