A Carboxy-terminal Inter-Helix Linker As the Site of Phosphatidylinositol 4,5-Bisphosphate Action on Kv7 (M-type) K+ Channels

Abstract

Members of the KCNQ (Kv7) family of voltage-gated K + channels underlie " M-type " K + currents in many different types of neurons, delayed-rectifi er currents of the heart, and K + transport channels of the inner ear and epithelia ( Jentsch, 2000 ;Robbins, 2001 ). Neuronal M currents play strong roles in regulating excitability and neuronal discharge, and their modulation by several receptors linked to the G q/11 class of G proteins endows them with powerful effects on the function of excitable cells ( Delmas and Brown, 2005 ). As for a plethora of other channels and transporters ( Gamper and Shapiro, 2007 ), M-type channels are very sensitive to the abundance of phosphatidylinositol 4,5-bisphosphate (PIP 2 ) in the membrane, and PIP 2 depletion is widely accepted as the mechanism of M current suppression by muscarinic receptor stimulation in sympathetic neurons ( Delmas and Brown, 2005 ;Suh et al., 2006 ;Brown et al., 2007 ;Suh and Hille, 2007 ).We have examined the activity of Kv7.2 -7.4 channels at the single-channel level and found these voltage-gated channels to have strikingly differential saturating open probabilities (P o ) in cell-attached patches ( Li et al., 2004 ).Correspondence to Mark S. Shapiro: s h a p i r o m @ u t h s c s a . e d u Abbreviations used in this paper: CaM, calmodulin; C ␣ RMS, carbon ␣ root mean squared; CHO, Chinese hamster ovary; diC8-PIP 2 , dioctanoyl-PIP 2 ; GPMI-P2, l -␣ -glycerophospho-d -myo-inositol 4,5-bisphosphate; Kv7, KCNQ; PIP 2 , phosphatidylinositol 4,5-bisphosphate; P o , open probabilities; RMSD, root mean square distance; wt, wild-type.The online version of this article contains supplemental material.In accord with the role of PIP 2 in regulating gating, channel activity rapidly runs down upon excision as inside-out patches, but activity can be fully restored by adding PIP 2 or an analogue to the cytoplasmically facing bath solution ( Zhang et al., 2003 ;Li et al., 2005 ). For all the channels studied, the P o is a function of the concentration of supplied PIP 2 , but with very different apparent affi nities among the channels. Thus, Kv7.3 homomultimers display a very high apparent affi nity, Kv7.2 and Kv7.4 homomultimers display apparent affi nities some one to two orders of magnitude lower, and Kv7.2/7.3 heteromultimers display an intermediate value, as expected for channels composed of both high-and low-affi nity PIP 2 -binding subunits ( Li et al., 2005 ). Such differential PIP 2 affi nities among the channels are also supported by whole cell experiments in which PIP 2 abundance was either tonically The regulation of M-type (KCNQ [Kv7]) K + channels by phosphatidylinositol 4,5-bisphosphate (PIP 2 ) has perhaps the best correspondence to physiological signaling, but the site of action and structural motif of PIP 2 on these channels have not been established. Using single-channel recordings of chimeras of Kv7.3 and 7.4 channels with highly differential PIP 2 sensitivities, we localized a carboxy-terminal inter-helix linker as the primary site of PIP 2 action....

Long QT mutations disrupt IKS regulation by PKA and PIP2 at the same KCNQ1 helix C-KCNE1 interface

“…Different regions endowed with clusters of basic residues have been proposed to interact with PIP 2 . Hernandez et al, identified a conserved cluster of basic residues within the linker connecting helix A and B in the C-terminus of KCNQ2-KCNQ4 subunits (Hernandez et al, 2008). This basic cluster was suggested to interact with PIP 2 through electrostatic and hydrogen-bonding networks (Hernandez et al, 2008).…”

“…Hernandez et al, identified a conserved cluster of basic residues within the linker connecting helix A and B in the C-terminus of KCNQ2-KCNQ4 subunits (Hernandez et al, 2008). This basic cluster was suggested to interact with PIP 2 through electrostatic and hydrogen-bonding networks (Hernandez et al, 2008). However, a recent study showed that this linker is not required for PIP 2 regulation of KCNQ2 (Aivar et al, 2012).…”

“…Accordingly, the spontaneous rundown of I KS channels observed in excised patches is markedly reduced by cytosolic application of PIP 2 (Loussouarn et al, 2003;Park et al, 2005). The site of PIP 2 binding on KCNQ2-KCNQ4 channels has been suggested to be located in the linker connecting helix A and B (Hernandez et al, 2008); however, a recent work indicates that this AB helix linker is not required for PIP 2 regulation of KCNQ2 (Aivar et al, 2012). The PIP 2 -binding site also remains elusive for KCNQ1.…”

Uncoupling PIP2-calmodulin regulation of Kv7.2 channels by an assembly destabilizing epileptogenic mutation

“…CaM regulatory mechanisms that change the gating behavior proceed through an effect on sensitivity to PIP 2 (Kosenko et al, 2012;Kosenko and Hoshi, 2013;Zaydman et al, 2013;Kang et al, 2014). The interlinker between helices A and B is not essential for function, although it plays a critical role in PIP 2 regulation for Kv7.3, but not in Kv7.1 and Kv7.2 channels (Hernandez et al, 2008;Aivar et al, 2012;Sachyani et al, 2014).…”

Dynamic PIP 2 interactions with voltage sensor elements contribute to KCNQ2 channel gating

“…Subsequent studies supported that PIP 2 also interacts directly with voltage-gated potassium (Kv) channels (6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19). Several positive residues that may be critical for PIP 2 activity have been identified (7,11,18,(20)(21)(22)(23)(24). Previous studies on Kv1.2 and Shaker channels showed that PIP 2 exerts opposing effects on Kv channels, up-regulating the current amplitude, while leading to a decrease in voltage sensitivity (7, 18).…”