Calcium signalling in muscle: a milestone for modulation studies.

Abstract

Calcium signals mediate metabolic switching in most cells and tissues. In skeletal and cardiac muscle, where they allow for excitation–contraction (EC) coupling, these signals reach their largest amplitudes and fastest rates. In fast twitch muscle the signalling goal is to cause near-saturation of troponin C and then make it Ca2+-free, all in milliseconds. This requires up to 1 mm Ca2+ to move from stores to cytosol. Because Ca2+ is a multiuse messenger, EC coupling is a high-wire act, which keeps fast muscle ‘near death’. This hyperbole (Carafoli, 2005) stresses that the signals must be exquisitely tuned for their role. Fast change is achieved by juxtaposition of two large fluxes (Ca2+ release and removal), much in the way that nimble cars are given large engines and power brakes. Fluxes up to 250 mm s−1 for release and 50 mm s−1 for removal are made possible by finely interspersed Ca2+ sources (release channels, or RyRs) and sinks (pumps and Ca2+-binding molecules). Top flux is reached in less than 1 ms and is turned off entirely within about 4 ms (Rome et al. 1996). This is achieved by the interplay of a number of mechanisms to amplify release and then abruptly end it. The processes start from voltage sensors (DHPRs) of the transverse tubule, and are thought to include interactions of channels with the permeant ion itself (Ca2+-induced Ca2+ release and Ca2+-dependent inactivation). In addition there are ancillary proteins, candidates for modulating further the function of the release channels. The list of such proteins is always growing. Some, including calmodulin (Balshaw et al. 2002), sorcin (Farrell et al. 2004), Homer (Ward et al. 2004) and the immunophilins, are in the cytosol or accessible from it, and hence may affect many cellular processes in addition to EC coupling. Triadin (Caswell et al. 1991; Guo & Campbell 1995), junctin (Jones et al. 1995), mg29 (Takeshima et al. 1998), the junctophilins (Takeshima et al. 2000) and junctate (Treves et al. 2000) are integral to the SR membrane. Calsequestrin (MacLennan & Wong, 1971), sarcalumenin (Leberer et al. 1989) and histidine-rich Ca2+-binding protein (Hofmann et al. 1989) reside in the SR lumen. The roles of these molecules, initially inferred from their biochemical properties, are increasingly the subject of functional studies. Interesting protocols have appeared, including construction of murine transgenics (Kirchhefer et al. 2001; Pan et al. 2002; Yoshida et al. 2005), viral transduction of mutant molecules (Viatchenko-Karpinski et al. 2004), direct application of molecules or their removal from channels in bilayers (Beard et al. 2002), and various pharmacological strategies to study the molecules accessible from the cytosol. In the current issue of The Journal of Physiology, Gouadon et al. (2006) now demonstrate a modulatory role of JP-45 (Zorato et al. 2000). This small protein, integral to the SR junctional membrane, is intriguing because it apparently interacts with both the DHPR and, inside the lumen of the SR, its main Ca2+ buffer, calsequestrin. Calsequestrin is linked to the RyR via triadin and junctin, to implement the reported influence of intra-SR [Ca2+] on RyR channel gating. Thus JP-45 is positioned with a finger on the voltage sensor and a toe dipped in the SR lumen, as if sampling critical links of the functional chain. The present study is unique on multiple counts. By using transient over-expression rather than radical genetic engineering, it avoids the developmental or compensatory effects of permanent changes imposed in transgenic mice. In myotubes over-expressing JP-45, a detailed biophysical study then defines the effect by evaluating the Ca2+ release flux induced by voltage, then deriving the time course of RyR permeability. To yield permeability, rather than flux, the technique must deal correctly with other variables that traditionally obscure these measurements, namely the load of Ca2+ in the storage compartment and the activity of the SR Ca2+ pump. Better yet, by measuring intramembranous charge movement, the study evaluates the ‘signal’ provided by the voltage sensor of the electrically excitable membrane, thus yielding specifically the ‘transfer function’ (Gonzalez & Rios, 1993) between signal (movement of the sensor) and response (permeability change). This transfer function is shown to be depressed by the over-expression of JP-45. While its precise parsing of physiological interactions makes the work remarkable, the study has limitations. The main problem is one that clouds most over-expression approaches: it is not clear whether a native molecule in excess will simply enhance the native function – there may be, after all, too much of a good thing. To complement their work, the researchers propose knockout approaches. Perhaps a transient knock-down, through fast-evolving siRNA technology, will prove a better option. In any case, through their approach Gouadon et al. (2006) show how to define a physiological effect of an ancillary protein with great precision, and clearly raise the bar for future studies of these intriguing modulators.