Role of PIP2 in regulating versus modulating Ca2+ channel activity

We would like to comment on an article by Gamper & Shapiro (2007) recently published in The Journal of Physiology entitled Target-specific PIP2 signalling: How might it work? In this paper, they contend that, ‘there is wide agreement that the Gq/11-coupled muscarinic modulation of both N-type Ca2+ and M-type K+ channels is mediated by receptor-induced depletion of PIP2’. We disagree with Gamper and Shapiro's portrayal of widespread acceptance of this model for Ca2+ channel modulation by Gq-coupled receptors. In this paradigm, loss of PIP2 association with Ca2+ channels is necessary and sufficient for current inhibition (Wu et al. 2002; Gamper et al. 2004; Michailidis et al. 2007). Activation of phospholipase C (PLC) by Gq-coupled receptors substantially lowers free PIP2 levels in the plasma membrane. As a consequence, when constitutively bound PIP2 dissociates and diffuses away from the channel rather than rebinding, channel activity is inhibited (Gamper et al. 2004). No other signalling events, e.g. kinases, phosphatases or additional lipases, participate in channel inhibition. This hypothesis arose from a similar model for M-current modulation by M1Rs, first proposed by Suh & Hille (2002) and later embraced by several labs examining Ca2+ current modulation by Gq-coupled receptors (Wu et al. 2002; Gamper et al. 2004; Robbins et al. 2006). We have offered an alternative hypothesis to Gamper et al. (2004) as to the signal cascade that confers Ca2+ current inhibition. Studies in our lab indicate that in addition to PLC, group IVa phospholipase A2 (cPLA2) activity is required in order to observe modulation of L- and N-type Ca2+ currents. Our conclusion is based on the following specific findings. (1) Arachidonic acid (AA) mimics the actions of M1R agonists in inhibiting channel activity (Liu & Rittenhouse, 2000, 2003b; Barrett et al. 2001; Liu et al. 2001, 2006). (2) The presence of bovine serum albumin (BSA), either in the pipette or in the bath solution, minimizes L- and N-current inhibition by Oxo-M (Liu & Rittenhouse, 2003b; Liu et al. 2006). Moreover, AA rather than a metabolite, mediates N-current modulation since blocking AA's metabolism has no effect on exogenous AA or Oxo-M's ability to inhibit current (Barrett et al. 2001; Liu et al. 2001; Liu & Rittenhouse, 2003b). (3) Antagonizing PLA2 with oleyloxyethyl phosphorylcholine (OPC) minimizes inhibition of both L- and N-currents (Liu & Rittenhouse, 2003a, b; Liu et al. 2004, 2006). Our OPC data contrast findings from Gamper et al. (2004) who reported no effect of OPC on N-current inhibition by Oxo-M. Similarly, Bannister et al. (2002) found that the PLA2 antagonist quinacrine had no effect on M1R inhibition of recombinant L-current (CaV1.2). However, both studies failed to provide controls demonstrating that the PLA2 antagonist was effectively blocking the enzyme, so that the respective conclusions appear premature. (4) Using antibodies as functional antagonists, we found that dialysing cells with antibodies to cPLA2, but not to sPLA2 or non-immunized antibody minimized L-current inhibition (Liu & Rittenhouse, 2003a; Liu et al. 2004, 2006). (5) Using a genetic approach we found that neurons from mice deficient in cPLA2 (cPLA2−\−) exhibited minimal L-current inhibition by Oxo-M (Liu et al. 2006). No significant differences in control current amplitude or magnitude of current inhibition by AA was observed between cPLA2+/+versus cPLA2−/− neurons, indicating normal channel activity in cPLA2−/− neurons. Moreover, M-current inhibition by Oxo-M remained normal, indicating no change in M1R, Gq, or PLC functioning in cPLA2−/− neurons. However, cPLA2−/− neurons exhibited decreased fatty acid release following exposure to Oxo-M compared to wild-type neurons, consistent with a requirement for cPLA2-dependent increases in free fatty acid levels in order to observe L- and N-current inhibition. (6) Using BSA as an AA scavenger to limit free fatty acid levels antagonized L- and N-current inhibition (Liu & Rittenhouse, 2003b; Liu et al. 2006). Taken together our findings indicate that lipid products downstream of PIP2 are required for Ca2+ current modulation, whereas M-current inhibition appears to occur with PIP2 breakdown by PLC. Most critically, the studies with cPLA2−/− neurons document that PLC activity alone is insufficient to mediate Ca2+ current inhibition. How to reconcile our previous findings that a fatty acid (probably AA) mediates Ca2+ current inhibition by M1R signalling with the PIP2 model remains unresolved (Liu & Rittenhouse, 2003b; Liu et al. 2004, 2006; Michailidis et al. 2007). Resolution will come with additional controls and experiments. For example the PIP2 analogue DiC8-PIP2 appears to minimize Ca2+ current inhibition; however, this analogue of PIP2 does not contain the normal fatty acid chains associated with PIP2, e.g. AA and stearic acid. It is possible that diC8-PIP2 acts as a substrate competitor with other phospholipids for cPLA2 decreasing the AA liberated and minimizing Ca2+ channel inhibition. Similarly, whether application of exogenous phospholipids swells membranes to such an extent that M1R signalling no longer functions needs to be tested and resolved. Most critical is the need to distinguish between questions that test how PIP2 functions as a regulator of channel activity versus its role in Ca2+ channel modulation by specific Gq-coupled receptors. For example, dephosphorylating PIP2 may cause PIP2 to dissociate from Ca2+ channels and lower activity; however, during muscarinic signalling, this mechanism may play no role in decreasing current amplitude. In recent work palmitoylated charged peptides, that sequester PIP2, were dialysed into neurons, decreasing both M- and N-current amplitudes. However, low concentrations of peptide, which only minimally decreased basal current amplitude, disrupted M- but not N-current modulation by M1Rs (Robbins et al. 2006). The different results underscore the notion that specific experiments testing roles in modulation as well as regulation are needed in order to properly define the function of PIP2 in ion channel regulation. Thus experiments outlined by Gamper & Shapiro (2007) that test how plasma membrane PIP2 levels are regulated contribute to our understanding of PIP2's functioning, but not necessarily its role in modulating Ca2+ channel activity. Thus, we strongly encourage caution when using these experiments as evidence that a simple dissociation of PIP2 from Ca2+ channels explains how M1Rs inhibit Ca2+ channel activity. Our model provides additional levels of control allowing more independent regulation of specific ion channel activity by M1R signalling.