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• W2012544852 abstract "Calmodulin (CaM) in complex with Ca2+ channels constitutes a prototype for Ca2+ sensors that are intimately colocalized with Ca2+ sources. The C-lobe of CaM senses local, large Ca2+ oscillations due to Ca2+ influx from the host channel, and the N-lobe senses global, albeit diminutive Ca2+ changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca2+ sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca2+ oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca2+ release from CaM combined with greater affinity of the channel for Ca2+-free versus Ca2+-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca2+ sources. Calmodulin (CaM) in complex with Ca2+ channels constitutes a prototype for Ca2+ sensors that are intimately colocalized with Ca2+ sources. The C-lobe of CaM senses local, large Ca2+ oscillations due to Ca2+ influx from the host channel, and the N-lobe senses global, albeit diminutive Ca2+ changes arising from distant sources. Though biologically essential, the mechanism underlying global Ca2+ sensing has remained unknown. Here, we advance a theory of how global selectivity arises, and we experimentally validate this proposal with methodologies enabling millisecond control of Ca2+ oscillations seen by the CaM/channel complex. We find that global selectivity arises from rapid Ca2+ release from CaM combined with greater affinity of the channel for Ca2+-free versus Ca2+-bound CaM. The emergence of complex decoding properties from the juxtaposition of common elements, and the techniques developed herein, promise generalization to numerous molecules residing near Ca2+ sources. Ca2+ constitutes a ubiquitous signal with wide-ranging biological impact (Berridge et al., 2000Berridge M.J. Lipp P. Bootman M.D. The versatility and universality of calcium signalling.Nat. Rev. Mol. Cell Biol. 2000; 1: 11-21Crossref PubMed Scopus (4221) Google Scholar). Despite the pervasive nature of Ca2+, its detection can be highly selective in space and time, as required for specificity in signaling to appropriate targets (Bootman et al., 2001Bootman M.D. Lipp P. Berridge M.J. The organisation and functions of local Ca(2+) signals.J. Cell Sci. 2001; 114: 2213-2222Crossref PubMed Google Scholar, Cullen, 2006Cullen P.J. Decoding complex Ca2+ signals through the modulation of Ras signaling.Curr. Opin. Cell Biol. 2006; 18: 157-161Crossref PubMed Scopus (25) Google Scholar, De Koninck and Schulman, 1998De Koninck P. Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.Science. 1998; 279: 227-230Crossref PubMed Scopus (1060) Google Scholar, Dolmetsch et al., 1998Dolmetsch R.E. Xu K. Lewis R.S. Calcium oscillations increase the efficiency and specificity of gene expression.Nature. 1998; 392: 933-936Crossref PubMed Scopus (1634) Google Scholar, Gu and Spitzer, 1995Gu X. Spitzer N.C. Distinct aspects of neuronal differentiation encoded by frequency of spontaneous Ca2+ transients.Nature. 1995; 375: 784-787Crossref PubMed Scopus (461) Google Scholar, Li et al., 1998Li W. Llopis J. Whitney M. Zlokarnik G. Tsien R.Y. Cell-permeant caged InsP3 ester shows that Ca2+ spike frequency can optimize gene expression.Nature. 1998; 392: 936-941Crossref PubMed Scopus (763) Google Scholar, Oancea and Meyer, 1998Oancea E. Meyer T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals.Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, Winslow and Crabtree, 2005Winslow M.M. Crabtree G.R. Immunology. Decoding calcium signaling.Science. 2005; 307: 56-57Crossref PubMed Scopus (20) Google Scholar). Among the most critical of these detection modes are those relating to Ca2+ sensors positioned in close proximity, i.e., within nanometers, of Ca2+ sources. This placement of sensors within such a “nanodomain” promotes rapid and privileged Ca2+ signaling (Augustine et al., 2003Augustine G.J. Santamaria F. Tanaka K. Local calcium signaling in neurons.Neuron. 2003; 40: 331-346Abstract Full Text Full Text PDF PubMed Scopus (427) Google Scholar, Bootman et al., 2001Bootman M.D. Lipp P. Berridge M.J. The organisation and functions of local Ca(2+) signals.J. Cell Sci. 2001; 114: 2213-2222Crossref PubMed Google Scholar, Catterall, 1999Catterall W.A. Interactions of presynaptic Ca2+ channels and snare proteins in neurotransmitter release.Ann. N Y Acad. Sci. 1999; 868: 144-159Crossref PubMed Scopus (227) Google Scholar). However, such proximity to a Ca2+ source challenges a sensor's ability to integrate Ca2+ signals from distant sources, which is essential for coordinated signaling at the whole-cell level. A prototype for coupled sensors and sources is the Ca2+ sensor calmodulin (CaM), in its regulation of the CaV1-2 family of Ca2+ channels (Dunlap, 2007Dunlap K. Calcium channels are models of self-control.J. Gen. Physiol. 2007; 129: 379-383Crossref PubMed Scopus (43) Google Scholar). CaM is continuously complexed with channels as a resident Ca2+ sensor (Erickson et al., 2001Erickson M.G. Alseikhan B.A. Peterson B.Z. Yue D.T. Preassociation of calmodulin with voltage-gated Ca(2+) channels revealed by FRET in single living cells.Neuron. 2001; 31: 973-985Abstract Full Text Full Text PDF PubMed Scopus (359) Google Scholar, Pitt et al., 2001Pitt G.S. Zuhlke R.D. Hudmon A. Schulman H. Reuter H. Tsien R.W. Molecular basis of calmodulin tethering and Ca2+-dependent inactivation of L-type Ca2+ channels.J. Biol. Chem. 2001; 276: 30794-30802Crossref PubMed Scopus (239) Google Scholar), and Ca2+ binding to the C- and N-terminal lobes of CaM can each induce a separate form of regulation on the same channel (DeMaria et al., 2001DeMaria C.D. Soong T.W. Alseikhan B.A. Alvania R.S. Yue D.T. Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.Nature. 2001; 411: 484-489Crossref PubMed Scopus (329) Google Scholar, Yang et al., 2006Yang P.S. Alseikhan B.A. Hiel H. Grant L. Mori M.X. Yang W. Fuchs P.A. Yue D.T. Switching of Ca2+-dependent inactivation of Ca(v)1.3 channels by calcium binding proteins of auditory hair cells.J. Neurosci. 2006; 26: 10677-10689Crossref PubMed Scopus (123) Google Scholar). Given the approximate diameter of Ca2+ channels (Wang et al., 2002Wang M.C. Velarde G. Ford R.C. Berrow N.S. Dolphin A.C. Kitmitto A. 3D structure of the skeletal muscle dihydropyridine receptor.J. Mol. Biol. 2002; 323: 85-98Crossref PubMed Scopus (40) Google Scholar), the resident CaM would be ∼10 nm from the channel pore, well within the channel nanodomain. Despite this proximity, each lobe responds selectively to distinct Ca2+ signals (cartooned in Figure 1A), which differ in both their spatial distribution (top row) and temporal characteristics (bottom row). Under physiological conditions, the composite Ca2+ signal (Figure 1A, left column) is the sum of two distinct components. First, Ca2+ inflow during channel openings produces a “local signal” component (Figure 1A, middle column) comprising brief yet intense local spikes of amplitude Caspike ∼100 μM (bottom row). These spikes are tightly synchronized with openings of the host channel, and localized to the nanodomain (top row, green hemisphere) (Neher, 1998Neher E. Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.Neuron. 1998; 20: 389-399Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar, Sherman et al., 1990Sherman A. Keizer J. Rinzel J. Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.Biophys. J. 1990; 58: 985-995Abstract Full Text PDF PubMed Scopus (78) Google Scholar) (see Supplemental Data [3], available online). Second, accumulation of Ca2+ from distant sources (e.g., other Ca2+ channels) generates a “global signal” component (Figure 1A, right column) consisting of a far smaller (∼5 μM) global pedestal (bottom row), which is spatially widespread (top row, green shading). In the CaV1-2 family of Ca2+ channels, regulation triggered by the C-lobe of CaM exploits channel proximity and responds almost maximally to the local Ca2+ signal alone (Liang et al., 2003Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). This “local selectivity” is schematized for a Ca2+-dependent inactivation process (CDI) triggered by the C-lobe (Figure 1B). Such CDI produces a strong decay of Ca2+ current during sustained voltage activation whether Ca2+ is buffered at physiological levels, or much more strongly (Figure 1B). Since high Ca2+ buffering eliminates the global pedestal while hardly affecting local spikes (Figure 1A, middle column; Supplemental Data [3]) (Neher, 1998Neher E. Vesicle pools and Ca2+ microdomains: new tools for understanding their roles in neurotransmitter release.Neuron. 1998; 20: 389-399Abstract Full Text Full Text PDF PubMed Scopus (833) Google Scholar), the sparing of CDI under this condition indicates that the local signal alone is sufficient. By contrast, N-lobe mediated regulation of all CaV2 channels somehow prefers the diminutive global pedestal over the far larger local spikes. The hallmark of this “global selectivity” is the presence of strong CDI in physiological buffering (Figure 1C, left), and its near absence in high buffering (Figure 1C, middle) (DeMaria et al., 2001DeMaria C.D. Soong T.W. Alseikhan B.A. Alvania R.S. Yue D.T. Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.Nature. 2001; 411: 484-489Crossref PubMed Scopus (329) Google Scholar, Liang et al., 2003Liang H. DeMaria C.D. Erickson M.G. Mori M.X. Alseikhan B.A. Yue D.T. Unified mechanisms of Ca2+ regulation across the Ca2+ channel family.Neuron. 2003; 39: 951-960Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Without this detection mode, Ca2+ feedback would be restricted to isolated complexes, and lack coordination over larger regions. Global selectivity is thus critical to the signaling repertoire of Ca2+ sensors positioned near Ca2+ sources. What are the mechanisms for the contrast in spatial Ca2+ selectivity of the lobes of CaM? The simplest explanation would presume that while the C-lobe resides within the nanodomain, the N-lobe lies outside this zone, where the local signal would be smaller than the global pedestal (Figure 1A, top row). However, each channel is constitutively complexed with a single CaM (Mori et al., 2004Mori M.X. Erickson M.G. Yue D.T. Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.Science. 2004; 304: 432-435Crossref PubMed Scopus (162) Google Scholar, Yang et al., 2007Yang P.S. Mori M.X. Antony E.A. Tadross M.R. Yue D.T. A single calmodulin imparts distinct N- and C-lobe regulatory processes to individual CaV1.3 channels (abstr.).Biophys. J. 2007; 20a: 1669Google Scholar), and the lobes of CaM are very close to one another (<6 nm), indicating that both lobes are likely within the nanodomain (Dunlap, 2007Dunlap K. Calcium channels are models of self-control.J. Gen. Physiol. 2007; 129: 379-383Crossref PubMed Scopus (43) Google Scholar, Stern, 1992Stern M.D. Buffering of calcium in the vicinity of a channel pore.Cell Calcium. 1992; 13: 183-192Crossref PubMed Scopus (252) Google Scholar). Hence, the N-lobe must be insensitive to Ca2+ intensity, and instead may respond to certain temporal features of nanodomain Ca2+ (Figure 1A, bottom row). Though some Ca2+-dependent mechanisms that favor specific temporal patterns of Ca2+ have been characterized (De Koninck and Schulman, 1998De Koninck P. Schulman H. Sensitivity of CaM kinase II to the frequency of Ca2+ oscillations.Science. 1998; 279: 227-230Crossref PubMed Scopus (1060) Google Scholar, Oancea and Meyer, 1998Oancea E. Meyer T. Protein kinase C as a molecular machine for decoding calcium and diacylglycerol signals.Cell. 1998; 95: 307-318Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar), none can respond to signals of low amplitude and frequency (global pedestal), while ignoring signals of high amplitude and frequency (local spikes). Hence, global selectivity must employ a thus far unknown mechanism. Here, theoretical and experimental advances explain how this unusual selectivity for global Ca2+ signals arises from the combination of two common elements: rapid Ca2+ release from CaM, together with greater channel affinity for Ca2+-free (apoCaM) versus Ca2+-bound CaM (Ca2+/CaM). Since our proposed mechanism requires CaM/channel interactions as present within intact channels, we develop the means to probe Ca2+ dynamics within this integrated setting, using channels engineered for enhanced opening, with a “voltage block” electrophysiological technique to precisely control nanodomain Ca2+. These tools resolve Ca2+ response characteristics clearly distinctive of the proposed mechanism. Combining this approach with manipulation of a recently identified CaM regulatory site (Dick et al., 2008Dick I.E. Tadross M.R. Liang H. Tay L.H. Yang W. Yue D.T. A modular switch for spatial Ca2+ selectivity in the calmodulin regulation of CaV channels.Nature. 2008; 451: 830-834Crossref PubMed Scopus (195) Google Scholar) enables quantitative confirmation of a key prediction—selectivity can be incrementally changed from global to local by adjusting the ratio of channel affinity for apoCaM versus Ca2+/CaM. Our findings generalize across CaV1–CaV2 channels, and likely extend to diverse Ca2+ sensors situated near Ca2+ sources. To explore how the CaM/channel complex could produce both local and global Ca2+ selectivity, we outlined a basic system comprised of the dominant conformations of this molecular assembly (Figure 2A). Several established features were considered. First, a 1:1 CaM/channel stoichiometry has been demonstrated (Mori et al., 2004Mori M.X. Erickson M.G. Yue D.T. Functional stoichiometry and local enrichment of calmodulin interacting with Ca2+ channels.Science. 2004; 304: 432-435Crossref PubMed Scopus (162) Google Scholar, Yang et al., 2007Yang P.S. Mori M.X. Antony E.A. Tadross M.R. Yue D.T. A single calmodulin imparts distinct N- and C-lobe regulatory processes to individual CaV1.3 channels (abstr.).Biophys. J. 2007; 20a: 1669Google Scholar). Second, two distinct types of CaM/channel interactions are known to exist: apoCaM binding, which anchors CaM to the channel as a resident sensor; and Ca2+/CaM binding, which here produces CDI. Finally, an appropriate first-order approximation was to separately consider the operation of the C- and N-lobes of CaM, each with simultaneous (un)binding of two Ca2+ ions (Linse et al., 1991Linse S. Helmersson A. Forsen S. Calcium binding to calmodulin and its globular domains.J. Biol. Chem. 1991; 266: 8050-8054Abstract Full Text PDF PubMed Google Scholar, Martin et al., 1985Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsen S. Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure.Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (116) Google Scholar). This single-lobe approximation was reinforced in our experiments by the use of mutant CaM molecules that restrict Ca2+ binding to one lobe or the other (DeMaria et al., 2001DeMaria C.D. Soong T.W. Alseikhan B.A. Alvania R.S. Yue D.T. Calmodulin bifurcates the local Ca2+ signal that modulates P/Q-type Ca2+ channels.Nature. 2001; 411: 484-489Crossref PubMed Scopus (329) Google Scholar, Peterson et al., 1999Peterson B.Z. DeMaria C.D. Adelman J.P. Yue D.T. Calmodulin is the Ca2+ sensor for Ca2+ -dependent inactivation of L- type calcium channels.Neuron. 1999; 22: 549-558Abstract Full Text Full Text PDF PubMed Scopus (689) Google Scholar). Based upon these features, four main conformations result (Figure 2A, valid for either the C- or N-lobe). State 1 represents apoCaM (yellow circle) bound to the channel preassociation site (round pocket). Direct Ca2+ binding to CaM in state 1 is not considered, as such interaction is unlikely according to an analogous apoCaM/peptide structure (Houdusse et al., 2006Houdusse A. Gaucher J.F. Krementsova E. Mui S. Trybus K.M. Cohen C. Crystal structure of apo-calmodulin bound to the first two IQ motifs of myosin V reveals essential recognition features.Proc. Natl. Acad. Sci. USA. 2006; 103: 19326-19331Crossref PubMed Scopus (107) Google Scholar). State 2 portrays apoCaM after it releases from the preassociation site, at which point it can bind Ca2+ to produce Ca2+/CaM (square) in state 3. We reason that a transiently dissociated lobe of CaM (state 2 or 3) does not diffuse away (retained within a channel alcove), because of the slow rate of exchange between perfused CaM and channel-associated CaM (Chaudhuri et al., 2005Chaudhuri D. Alseikhan B.A. Chang S.Y. Soong T.W. Yue D.T. Developmental activation of calmodulin-dependent facilitation of cerebellar P-type Ca2+ current.J. Neurosci. 2005; 25: 8282-8294Crossref PubMed Scopus (50) Google Scholar). Finally, in state 4, Ca2+/CaM binds the channel effector site (square pocket), which triggers CDI. The effector site is unlikely to bind apoCaM, because CDI is absent without Ca2+. Further arguments in support of this basic four-state configuration appear in the Discussion and Supplemental Data (1F). How might local Ca2+ selectivity of the C-lobe arise? This lobe is known to release Ca2+ slowly compared to the millisecond duration of Ca2+ channel closings (Bayley et al., 1984Bayley P. Ahlstrom P. Martin S.R. Forsen S. The kinetics of calcium binding to calmodulin: Quin 2 and ANS stopped-flow fluorescence studies.Biochem. Biophys. Res. Commun. 1984; 120: 185-191Crossref PubMed Scopus (97) Google Scholar, Black et al., 2005Black D.J. Halling D.B. Mandich D.V. Pedersen S.E. Altschuld R.A. Hamilton S.L. Calmodulin interactions with IQ peptides from voltage-dependent calcium channels.Am. J. Physiol. Cell Physiol. 2005; 288: C669-C676Crossref PubMed Scopus (27) Google Scholar, Chaudhuri et al., 2007Chaudhuri D. Issa J.B. Yue D.T. Elementary Mechanisms Producing Facilitation of Cav2.1 (P/Q-type) Channels.J. Gen. Physiol. 2007; 129: 385-401Crossref PubMed Scopus (43) Google Scholar, Imredy and Yue, 1994Imredy J.P. Yue D.T. Mechanism of Ca2+-sensitive inactivation of L-type Ca2+ channels.Neuron. 1994; 12: 1301-1318Abstract Full Text PDF PubMed Scopus (208) Google Scholar, Martin et al., 1985Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsen S. Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure.Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (116) Google Scholar), yielding a “slow CaM” mechanism (Figure 2B). Given this scheme, numerical simulations readily exhibit local selectivity, with strong CDI produced by Ca2+ signals appropriate for both physiological Ca2+ buffering (Figure 2Ca) and high buffering (Figure 2Cb, see legend for simulation details). This outcome is plausible because once CaM binds Ca2+ during a channel opening (Figure 2A, state 3), CaM will likely retain Ca2+ during millisecond closures, causing accumulation in state 3 that subsequently drives entry into state 4 (CDI) (Supplemental Data [2]). For analytical insight, we define a graphical “input-output” relation for Ca2+ detection (Figure 2D). The “output” is taken as the extent of CDI reached at steady-state (CDI(∞)), and the “input” as the channel open probability PO, both as observed under high Ca2+ buffering. In this regime, PO specifies the fraction of time Ca2+ is present (the “fractional presence” of Ca2+). This quantity is useful because nanodomain Ca2+ will achieve a steady concentration of Caspike within microseconds of channel opening, and rapidly decay to zero upon channel closing (Figure 2Cb, top; Supplemental Data [3]) (Sherman et al., 1990Sherman A. Keizer J. Rinzel J. Domain model for Ca2(+)-inactivation of Ca2+ channels at low channel density.Biophys. J. 1990; 58: 985-995Abstract Full Text PDF PubMed Scopus (78) Google Scholar) . Accordingly, PO becomes directly proportional to Ca2+ entry, and thereby serves as shorthand for the Ca2+ input. A first beneficial outcome of this format is a visual definition of local Ca2+ selectivity. Specifically, local selectivity maintains near maximal CDI under high buffering, with inputs spanning the range of naturally occurring PO (∼0.1–0.6). Therefore, the CDI(∞)–PO relation for all processes with local selectivity must intersect the red zone in Figure 2D, as exemplified by the data point (round symbol) corresponding to our numerical simulation (Figure 2Cb). As a second advantage, we deduce a simple formula for the CDI(∞)–PO response of the slow CaM mechanism. Under high buffering, slow Ca2+ release from the C-lobe allows the time-varying rate constant for transitions from state 2 to 3 (= kon·Ca2(t)) to be replaced by a fixed value equal to kon · Caspike2 · PO (Supplemental Data [2A]). This yields the equivalent system in Figure 2E, from which algebraic manipulation gives the analytic CDI(∞)–PO relation, with CDI(∞) proportional to steady-state occupancy of state 4.CDI(∞)=G⋅POPO+Keff.(Equation 1) This relation resembles the Michaelis-Menton equation, with PO analogous to substrate concentration. G incorporates the affinity of the channel for the Ca2+-bound C-lobe, and Keff reflects the balance between Caspike and CaM affinity for Ca2+ (Supplemental Data [2B]). Though Equation 1 is defined for high buffering, the strong CDI characteristic of Ca2+-detection processes under physiological Ca2+ buffering constrains G ∼1 and Keff ≪ 1 (Supplemental Data [2C]). Hence, any slow CaM mechanism will invariably produce local selectivity, with saturating CDI(∞)–PO relations that intersect the red zone (Figure 2D, smooth curve). How might global Ca2+ selectivity of the N-lobe arise? For this lobe, Ca2+ binding and unbinding should be rapid enough to achieve steady state within the millisecond durations of channel openings and closings (Bayley et al., 1984Bayley P. Ahlstrom P. Martin S.R. Forsen S. The kinetics of calcium binding to calmodulin: Quin 2 and ANS stopped-flow fluorescence studies.Biochem. Biophys. Res. Commun. 1984; 120: 185-191Crossref PubMed Scopus (97) Google Scholar, Black et al., 2005Black D.J. Halling D.B. Mandich D.V. Pedersen S.E. Altschuld R.A. Hamilton S.L. Calmodulin interactions with IQ peptides from voltage-dependent calcium channels.Am. J. Physiol. Cell Physiol. 2005; 288: C669-C676Crossref PubMed Scopus (27) Google Scholar, Chaudhuri et al., 2007Chaudhuri D. Issa J.B. Yue D.T. Elementary Mechanisms Producing Facilitation of Cav2.1 (P/Q-type) Channels.J. Gen. Physiol. 2007; 129: 385-401Crossref PubMed Scopus (43) Google Scholar, Imredy and Yue, 1994Imredy J.P. Yue D.T. Mechanism of Ca2+-sensitive inactivation of L-type Ca2+ channels.Neuron. 1994; 12: 1301-1318Abstract Full Text PDF PubMed Scopus (208) Google Scholar, Martin et al., 1985Martin S.R. Andersson Teleman A. Bayley P.M. Drakenberg T. Forsen S. Kinetics of calcium dissociation from calmodulin and its tryptic fragments. A stopped-flow fluorescence study using Quin 2 reveals a two-domain structure.Eur. J. Biochem. 1985; 151: 543-550Crossref PubMed Scopus (116) Google Scholar). Thus, the system would switch “quickly” between states 3 and 2, in sync with channel openings and closings. A second difference from the C-lobe regime is that the kinetics of entry and exit from the outermost states become important. Switching between states 3 and 4 must be “slow” because experimentally, both CDI onset and recovery take much longer than milliseconds (Schnee and Ricci, 2003Schnee M.E. Ricci A.J. Biophysical and pharmacological characterization of voltage-gated calcium currents in turtle auditory hair cells.J. Physiol. 2003; 549: 697-717Crossref PubMed Scopus (60) Google Scholar). If switching between states 1 and 2 is similarly slow, the resulting SQS mechanism (“slow quick slow” in Figure 2F) can produce global selectivity—simulated CDI is strong under physiological buffering (Figure 2Ga), but absent in high buffering (Figure 2Gb and legend). A more quantitative definition of the kinetic constraints for the SQS regime appears in Supplemental Data (1A). This mechanism achieves global selectivity by combining two factors which collaborate to resist inactivation in response to transient and high-amplitude local Ca2+ spikes. Consider the case where only local spikes are present, such as in high buffering (Figure 2Gb, top). First, since the preassociated form of apoCaM (Figure 2A, state 1) binds Ca2+ poorly, the long lifetimes in state 1 can “protect” CaM from binding Ca2+ during brief Ca2+ spikes of high intensity. Second, should this first line of protection fail and state 3 be reached, rapid Ca2+ release ensures prompt return to state 2 during channel closures, thus preventing CDI (state 4) and favoring return to the apoCaM preassociated site (state 1). Under physiological buffering, however, global signals would bypass both protective measures. The persistence of a ∼5 μM global Ca2+ pedestal (Figure 2Ga, top), whose amplitude exceeds the micromolar Ca2+ affinity of the N-lobe, would outlast lifetimes in protected state 1 (Figure 2A) and subsequently allow state 2 to bind Ca2+ and accumulate in state 3, thus driving strong CDI (state 4). Likewise, even under high buffering, if a channel had a PO of 1, the continuous local Ca2+ entry would produce a ∼100 μM Ca2+ pedestal (Figure 2Gc, top). This sustained Ca2+ signal would again overwhelm both measures of protection, resulting in strong CDI (bottom). This CDI is only slightly stronger than seen at the native channel PO under physiological buffering (compare Figures 2Gc and 2Ga), illustrating that the SQS mechanism is relatively insensitive to Ca2+ intensity. Instead, the contrast in CDI produced by different PO values under high buffering (compare Figures 2Gb and 2Gc) implicates a striking preference for the fractional presence of Ca2+. Viewing these events in the graphical CDI(∞)–PO format aids analytical understanding of this mechanism (Figure 2H). For orientation, the red zone associated with local selectivity is reproduced from Figure 2D. Additionally, a corresponding green zone for global Ca2+ selectivity can be specified, because all processes with global Ca2+ selectivity, regardless of mechanism, must exhibit near elimination of CDI by high Ca2+ buffering. Indeed, mapping the high-buffering SQS simulation (Figure 2Gb) onto this graph (Figure 2H, round symbol) confirms the ability of the SQS mechanism to intersect this global zone (Figure 2H, green shading). This graphical framework motivates deduction of the closed-form CDI(∞)–PO solution, obtained as follows (Supplemental Data [1A]). Because multiple transitions will occur between states 2 and 3 before exiting to either state 1 or 4, a number of simplifications can be made according to the analysis of Neher and Steinbach (Neher and Steinbach, 1978Neher E. Steinbach J.H. Local anaesthetics transiently block currents through single acetylcholine-receptor channels.J. Physiol. 1978; 277: 153-176PubMed Google Scholar). States 2 and 3 may be combined into a single state “2-3,” with all rate constants replaced by time-independent equivalents (Figure 2I). Since transitions to state 4 can only occur from state 3, which is only occupied during channel openings, the rate constant from compound state “2-3” to state 4 becomes α · PO. Similarly, the rate constant from compound state “2-3” to state 1 is a · (1 − PO). This equivalent system (Figure 2I) yields:CDI(∞)=CDImax⋅PO⋅rPO⋅(r−1)+1/ɛ+1(Equation 2) with individual rate constants (a, b, α, β) defined in Figure 2A, γ = α / β, ɛ = a / b, and r = γ / ɛ. CDImax accounts for the small PO of inactivated channels (Imredy and Yue, 1994Imredy J.P. Yue D.T. Mechanism of Ca2+-sensitive inactivation of L-type Ca2+ channels.Neuron. 1994; 12: 1301-1318Abstract Full Text PDF PubMed Scopus (208) Google Scholar). Importantly, Equation 2 suggests that the SQS mechanism need not always produce a global Ca2+ selectivity. Rather, N-lobe selectivity is predicted to be transformable between global and local extremes by adjusting the ratio of channel affinity for Ca2+/CaM versus apoCaM (parameter r). To begin, we note that CDI at PO = 1 (square in Figures 2Gc and 2H) will always exceed the strong CDI present in physiological buffering (arrow in Figures 2Ga and 2H). This is true because the Ca2+ input from a channel with PO = 1 under high buffering (Figure 2Gc, top) is larger than the input from a channel with native PO under physiological buffering (Figure 2Ga, top). Hence, we need only consider parameters yielding CDI(∞)–PO relations with strong CDI at PO = 1. Given this constraint, we first evaluate the case where r = 1/10 in Equation 2, as for the simulations in Figure 2G. Here, the equation produces a CDI(∞)–PO relation that intersects the global zone, as well as the numerically simulated data points (Figure 2H, green curve). Because r < 1, the denominator of Equation 2 now differs from a Michaelis-Menton relation. This yields an upward curvature indicating high sensitivity to the fractional presence of Ca2+, as represented by PO. However, this is not the only mode of operation. Setting r = 10 yields a saturating relation that intersects the local selectivity region (Figure 2H, red curve). Finally, if r = 1, the relationship becomes a straight" @default.
• W2012544852 created "2016-06-24" @default.
• W2012544852 creator A5006064421 @default.
• W2012544852 creator A5045137391 @default.
• W2012544852 creator A5078373067 @default.
• W2012544852 date "2008-06-01" @default.
• W2012544852 modified "2023-10-14" @default.
• W2012544852 title "Mechanism of Local and Global Ca2+ Sensing by Calmodulin in Complex with a Ca2+ Channel" @default.
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