Abstract
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.
Highlights
No significant effects were observed on the activation mechanism by any of these treatments
We explored the PIP2 regulation of Kv2.1 channels expressed in HEK293 cells by employing different strategies to manipulate PIP2 levels in excised patches and intact cells
It is likely that the rundown observed in the inside-out patch recordings at a holding potential of −80 mV was due to an accumulation of inactivation of Kv2.1 channels, as rundown was not observed at a holding potential of −120 mV, where inactivation does not take place
Summary
No significant effects were observed on the activation mechanism by any of these treatments. Voltage-gated potassium (Kv) channels are integral membrane proteins that enable the passage of potassium ions (K+) across cell membranes. Kv channels transition from a resting (closed) to an activated (open) state, during prolonged depolarizations, Kv channels switch to an inactivated (open non-conductive) state[2] Transitions between these three states (gating) of Kv channels can be modulated by different stimuli. Phosphoinositides, phosphatidylinositol 4,5-bisphosphate (PIP2), have been shown to modulate the gating mechanism of several Kv channels[10,11,12], some of these results have been debated[13,14]. The reasons why some Kv channels (e.g. Kv2.1) show PIP2 sensitivity in excised patches, but not in intact cells still remains poorly understood and deserves further investigation
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