A bilobal model of Ca2+-dependent inactivation to probe the physiology of L-type Ca2+ channels
JournalJournal of General Physiology
PublisherRockefeller University Press
MetadataShow full item record
AbstractL-type calcium channels (LTCCs) are critical elements of normal cardiac function, playing a major role in orchestrating cardiac electrical activity and initiating downstream signaling processes. LTCCs thus use feedback mechanisms to precisely control calcium (Ca2+) entry into cells. Of these, Ca2+-dependent inactivation (CDI) is significant because it shapes cardiac action potential duration and is essential for normal cardiac rhythm. This important form of regulation is mediated by a resident Ca2+ sensor, calmodulin (CaM), which is comprised of two lobes that are each capable of responding to spatially distinct Ca2+ sources. Disruption of CaM-mediated CDI leads to severe forms of long-QT syndrome (LQTS) and life-threatening arrhythmias. Thus, a model capable of capturing the nuances of CaM-mediated CDI would facilitate increased understanding of cardiac (patho)physiology. However, one critical barrier to achieving a detailed kinetic model of CDI has been the lack of quantitative data characterizing CDI as a function of Ca2+. This data deficit stems from the experimental challenge of uncoupling the effect of channel gating on Ca2+ entry. To overcome this obstacle, we use photo-uncaging of Ca2+ to deliver a measurable Ca2+ input to CaM/LTCCs, while simultaneously recording CDI. Moreover, we use engineered CaMs with Ca2+ binding restricted to a single lobe, to isolate the kinetic response of each lobe. These high-resolution measurements enable us to build mathematical models for each lobe of CaM, which we use as building blocks for a full-scale bilobal model of CDI. Finally, we use this model to probe the pathogenesis of LQTS associated with mutations in CaM (calmodulinopathies). Each of these models accurately recapitulates the kinetics and steady-state properties of CDI in both physiological and pathological states, thus offering powerful new insights into the mechanistic alterations underlying cardiac arrhythmias. Copyright 2018 Limpitikul et al.
SponsorsDavid T. Yue, who originally conceptualized this work, passed away on December 23, 2014. His mentorship, wisdom, and kindness are greatly missed. We thank Dr. Shin Rong Lee for advice and training with the photolysis system and thank Wanjun Yang for dedicated technical support. We also thank Dr. Gordon Tomaselli for providing valuable advice and discussions, and Dr. Manu Ben-Johny and members of the Calcium Signals Lab for ongoing feedback and support. This work was supported by grants: American Heart Association Pre-doctoral Fellowship (W.B. Limpitikul) and National Institutes of Health R01HL105239 (J.L. Greenstein and R.L. Winslow) and R01MH065531 (W.B. Limpitikul, D.T. Yue, and I.E. Dick). The authors declare no competing financial interests. Author contributions: W.B. Limpitikul collected and analyzed data and wrote and edited the manuscript. J.L. Greenstein, R.L. Winslow, and I.E. Dick supervised the overall project and wrote and edited the manucript. D.T. Yue originally envisioned and conceptualized this work
Identifier to cite or link to this itemhttps://www.scopus.com/inward/record.uri?eid=2-s2.0-85057770365&doi=10.1085%2fjgp.201812115&partnerID=40&md5=ed8b174e898fa2892decf477620e0de7; http://hdl.handle.net/10713/9046