Medicine and Health Sciences Commons^™

3d Mapping Of The Spry2 Domain Of Ryanodine Receptor 1 By Single-Particle Cryo-Em, Alex Peralvaerz-Marin, Hanshen Tae, Phillip G. Board, Marco G. Casarotto, Angela F. Dulhunty, Montserrat Samsó

Physiology and Biophysics Publications

The type 1 skeletal muscle ryanodine receptor (RyR1) is principally responsible for Ca2+release from the sarcoplasmic reticulum and for the subsequent muscle contraction. The RyR1 contains three SPRY domains. SPRY domains are generally known to mediate protein-protein interactions, however the location of the three SPRY domains in the 3D structure of the RyR1 is not known. Combining immunolabeling and single-particle cryo-electron microscopy we have mapped the SPRY2 domain (S1085-V1208) in the 3D structure of RyR1 using three different antibodies against the SPRY2 domain. Two obstacles for the image processing procedure; limited amount of data and signal dilution introduced by the …

State-Dependent Camp Binding To Functioning Hcn Channels Studied By Patch-Clamp Fluorometry, Shengjun Wu, Zhanna V. Vysotskaya, Xinping Xu, Changan Xie, Qinglian Liu, Lei Zhou

Physiology and Biophysics Publications

Abstract

One major goal of ion channel research is to delineate the molecular events from the detection of the stimuli to the movement of channel gates. For ligand-gated channels, it is challenging to separate ligand binding from channel gating. Here we studied the cyclic adenosine monophosphate (cAMP)-dependent gating in hyperpolarization-activated cAMP-regulated (HCN) channel by simultaneously recording channel opening and ligand binding, using the patch-clamp fluorometry technique with a unique fluorescent cAMP analog that fluoresces strongly in the hydrophobic binding pocket and exerts regulatory effects on HCN channels similar to those imposed by cAMP. Corresponding to voltage-dependent channel activation, we observed …

Cholesterol Sensitivity Of Kir2.1 Is Controlled By A Belt Of Residues Around The Cytosolic Pore, Avia Rosenhouse-Dantsker, Diomedes E. Logothetis, Irena Levitan

Physiology and Biophysics Publications

Abstract

Kir channels play an important role in setting the resting membrane potential and modulating membrane excitability. A common feature of several Kir channels is that they are regulated by cholesterol. Yet, the mechanism by which cholesterol affects channel function is unclear. We recently showed that the cholesterol sensitivity of Kir2 channels depends on several CD-loop residues. Here we show that this cytosolic loop is part of a regulatory site that also includes residues in the G-loop, the N-terminus, and the connecting segment between the C-terminus and the inner transmembrane helix. Together, these residues form a cytosolic belt that surrounds …