Calexcitin: A signaling protein that binds calcium and GTP, inhibits potassium channels, and enhances membrane excitability

^*Laboratory of Adaptive Systems and ^¶Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20892; ^‡Harvard Medical School, McLean Hospital, 115 Mill Street, Belmont, MA 02178; ^§Protein Engineering Group, Korea Research Institute of Bioscience and Biotechnology, Korea Institute of Science and Technology, Yusong, Taejon 305-600, South Korea; and ^‖George Mason University, Fairfax, VA 22030

Abstract

A previously uncharacterized 22-kDa Ca²⁺-binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EF-hands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca²⁺, calexcitin bound GTP and possessed GTPase activity. Calexcitin is also a high affinity substrate for protein kinase C. Application of calexcitin to the inner surface of inside-out patches of human fibroblast membranes, in the presence of Ca²⁺ and the absence of endogenous Ca²⁺/calmodulin kinase type II or protein kinase C activity, reduced the mean open time and mean open probability of 115 ± 6 pS K⁺ channels. Calexcitin thus appears to directly regulate K⁺ channels. When microinjected into molluscan neurons or rabbit cerebellar Purkinje cell dendrites, calexcitin was highly effective in enhancing membrane excitability. Because calexcitin translocates to the cell membrane after phosphorylation, calexcitin could serve as a Ca²⁺-activated signaling molecule that increases cellular excitability, which would in turn increase Ca²⁺ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca²⁺.

Footnotes

↵ † To whom reprint requests should be addressed. e-mail: tjnelson{at}las1.ninds.nih.gov.
Julius Axelrod, National Institute of Mental Health, Bethesda, MD
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Abbreviations: PKC, protein kinase C; CE, calexcitin; GST, glutathione S-transferase; ACSF, artificial cerebrospinal fluid.

Data deposition: The sequence reported in this paper has been deposited in the GenBank data base (accession no. U49390U49390).