Reply from N. Gamper and M. S. Shapiro

Abstract

Ours is one of a number of groups that have shown N- and P/Q-type Ca2+ channels to be sensitive to the abundance of PIP2 in the plasma membrane (Wu et al. 2002; Gamper et al. 2004; Lechner et al. 2005; Robbins et al. 2006; Suh et al. 2006). The evidence comes from cloned channels expressed in Xenopus oocytes studied in inside-out patches and from endogenous channels of sympathetic neurons in which [PIP2] was manipulated with phosphoinositide (PI) phosphatases, blockers of PI kinases, PIP2 scavengers and by PIP2 dialysis from whole-cell pipettes. All these experiments strongly suggest that depletion of PIP2 from plasma membrane does induce suppression of ICa of N- and P/Q-types. Of particular note is the N-type ICa depression observed upon PIP2 depletion produced by rapamycin-induced plasma membrane recruitment of a PIP2 5-phosphatase in mammalian cells (Suh et al. 2006). This is a receptor- and PLC-free experiment in which no further second messengers are produced. On the other hand, much evidence also indicates that [PIP2] can be strongly depleted in the neurons by stimulation of Gq/11-coupled M1R stimulation (Suh & Hille, 2002; Zhang et al. 2003; Horowitz et al. 2005; Winks et al. 2005; Hughes et al. 2007). Taken together, these two lines of evidence make M1R-mediated suppression of ICa by depletion of [PIP2] a mechanism logical to most investigators. In spite of all this, however, we accept a role for arachadonic acid (AA) in tuning the sensitivity of the Ca2+ channels to small or local depletions of PIP2, as is plausible given that AA is produced downstream of PIP2 hydrolysis and consequential diacylglycerol production via the actions of diacylglycerol lipase. Indeed, we explicitly consider such a mechanism for ensuring fidelity in spatiotemporally limited receptor signalling (Gamper et al. 2004; Delmas et al. 2005). Finally, our review article in the special issue of The Journal of Physiology (Gamper & Shapiro, 2007), dedicated to the question of the regulation of ion channels by phosphoinositides, is not focused on voltage-gated Ca2+ channels (particularly, we do not discuss L-type channels at all) but rather on a few difficult issues in phosphoinositide signalling, such as receptor specificity, the affinity of PIP2-binding sites and technical problems in phosphoinositide detection and manipulation, which are often left behind in such discussions. In this sense, we whole-heartedly agree with the Rittenhouse lab: the biochemistry of phosphoinositide interactions with membrane proteins is not ‘simple’ and we do not hold a ‘PIP2-centric’ view of G protein-mediated signalling. Nevertheless, the widespread and powerful role of phosphoinositide signals in regulation of ion channels and transporters cannot be ignored, as the recent special issue of The Journal of Physiology (Robertson, 2007) clearly shows.