The secret lives of voltage sensors

Voltage-gated ion channels serve a wide array of biological purposes. In one well-known example, they orchestrate the action potential in the excitable cells of nervous tissue thus generating excitability throughout the nervous system. This diverse family of transmembrane proteins is exquisitely sensitive to membrane potential and many of its members are modulated by a growing number of other stimuli including, but not limited to, G-proteins, phosphorylation, temperature, sumoylation and inositol phosphates. In the current issue of The Journal of Physiology, Murata & Okamura (2007) characterize a recent addition to the voltage-gated family termed Ci-VSP, the voltage sensitive phosphatase. Cloned from the ascidian Ciona intestinalis, Ci-VSP consists of four transmembrane segments homologous to the voltage sensor from voltage-gated ion channels concatenated to a cytosolic domain homologous to the phosphatase and tumour suppressor protein PTEN (phosphatase and tensin homologue deleted from chromosome 10) (Maehama & Dixon, 1999). Catalytic activity is not without precedent in voltage-gated channel complexes as both potassium and calcium channel β-subunits have such domains embedded in their sequences (Takahashi et al. 2004; Heinemann & Hoshi, 2006). Furthermore, the burgeoning family of putative voltage gated phosphatases have associated members from H. sapiens to X. laevis suggesting that they represent more than a curious evolutionary dead-end.

The authors have shown previously that Ci-VSP is capable of generating high density gating currents similar to those produced by voltage-gated channels and its phophatidylinositol 3,4,5-bisphosphate (PIP₃) phosphatase activity was verified in vitro by malachite assay (Murata et al. 2005). When coexpressed in Xenopus oocytes, Ci-VSP can positively modulate GIRK2 or KCNQ2/3 currents, two channels activated by phophatidylinositol-4,5-bisphosphate (PIP₂) (Rohacs et al. 1999; Zhang et al. 2003). One interesting observation to come from these early experiments was that the ionic currents for both GIRK2 and KCN2/3 channels increased with hyperpolarization suggesting that the phosphatase activity runs with ‘reversed’ polarity, i.e. Ci-VSP dephosphorylates PIP₃ to PIP₂ upon membrane hyperpolarization. This reasonable conclusion was based, at least partially, on the assumption that PTEN predominately dephosphorylates PIP₃ (Maehama & Dixon, 1998).

Armed with a bevy of novel methods to measure changes in voltage-driven PIP₃ and PIP₂ levels, the authors set out to further characterize Ci-VSP. The first hint that we have entered interesting territory arrives with the use of two different GFP tagged pleckstrin homology (PH) domains whose surface targeting relies on the cytosolic levels of PIP₂ and PIP₃, respectively (Zhang et al. 2003; also see Suh & Hille, 2007). Surprisingly, repeated depolarization of oocytes coexpressing Ci-VSP and either PH domain results in decreased surface expression of the PH domain as measured by lowered GFP fluorescence at the surface of the oocyte. Conversely, surface expression increases with hyperpolarization and in both cases catalytic integrity of the PTEN domain is required. Put together, these results suggest that Ci-VSP dephosphorylates both PIP₂ and PIP₃. With fresh eyes, and with these new results in hand the authors then re-examine the effects of Ci-VSP on GIRK2 currents, and propose that Ci-VSP actually gates in a ‘forward’ direction through which depolarization activates the phosphatase. Why, then, do PIP₂-sensitive GIRK2 and KCNQ2/3 currents get larger with hyperpolarization when the phosphatase is shut off? One possibility may be that prolonged depolarization depletes both PIP₃ and PIP₂, and upon hyperpolarization endogenous PIP 5- and 4-kinases are allowed to replenish the balance of PIP₂ and PIP₃. Future experiments verifying the ability of Ci-VSP's PTEN domain to dephosphorylate PIP₂in vitro and the use of specific inositol 4- and 5-kinase inhibitors in vivo will be helpful in exploring these possibilities.

It is worth noting that changes in local PIP₂ concentration can elicit a rapid response from KCNQ2/3 channels, on the order of seconds (Suh et al. 2006), and a striking example of this phenomenon is presented here. KCNQ2/3 channels coexpressed with Ci-VSP are purported to undergo a rapid (τ∼50 ms at +100 mV) inactivation due to Ci-VSP catalysed PIP₂ depletion. This effect saturates at potentials above +100 mV, like the charge movement from Ci-VSP, and is entirely absent with the catalytically inactive mutant C363S. Furthermore, voltage-sensor mutations which cause shifts in charge movement have similar effects on voltage dependence of PIP₂ depletion. While the possibility remains that the rapid time course of PIP₂ depletion and its putative effects on inactivation results from the over-expression of Ci-VSP in a heterologous expression system, this result suggests that the phosphatase, kinases and the channel might be in exceptionally close quarters.

The recent cloning and expression of a proton channel comprising a single voltage-sensing domain (Ramsey et al. 2006) and crystallographic studies on Kv1.2 (Long et al. 2005) suggest that the ion channel voltage sensing domain is modular in nature. Barring the presence of an endogenous pore domain surrogate in oocytes or cooperative interactions between voltage sensors, the robust gating currents generated by Ci-VSP provides further support for this possibility. Future experiments focusing on the mechanistic details of phosphatase–voltage coupling and Ci-VSP stoichiometry therefore will not only help decode the mysteries of Ci-VSP but may also shed light on the structure–function relationships relevant to all voltage-gated channels. While many such questions persist, the real surprises may be revealed when our attention is turned to the unknown physiology of Ci-VSP.