Mechanism of Spatial Ca2+ Selectivity of a Ca2+ Sensor in Complex with a Ca2+ Source

Calmodulin (CaM) in complex with Ca2+ channels constitutes a prototype for Ca2+ sensors that are intimately co-localized 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 of global Ca2+ sensing has defied explanation. Here, we advance a theory of how global selectivity arises, and validate this proposal with new experimental tools enabling millisecond control of Ca2+ oscillations within nanometers of channels. 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., 2000). 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., 2001; Cullen, 2006; De Koninck and Schulman, 1998; Dolmetsch et al., 1998; Gu and Spitzer, 1995; Li et al., 1998; Oancea and Meyer, 1998; Winslow and Crabtree, 2005). 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 in the ‘nanodomain’ of sources promotes rapid and privileged Ca2+ signaling (Augustine et al., 2003; Bootman et al., 2001; Catterall, 1999). 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. © 2008 Elsevier Inc. All rights reserved. To whom correspondence should be addressed Correspondence and requests for materials should be addressed to DTY (dyue@bme.jhu.edu). voice: (410) 955-0078 fax: (410) 955-0549. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author Manuscript Cell. Author manuscript; available in PMC 2014 December 10. Published in final edited form as: Cell. 2008 June 27; 133(7): 1228–1240. doi:10.1016/j.cell.2008.05.025. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript 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, 2007). CaM is continuously complexed with channels as a resident Ca2+ sensor (Erickson et al., 2001; Pitt et al., 2001), and Ca2+ binding to the Cand N-terminal lobes of CaM can each induce a separate form of regulation on the same target channel (DeMaria et al., 2001; Yang et al., 2006). Given the approximate diameter of Ca2+ channels (Wang et al., 2002), the resident CaM would be positioned ~10 nm from the channel pore, in the nanodomain of these channels. As such, it is intriguing that 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, 1998; Sherman et al., 1990) (Supplementary Information 3). 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., 2003). This ‘local selectivity’ is schematized for a Ca2+-dependent inactivation process (CDI) triggered by the C-lobe (Figure 1B). 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; Supplementary Information 3) (Neher, 1998), 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, right) (DeMaria et al., 2001; Liang et al., 2003). 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 Ca2+ 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 local preference of the C-lobe might be expected, since this lobe responds to the component of greater intensity. However, the global selectivity of the N-lobe is difficult to imagine. 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 known to constitutively associate with a single CaM (Mori et al., 2004; Yang et al., 2007), and the lobes of CaM are very close to one another (< 6 nm), indicating that both lobes are likely within the nanodomain (Dunlap, 2007; Stern, 1992). 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 Tadross et al. Page 2 Cell. Author manuscript; available in PMC 2014 December 10. N IH -P A A uhor M anscript N IH -P A A uhor M anscript N IH -P A A uhor M anscript patterns of Ca2+ have been characterized (De Koninck and Schulman, 1998; Oancea and Meyer, 1998 ), none can respond to signals of low amplitude and frequency (global pedestal), while ignoring signals of high amplitude and frequency (local spikes). Thus, global selectivity must employ a novel Ca2+ sensing mechanism. Here, theoretical and experimental advances explain how global selectivity could be produced by a mechanism that favors persistent, rather than intense Ca2+ signals. This unusual property emerges 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 novel ‘voltage block’ electrophysiological technique to precisely control nanodomain Ca2+. These tools resolved Ca2+ response characteristics clearly distinctive of the proposed mechanism. Combining this approach with manipulation of a recently identified CaM regulatory site (Dick et al., 2008) enables quantitative confirmation of a key prediction—selectivity can be incrementally changed from global to local by adjusting the ratio of apoCaM versus Ca2+/CaM affinities for a given channel. Our findings generalize across CaV1–CaV2 channels, and likely extend to diverse Ca2+ sensors situated near Ca2+ sources.