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

Abstract

A previously uncharacterized 22-kDa Ca 2؉ -binding protein that also binds guanosine nucleotides was characterized, cloned, and analyzed by electrophysiological techniques. The cloned protein, calexcitin, contains two EFhands and also has homology with GTP-binding proteins in the ADP ribosylation factor family. In addition to binding two molecules of Ca 2؉ , 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 Ca 2؉ and the absence of endogenous Ca 2؉ ͞cal-modulin 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 Ca 2؉ -activated signaling molecule that increases cellular excitability, which would in turn increase Ca 2؉ influx through the membrane. This is also the first known instance of a GTP-binding protein that binds Ca 2؉ .In neuronal cells, ion channel conductance is regulated by ligand binding, direct interaction with G proteins, or phosphorylation (1). K ϩ and Ca 2ϩ channels, for example, can be phosphorylated by Ca 2ϩ ͞calmodulin-dependent kinase (2, 3) and͞or protein kinase C (PKC) (4-8); however, other elements of Ca 2ϩ signaling cascades might also regulate ion channels directly. Such a protein was suggested by a previous study in which a low molecular weight protein, designated cp20, reduced two voltage-dependent K ϩ currents, i A and i Ca-Kϩ , in identified molluscan neurons (9-11). These same currents were reduced in the same neurons in molluscs (Hermissenda crassicornis) exposed to a Pavlovian conditioning paradigm (9, 12). To investigate this protein, we purified calexcitin (CE) from squid optic lobe and used microsequencing and PCR techniques to obtain a DNA probe which was used to screen a cDNA library. The cloned protein had separate regions that are homologous to Ca 2ϩ -binding proteins and to GTPbinding proteins. Functionally, this protein binds both Ca 2ϩ and GTP, is phosphorylated by PKC, and regulates K ϩ channels in human fibroblasts and membrane excitability in mammalian and molluscan neurons. CE, therefore, offers a molecular link between PKC, intraneuronal Ca 2ϩ , and persistent changes of membrane excitability that correlate with associative memory storage. METHODSPurification of CE. Squid optic lobes were homogenized in a high speed homogenizer with 1 ml of buffer (10 mm Tris⅐HCl, pH 7.4͞20 g/ml leupeptin͞50 g/ml pepstatin͞50 mM NaF͞1 mM EDTA͞1 mM EGTA) containing 1 M DTT and 0.1 mM phenylmethylsulfonyl fluoride (PMSF), followed by sonication with a 20-W probe sonicator. The homogenate was centrifuged at ...

Crystallization and preliminary X-ray diffraction analysis of calexcitin fromLoligo pealei: a neuronal protein implicated in learning and memory

“…Electrophysiological studies showed that low concentrations of squid calexcitin had a strong inhibitory effect on human K + channels (Nelson et al, 1996). These findings suggest that a functionally related protein exists in mammals and may play a similar role in the regulation of potassium channels.…”

“…Cloning and sequencing of squid calexcitin cDNA showed that the protein consists of 191 amino acids and has sequence similarity with several sarcoplasmic calcium-binding proteins (SCPs) and some elements of sequence similarity with GTP-binding proteins of the ADPribosylation factor family (Nelson et al, 1996;Gombos et al, 2001). Squid calexcitin has significant sequence identity (28%) with an invertebrate SCP of known X-ray structure, referred to as ASCP (an SCP from the proto-vertebrate marine organism Amphioxus; Cook et al, 1993).…”

“…Calexcitin has also been isolated from the optic lobe of the longfinned squid Loligo pealei (Nelson et al, 1994(Nelson et al, , 1996. Cloning and sequencing of squid calexcitin cDNA showed that the protein consists of 191 amino acids and has sequence similarity with several sarcoplasmic calcium-binding proteins (SCPs) and some elements of sequence similarity with GTP-binding proteins of the ADPribosylation factor family (Nelson et al, 1996;Gombos et al, 2001).…”

Bryostatin Enhancement of Memory inHermissenda

“…In Hermissenda, this activation causes PKC to translocate to the cell membrane (McPhie et al, 1993). Subsequently, this translocation induces secondary pathway cascades that lead to the activation and increased production of the GTP/Ca 2ϩ -binding protein calexcitin (Nelson et al, , 1996(Nelson et al, , 1999Ascoli et al, 1997;Kuzirian et al, 2001), the release of internal calcium from the endoplasmic reticulum, and the reduction of outward K ϩ currents that triggers a rise in the resting potential and the establishment of a long-lasting depolarization. Early gene activation is also associated with PKC up-regulation caused by these same associative conditioning regimes .…”

Involvement of the Ryanodine Receptor in Morphologic Modification ofHermissendaType B Photoreceptors After In Vitro Conditioning

“…It was recently reported that actin-related protein is associated with intermediate memory in Hermissenda (Crow and Xue-Bian 2002). Calexcitin, extracted from the conditioned Hermissenda type B photoreceptors, is a potent signaling protein that binds calcium and GTP, inhibits potassium channels due to phosphorylation by PKC, and enhances membrane excitability (Nelson et al 1996). Thus activation of the conditioning specific calcium sensor G-proteins, calexcitin, might contribute to contraction at the B cell terminal branches.…”