Detection of channel proximity by nanoparticle-assisted delaying of toxin binding; a combined patch-clamp and flow cytometric energy transfer study.
Gold nanoparticles of 30 nm diameter bound to cell-surface receptor major histocompatibility complex glycoproteins (MHCI and MHCII), interleukin-2 receptor alpha subunit (IL-2Ralpha), very late antigen-4 (VLA-4) integrin, transferrin receptor, and the receptor-type protein tyrosin phosphatase CD45 are shown by the patch-clamp technique to selectively modulate binding characteristics of Pi(2) toxin, an efficient blocker of K(v)1.3 channels. After correlating the electrophysiological data with those on the underlying receptor clusters obtained by simultaneously conducted flow cytometric energy transfer measurements, the modulation was proved to be sensitive to the density and size of the receptor clusters, and to the locations of the receptors as well. Based on the observation that engagement of MHCII by a monoclonal antibody down-regulates channel current and based on the close nanometer-scale proximity of the MHCI and MHCII glycoproteins, an analogous experiment was carried out when gold nanoparticles bound to MHCI delayed down-regulation of the K(v)1.3 current initiated by ligation of MHCII. Localization of K(v)1.3 channels in the nanometer-scale vicinity of the MHC-containing lipid rafts is demonstrated for the first time. A method is proposed for detecting receptor-channel or receptor-receptor proximity by observing nanoparticle-induced increase in relaxation times following concentration jumps of ligands binding to channels or to receptors capable of regulating channel currents.
Rubovszky
Bálint
B
Cell Biophysics Research Group, Hungarian Academy of Sciences, Debrecen.
Hajdú
Péter
P
Krasznai
Zoltán
Z
Gáspár
Rezsõ
R
Waldmann
Thomas A
TA
Damjanovich
Sándor
S
Bene
László
L
D002460:Cell Line; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015640:Ion Channel Gating / Q000187:drug effects / Q000502:physiology*; D051664:Kv1.3 Potassium Channel; D043942:Nanotubes / Q000737:chemistry; D010316:Particle Size; D018408:Patch-Clamp Techniques / Q000379:methods*; D024642:Potassium Channels, Voltage-Gated / Q000187:drug effects / Q000502:physiology*; D011485:Protein Binding; D012604:Scorpion Venoms / Q000493:pharmacokinetics*; D013601:T-Lymphocytes / Q000187:drug effects / Q000502:physiology*
C496973:KCNA3 protein, human; D051664:Kv1.3 Potassium Channel; C101360:Pi2 toxin; D024642:Potassium Channels, Voltage-Gated; D012604:Scorpion Venoms