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Voltage-gated K + (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4–S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP 2 depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP 2 . Gating current measurements revealed that PIP 2 constrains the movement of the sensor through interactions with the S4–S5 linker. Thus, PIP 2 controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them.

Over the past 16 years, there has been an impressive number of ion channels shown to be sensitive to the major phosphoinositide in the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PIP2). Among them are voltage-gated channels, which are crucial for both neuronal and cardiac excitability. Voltage-gated calcium (Cav) channels were shown to be regulated bidirectionally by PIP2. On one hand, PIP2 stabilized their activity by reducing current rundown but on the other hand it produced a voltage-dependent inhibition by shifting the activation curve to more positive voltages. For voltage-gated potassium (Kv) channels PIP2 was first shown to prevent N-type inactivation regardless of whether the fast inactivation gate was part of the pore-forming α subunit or of an accessory β subunit. Careful examination of the effects of PIP2 on the activation mechanism of Kv1.2 has shown a similar bidirectional regulation as in the Cav channels. The two effects could be distinguished kinetically, in terms of their sensitivities to PIP2 and by distinct molecular determinants. The rightward shift of the Kv1.2 voltage dependence implicated basic residues in the S4–S5 linker and was consistent with stabilization of the inactive state of the voltage sensor. A third type of a voltage-gated ion channel modulated by PIP2 is the hyperpolarization-activated cyclic nucleotide-gated (HCN) channel. PIP2 has been shown to enhance the opening of HCN channels by shifting their voltage-dependent activation toward depolarized potentials. The sea urchin HCN channel, SpIH, showed again a PIP2-mediated bidirectional effect but in reverse order than the depolarization-activated Cav and Kv channels: a voltage-dependent potentiation, like the mammalian HCN channels, but also an inhibition of the cGMP-induced current activation. Just like the Kv1.2 channels, distinct molecular determinants underlied the PIP2 dual effects on SpIH, with the proximal C-terminus implicated in the inhibitory effect. The dual regulation of these very different ion channels, all of which are voltage-dependent, points to conserved mechanisms of regulation of these channels by PIP2.

Objective We identified a novel de novo SCN2A variant (M1879T) associated with infantile‐onset epilepsy that responded dramatically to sodium channel blocker antiepileptic drugs. We analyzed the functional and pharmacological consequences of this variant to establish pathogenicity, and to correlate genotype with phenotype and clinical drug response. Methods The clinical and genetic features of an infant boy with epilepsy are presented. We investigated the effect of the variant using heterologously expressed recombinant human NaV1.2 channels. We performed whole‐cell patch clamp recording to determine the functional consequences and response to carbamazepine. Results The M1879T variant caused disturbances in channel inactivation including substantially depolarized voltage dependence of inactivation, slower time course of inactivation, and enhanced resurgent current that collectively represent a gain‐of‐function. Carbamazepine partially normalized the voltage dependence of inactivation and produced use‐dependent block of the variant channel at high pulsing frequencies. Carbamazepine also suppresses resurgent current conducted by M1879T channels, but this effect was explained primarily by reducing the peak transient current. Molecular modeling suggests that the M1879T variant disrupts contacts with nearby residues in the C‐terminal domain of the channel. Interpretation Our study demonstrates the value of conducting functional analyses of SCN2A variants of unknown significance to establish pathogenicity and genotype–phenotype correlations. We also show concordance of in vitro pharmacology using heterologous cells with the drug response observed clinically in a case of SCN2A‐associated epilepsy.

The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP 2 ) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP 2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP 2 -dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP 2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP 2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP 2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease.

Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP 2 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 PIP 2 remains unclear. Here, we investigate the effects of PIP 2 on Kv2.1 channels by applying exogenous PIP 2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP 2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP 2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP 2 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 PIP 2 depletion facilitates inactivation of Kv2.1. We propose that PIP 2 regulates Kv2.1 channels by interfering with the inactivation mechanism.

Ang II receptor activation increases cytosolic Ca2+ levels to enhance the synthesis and secretion of aldosterone, a recently identified early pathogenic stimulus that adversely influences cardiovascular homeostasis. Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a downstream effector of the Ang II-elicited signaling cascade that serves as a key intracellular Ca2+ sensor to feedback-regulate Ca2+ entry through voltage-gated Ca2+ channels. However, the molecular mechanism(s) by which CaMKII regulates these important physiological targets to increase Ca2+ entry remain unresolved. We show here that CaMKII forms a signaling complex with alpha1H T-type Ca2+ channels, directly interacting with the intracellular loop connecting domains II and III of the channel pore (II-III loop). Activation of the kinase mediated the phosphorylation of Ser1198 in the II-III loop and the positive feedback regulation of channel gating both in intact cells in situ and in cells of the native adrenal zona glomerulosa stimulated by Ang II in vivo. These data define the molecular basis for the in vivo modulation of native T-type Ca2+ channels by CaMKII and suggest that the disruption of this signaling complex in the zona glomerulosa may provide a new therapeutic approach to limit aldosterone production and cardiovascular disease progression.

Voltage-gated K + (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4–S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP 2 ) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP 2 depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP 2 . Gating current measurements revealed that PIP 2 constrains the movement of the sensor through interactions with the S4–S5 linker. Thus, PIP 2 controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them. Note: By “distinct molecular determinants,” we mean that the two effects of PIP 2 (i.e., on voltage sensitivity and open probability) showed dependence on distinct amino acids. Thus, although mutation of K322 affected both voltage sensitivity and open probability, mutation of the N-terminal residue R147 controlled voltage sensitivity but not open probability, whereas mutation of R326 controlled open probability but not voltage sensitivity. Our data support a model in which electropositive residues in the N terminus and the S4–S5 linker interact with PIP 2 and exert two distinct effects. First, PIP 2 stabilizes the voltage sensor of Kv channels in a state of decreased sensitivity [charge–voltage (Q–V) and conductance–voltage (G–V) curves are shifted to the right], favoring the closed state of the channel. Second, PIP 2 stabilizes the Kv channel pore in the conducting state. These two apparently contradictory effects could be distinguished by differences in kinetics, sensitivities to PIP 2 , and molecular determinants. This study, which provides molecular evidence linking PIP 2 to the activation mechanism of Kv channels, does not explain why PIP 2 has the dual effect we have described in certain Kv channels (e.g., Kv1, CaV, HCN) but not in others (e.g., KCNQ, HERG, TRP, K2P). The molecular and physiological reasons for this differential design in voltage-gated channel regulation remain to be clarified. We identified three amino acids residues (K322 and R326 in the S4–S5 linker and R147Q in the N terminus) that are important for interactions between the channel protein and PIP 2 . R147Q and R326Q affect only voltage sensitivity and current amplitude, respectively, whereas K322Q affects both. Using computational analysis of the partial crystal structures of Kv1.2, where we could dock PIP 2 to a model of the closed and open conformations of Kv1.2 ( 2 , 4 ), suggested that PIP 2 can engage all three residues only in the closed state. We used single-channel recording to determine the mechanism of reduction of current amplitude and observed a reduction in the probability of channel opening (Po) after depleting PIP 2 . On the other hand, gating current recordings of Kv channels in control conditions and after wortmannin treatment showed a hyperpolarizing shift in the charge–voltage relationship after enzymatic depletion of PIP 2 , suggesting that PIP 2 modulates the voltage sensor directly, thereby constraining its movement. Application of a PIP 2 antibody to inside–out patches also induced a shift in the voltage dependence of activation and a decrease in current amplitude, effects that were reversed by application of exogenous PIP 2 . We also examined the effects of reducing PIP 2 levels in the plasma membrane of intact oocytes and recorded them with the two-electrode voltage-clamp technique. PIP 2 levels were depleted using an inhibitor (wortmannin) of type-III phosphatidylinositol 4-kinase or the voltage-sensitive phosphatase Ciona intestinalis VSP (Ci-VSP). Similar to observations in the excised patches, wortmannin and Ci-VSP affected current amplitude and voltage dependency. The differences in kinetics of both effects also were revealed in the Ci-VSP experiments. Taken together, these results suggest that two mechanisms are involved in determining how PIP 2 modulates the current amplitude and voltage dependence of its activation of Kv channels. Dose–response curves with the soluble eight-carbon-long acyl chain PIP 2 (diC8-PIP 2 ) revealed that the current amplitude and voltage dependence of activation also are distinctly sensitive to PIP 2 . Kv channels can be modulated by phospholipids that are interspersed between the VSD and PD. The phospholipid PIP 2 represents one such modulator. PIP 2, although a minor component of the inner membrane leaflet, modulates the activity of most ion channels and transporters ( 3 ). PIP 2 acts as a signaling molecule through its own direct interactions with a myriad of target proteins ( 3 ). Using electrophysiological techniques, such as the inside–out patch mode of the patch-clamp technique or the two-electrode voltage-clamp technique ( 1 ), we studied the effects of depleting PIP 2 on the activity of the Kv1.2 channel in Xenopus oocytes. Upon patch excision, a decrease of Kv currents developed within several minutes, and two effects were observed: a hyperpolarizing shift in the voltage dependence of activation and a decrease in the amplitude of current. Moreover, both effects exhibited distinct kinetics, suggesting that PIP 2 modulates the activity by two different mechanisms. Here, we used a combination of electrophysiological and mutagenesis techniques together with structural modeling and molecular dynamics simulations to study the mechanism by which the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) modulates Kv channels. We show that PIP 2 depletion exerted two kinetically distinct effects on Kv channels: an increase in sensitivity to voltage and a concomitant decrease in the amplitude of the current. These effects were reversible, exhibiting distinct molecular determinants and sensitivities to PIP 2 . Our results revealed that PIP 2 controls both the movement of its VSD and the stability of its open pore through interactions with the S4–S5 linker that connects them ( Fig. P1 ). Voltage-gated potassium (Kv) channels are involved in diverse physiological processes, including action potential repolarization, secretion of hormones and neurotransmitters, and contraction of skeletal muscle ( 1 ). These channels regulate the flow of potassium ions across the plasma membrane in response to changes in membrane potential and are regulated by several factors, such as posttranslational modifications and membrane lipids. Kv channels are homotetrameric, with each subunit containing six segments (S1–S6) that cross the cell membrane ( Fig. P1 ). Segments S1–S4 form the voltage-sensing domain (VSD), whereas segments S5 and S6 form the pore domain (PD). The VSD of Kv channel subunits harbors within its S4 helix several positively charged residues that respond directly to changes in membrane voltage. The movement of these charges can be monitored by the gating current they produce, and the opening of the pore can be monitored by the ionic current that follows. The S4–S5 linker couples the movement of the voltage sensor to the opening of the pore ( 2 ).

Voltage-gated K+ (Kv) channels couple the movement of a voltage sensor to the channel gate(s) via a helical intracellular region, the S4-S5 linker. A number of studies link voltage sensitivity to interactions of S4 charges with membrane phospholipids in the outer leaflet of the bilayer. Although the phospholipid phosphatidylinositol-4,5-bisphosphate (PIP...) in the inner membrane leaflet has emerged as a universal activator of ion channels, no such role has been established for mammalian Kv channels. Here we show that PIP... depletion induced two kinetically distinct effects on Kv channels: an increase in voltage sensitivity and a concomitant decrease in current amplitude. These effects are reversible, exhibiting distinct molecular determinants and sensitivities to PIP... Gating current measurements revealed that PIP... constrains the movement of the sensor through interactions with the S4-S5 linker. Thus, PIP... controls both the movement of the voltage sensor and the stability of the open pore through interactions with the linker that connects them. (ProQuest: ... denotes formulae/symbols omitted.)