Targeting Calcium Release–activated Calcium Channel Is Not Sufficient to Prevent Rejection in Nonhuman Primate Kidney Transplantation

The calcium release-activated calcium channel Orai regulates Ca2+ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca2+ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou et al., 2018). Here we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-electron microscopy. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel’s gate. Structured M1–M2 turrets, not evident previously, form the channel’s extracellular entrance.

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On the origin of rhythmic calcium transients in the ICC-MP of the mouse small intestine

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00077.2011 ◽

2011 ◽

Vol 301 (5) ◽

pp. G835-G845 ◽

Cited By ~ 28

Author(s):

Bobbi-Jo Lowie ◽

Xuan-Yu Wang ◽

Elizabeth J. White ◽

Jan D. Huizinga

Keyword(s):

Small Intestine ◽

Calcium Channel ◽

Calcium Release ◽

Imaging Study ◽

Cyclopiazonic Acid ◽

Calcium Oscillations ◽

Calcium Transients ◽

Pacemaker Activity ◽

Type I ◽

Endoplasmic Reticulum Calcium

Interstitial cells of Cajal associated with the myenteric plexus (ICC-MP) are pacemaker cells of the small intestine, producing the characteristic omnipresent electrical slow waves, which orchestrate peristaltic motor activity and are associated with rhythmic intracellular calcium oscillations. Our objective was to elucidate the origins of the calcium transients. We hypothesized that calcium oscillations in the ICC-MP are primarily regulated by the sarcoplasmic reticulum (SR) calcium release system. With the use of calcium imaging, study of the effect of T-type calcium channel blocker mibefradil revealed that T-type channels did not play a major role in generating the calcium transients. 2-Aminoethoxydiphenyl borate, an inositol 1,4,5 trisphosphate receptor (IP3R) inhibitor, and U73122, a phospholipase C inhibitor, both drastically decreased the frequency of calcium oscillations, suggesting a major role of IP3 and IP3-induced calcium release from the SR. Immunohistochemistry proved the expression of IP3R type I (IP3R-I), but not type II (IP3R-II) and type III (IP3R-III) in ICC-MP, indicating the involvement of the IP3R-I subtype in calcium release from the SR. Cyclopiazonic acid, a SR/endoplasmic reticulum calcium ATPase pump inhibitor, strongly reduced or abolished calcium oscillations. The Na-Ca exchanger (NCX) in reverse mode is likely involved in refilling the SR because the NCX inhibitor KB-R7943 markedly reduced the frequency of calcium oscillations. Immunohistochemistry revealed 100% colocalization of NCX and c-Kit in ICC-MP. Testing a mitochondrial NCX inhibitor, we were unable to show an essential role for mitochondria in regulating calcium oscillations in the ICC-MP. In summary, ongoing IP3 synthesis and IP3-induced calcium release from the SR, via the IP3R-I, are the major drivers of the calcium transients associated with ICC pacemaker activity. This suggests that a biochemical clock intrinsic to ICC determines the pacemaker frequency, which is likely directly linked to kinetics of the IP3-activated SR calcium channel and IP3 metabolism.

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Spontaneous oscillations in cytoplasmic calcium concentration in vascular smooth muscle

AJP Cell Physiology ◽

10.1152/ajpcell.1989.256.5.c951 ◽

1989 ◽

Vol 256 (5) ◽

pp. C951-C957 ◽

Cited By ~ 30

Author(s):

P. L. Weissberg ◽

P. J. Little ◽

A. Bobik

Keyword(s):

Smooth Muscle ◽

Sarcoplasmic Reticulum ◽

Calcium Channel ◽

Intracellular Calcium ◽

Calcium Concentration ◽

Calcium Release ◽

Extracellular Calcium ◽

Fluorescence Measurement ◽

Cytoplasmic Calcium ◽

Spontaneous Oscillations

Fluorescence measurement of fura-2 and quin2 signals from confluent primary cultures of serum-deprived rat aortic smooth muscle cells have revealed spontaneous oscillations in intracellular calcium concentration ([Ca2+]i). The transients consist of a rapid increase in [Ca2+]i that averages 60 nM and lasts approximately 30 s. They are caused by intracellular calcium release and an influx of extracellular calcium. Exposure of cells to the calcium-channel antagonists verapamil and diltiazem or incubation in nominally calcium-free medium reduced both the duration and amplitude of the transients; in contrast, the calcium-channel agonist (-)BAY K 8644 increased their duration. The transients were abolished by caffeine and 8-(N,N-diethylamino)octyl 3,4,5-trimethoxybenzoate, agents that interfere with calcium release from the sarcoplasmic reticulum. These findings demonstrate that the sarcoplasmic reticulum is a primary source for the spontaneous oscillations in cytoplasmic calcium and is closely associated with the influx of extracellular calcium. Although the function of these transients is unclear, they may be involved in the spontaneous contractions observed in some vessels and in the regulation of vascular resistance.

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