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Macroscopic Currents (macroscopic + current)

Distribution by Scientific Domains

Life Sciences	75%

Selected Abstracts

Dissecting the pathogenic mechanisms of mutations in the pore region of the human cone photoreceptor cyclic nucleotide-gated channel,

HUMAN MUTATION, Issue 7 2010
Katja Koeppen
Abstract The CNGA3 gene encodes the A3 subunit of the cone photoreceptor cyclic nucleotide-gated (CNG) channel, an essential component of the phototransduction cascade. Certain mutations in CNGA3 cause autosomal recessive achromatopsia, a retinal disorder characterized by severely reduced visual acuity, lack of color discrimination, photophobia, and nystagmus. We identified three novel mutations in the pore-forming region of CNGA3 (L363P, G367V, and E376K) in patients diagnosed with achromatopsia. We assessed the expression and function of channels with these three new and two previously described mutations (S341P and P372S) in a heterologous HEK293 cell expression system using Western blot, subcellular localization on the basis of immunocytochemistry, calcium imaging, and patch clamp recordings. In this first comparative functional analysis of disease-associated mutations in the pore of a CNG channel, we found impaired surface expression of S341P, L363P, and P372S mutants and reduced macroscopic currents for channels with the mutations S341P, G367V, and E376K. Calcium imaging and patch clamp experiments after incubation at 37°C revealed nonfunctional homo- and heteromeric channels in all five mutants, but incubation at 27°C combined with coexpression of the B3 subunit restored residual function of channels with the mutations S341P, G367V, and E376K. Hum Mutat 31:830,839, 2010. © 2010 Wiley-Liss, Inc. [source]

Selectivity of lynx proteins on insect nicotinic acetylcholine receptors in the brown planthopper, Nilaparvata lugens

INSECT MOLECULAR BIOLOGY, Issue 3 2010
B. Yang
Abstract Neuronal nicotinic acetylcholine receptors (nAChRs) are major excitatory neurotransmitter receptors in both vertebrates and invertebrates. Two lynx proteins (Nl-lynx1 and Nl-lynx2) have been identified in the brown planthopper, Nilaparvata lugens, which act as modulators on insect nAChRs. In the present study, two lynx proteins were found to act on the triplet receptor Nl,1/Nl,2/,2 expressed in Xenopus oocytes, increasing agonist-evoked macroscopic currents, but not changing agonist sensitivity and desensitization properties. Nl-lynx1 and Nl-lynx2 increased Imax (maximum responses) of acetylcholine to 4.85-fold and 2.40-fold of that of Nl,1/Nl,2/,2 alone, and they also increased Imax of imidacloprid to 2.57-fold and 1.25-fold. Although, on another triplet nAChRs Nl,3/Nl,8/,2, Nl-lynx2 increased Imax of acetylcholine and imidacloprid to 3.63-fold and 2.16-fold, Nl-lynx1 had no effects on Imax of either acetylcholine or imidacloprid. The results demonstrate the selectivity of lynx proteins for different insect nAChR subtypes. This selectivity was also identified in native N. Lugens. Co-immunoprecipitation was found between Nl,1/Nl,2-containing receptors and both Nl-lynx1 and Nl-lynx2, but was only found between Nl,3/Nl,8-containing receptors and Nl-lynx2. When the previously identified Nl,1Y151S and Nl,3Y151S mutations were included (Nl,1Y151S/Nl,2/,2 and Nl,3Y151S/Nl,8/,2), the increase in Imax of imidacloprid, but not acetylcholine, caused by co-expression of Nl-lynx1 and Nl-lynx2 was more noticeable than that of their wildtype counterparts. Taken together, these data suggest that two modulators, Nl-lynx1 and Nl-lynx2, might serve as an influencing factor in target site insensitivity in N. lugens, such as Y151S mutation. [source]

Subunit-specific desensitization of heteromeric kainate receptors

THE JOURNAL OF PHYSIOLOGY, Issue 4 2010
David D. Mott
Kainate receptor subunits can form functional channels as homomers of GluK1, GluK2 or GluK3, or as heteromeric combinations with each other or incorporating GluK4 or GluK5 subunits. However, GluK4 and GluK5 cannot form functional channels by themselves. Incorporation of GluK4 or GluK5 into a heteromeric complex increases glutamate apparent affinity and also enables receptor activation by the agonist AMPA. Utilizing two-electrode voltage clamp of Xenopus oocytes injected with cRNA encoding kainate receptor subunits, we have observed that heteromeric channels composed of GluK2/GluK4 and GluK2/GluK5 have steady state concentration,response curves that were bell-shaped in response to either glutamate or AMPA. By contrast, homomeric GluK2 channels exhibited a monophasic steady state concentration,response curve that simply plateaued at high glutamate concentrations. By fitting several specific Markov models to GluK2/GluK4 heteromeric and GluK2 homomeric concentration,response data, we have determined that: (a) two strikingly different agonist binding affinities exist; (b) the high-affinity binding site leads to channel opening; and (c) the low-affinity agonist binding site leads to strong desensitization after agonist binding. Model parameters also approximate the onset and recovery kinetics of desensitization observed for macroscopic currents measured from HEK-293 cells expressing GluK2 and GluK4 subunits. The GluK2(E738D) mutation lowers the steady state apparent affinity for glutamate by 9000-fold in comparison to GluK2 homomeric wildtype receptors. When this mutant subunit was expressed with GluK4, the rising phase of the glutamate steady state concentration,response curve overlapped with the wildtype curve, whereas the declining phase was right-shifted toward lower affinity. Taken together, these data are consistent with a scheme whereby high-affinity agonist binding to a non-desensitizing GluK4 subunit opens the heteromeric channel, whereas low-affinity agonist binding to GluK2 desensitizes the whole channel complex. [source]

Direct interaction of Na-azide with the KATP channel

BRITISH JOURNAL OF PHARMACOLOGY, Issue 6 2000
Stefan Trapp
The effects of the metabolic inhibitor sodium azide were tested on excised macropatches from Xenopus oocytes expressing cloned ATP-sensitive potassium (KATP) channels of the Kir6.2/SUR1 type. In inside-out patches from oocytes expressing Kir6.2,C36 (a truncated form of Kir6.2 that expresses in the absence of SUR), intracellular Na-azide inhibited macroscopic currents with an IC50 of 11 mM. The inhibitory effect of Na-azide was mimicked by the same concentration of NaCl, but not by sucrose. Na-azide and NaCl blocked Kir6.2/SUR1 currents with IC50 of 36 mM and 19 mM, respectively. Inhibition was abolished in the absence of intracellular Mg2+. In contrast, Kir6.2,C36 currents were inhibited by Na-azide both in the presence or absence of intracellular Mg2+. Kir6.2/SUR1 currents were less sensitive to 3 mM Na-azide in the presence of MgATP. This apparent reduction in sensitivity is caused by a small activatory effect of Na-azide conferred by SUR. We conclude that, in addition to its well-established inhibitory effect on cellular metabolism, which leads to activation of KATP channels in intact cells, intracellular Na-azide has direct effects on the KATP channel. Inhibition is intrinsic to Kir6.2, is mediated by Na+, and is modulated by SUR. There is also a small, ATP-dependent, stimulatory effect of Na-azide mediated by the SUR subunit. The direct effects of 3 mM Na-azide on KATP channels are negligible in comparison to the metabolic activation produced by the same Na-azide concentration. British Journal of Pharmacology (2000) 131, 1105,1112; doi:10.1038/sj.bjp.0703680 [source]