Voltage-gated ion channels generate dynamic ionic currents that are vital to the physiological functions of many tissues. These proteins contain separate voltage-sensing domains, which detect changes in transmembrane voltage, and pore domains, which conduct ions. Coupling of voltage sensing and pore opening is critical to the channel function and has been modeled as a protein-protein interaction between the two domains. Here, we show that coupling in Kv7.1 channels requires the lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP 2 . This result is due to loss of coupling because PIP 2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP 2 -dependent coupling at the interface between the voltage-sensing domain and the pore domain. This site is actually a conserved lipid-binding site among different K + channels, suggesting that lipids play an important role in coupling in many ion channels.V oltage-gated ion channels are integral membrane proteins that sense membrane voltage and respond by opening or closing a transmembrane pore. Ionic currents carried by voltage-gated ion channels control contraction in muscle, encode information in the nervous system, and trigger secretion in neurohormonal tissues. Voltage-gated ion channels contain four voltage-sensing domains (VSDs) and a central pore domain (PD) that are structurally distinct (1, 2). In voltage-gated potassium (Kv) channels, the first four transmembrane segments (S1-S4) of each α-subunit forms a VSD. In response to changes in transmembrane voltage, the VSD undergoes a conformational change, called activation, during which membrane depolarization moves the S4 segment outward (3). The PD is formed by the last two transmembrane segments (S5, S6) from four α-subunits and undergoes a mainly voltageindependent conformational change during which the intracellular ends of the S6 segments bend, opening the ionic pore (4, 5). Interestingly, the PD and VSD can exist in pore-only (6) and voltage sensor-only proteins, respectively, where they function independently (7,8). Confining sensitivity to voltage, or to other stimuli, within a domain diversifies the ion channel properties that can be achieved by partnering different pore and sensor domains. However, this modular architecture also raises a fundamental question as to how VSD activation is transmitted to the PD. Previous studies of this coupling process have revealed the importance of direct protein-protein interactions at the VSD-PD interface (9-13); however, the possible role of membrane lipids in VSD-PD coupling remains undetermined.The membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP 2 ) modulates the activity of many ion channels, including some voltage-gated channels (14). Notably, all members of the Kv7 family (Kv7.1-Kv7.5), which play important physiological roles in the cardiac (15) or the nervous (16) systems, require PIP 2 to be opened by...

Summary Ca2+-activated BK channels modulate neuronal activities including spike frequency adaptation and synaptic transmission. Previous studies found that Ca2+ binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca2+ binding opens the gate over this distance remains unknown. By studying an Asp to Gly mutation (D434G) associated with human syndrome of generalized epilepsy and paroxysmal dyskinesia (GEPD), we show that a cytosolic motif immediately following the activation gate S6 helix, known as the AC region, mediates the allosteric coupling between Ca2+ binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca2+ sensitivity is enhanced by increases in solution viscosity that reduce protein dynamics. The GEPD mutation alters such a response, suggesting that a less flexible AC region may be more effective in coupling Ca2+ binding to channel opening.

The voltage sensor domain (VSD) and the ligand sensor (cytoplasmic domain) of BK channels synergistically control channel activities, thereby integrating electrical and chemical signals for cell function. Studies show that intracellular Mg2+ mediates the interaction between these sensory domains to activate the channel through an electrostatic interaction with the VSD. Here we report that Mg2+ binds to a site that consists of amino acid side-chains from both the VSD (Asp99 and Asn172) and the cytoplasmic domain (Glu374 and Glu399). For each Mg2+ binding site the residues in the VSD and those in the cytoplasmic domain come from neighboring subunits. These results suggest that the VSD and the cytoplasmic domains from different subunits may interact during channel gating, and the packing of VSD or the RCK1 domain to the pore in BK channels differ from that in Kv1.2 or MthK channels.

The voltage-sensor domain (VSD) of voltage-dependent ion channels and enzymes is critical for cellular responses to membrane potential. The VSD can also be regulated by interaction with intracellular proteins and ligands, but how this occurs is poorly understood. Here, we show that the VSD of the BK-type K ؉ channel is regulated by a state-dependent interaction with its own tethered cytosolic domain that depends on both intracellular Mg 2؉ and the open state of the channel pore. Mg 2؉ bound to the cytosolic RCK1 domain enhances VSD activation by electrostatic interaction with Arg-213 in transmembrane segment S4. Our results demonstrate that a cytosolic domain can come close enough to the VSD to regulate its activity electrostatically, thereby elucidating a mechanism of Mg 2؉ -dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.oltage-dependent ion channels are modular proteins (1) containing four voltage-sensor domains (VSDs) that control the opening and closing of a central pore domain. Each VSD contains charged residues in transmembrane segments that sense changes in membrane potential. VSDs are important for controlling not only ion channel pores but also enzymatic activity (2) and can serve as stand-alone proton channels in the absence of a separate pore domain (3, 4). Because VSDs impact multiple aspects of cellular function, it is important to understand how VSD function is regulated. Many intracellular factors such as ligand binding, posttranslational modification, and the presence of cytosolic domains, accessory proteins, or subunits can alter the function of voltagedependent ion channels (5). In some cases such modulation is thought to involve the voltage sensor (6-10). However, the mechanisms by which intracellular factors and VSDs interact are poorly understood. BK channels are activated by membrane depolarization, intracellular Ca 2ϩ , and Mg 2ϩ (11) and are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing (12, 13). Like other voltagedependent K ϩ (Kv) channels, BK channels possess a VSD where the S4 segment contains multiple Arg residues (14, 15). However, in BK channels, only one of these, R213, contributes to voltage sensing (15-18). Interestingly, neutralizing R213 by mutation (R213Q) not only alters voltage-dependent activation but also abolishes Mg 2ϩ -dependent activation of BK channels, revealing that the VSD contributes to Mg 2ϩ sensitivity, although the mechanism is unknown (18). Here, we investigate the mechanism of Mg 2ϩ action to determine whether and how the VSD interacts with Mg 2ϩ ions that are bound to the BK channel's COOH-terminal cytosolic domain. ResultsMg 2؉ May Activate the VSD by Electrostatic Interaction. Physiological concentrations of Mg 2ϩ in the low millimolar range activate BK channels, independent of the effects of micromolar Ca 2ϩ , by binding to a site that includes residues E374 and E399 (19-21). The put...

The voltage-sensing domain of voltage-gated channels is comprised of four transmembrane helices (S1–S4), with conserved positively charged residues in S4 moving across the membrane in response to changes in transmembrane voltage. Although it has been shown that positive charges in S4 interact with negative countercharges in S2 and S3 to facilitate protein maturation, how these electrostatic interactions participate in channel gating remains unclear. We studied a mutation in Kv7.1 (also known as KCNQ1 or KvLQT1) channels associated with long QT syndrome (E1K in S2) and found that reversal of the charge at E1 eliminates macroscopic current without inhibiting protein trafficking to the membrane. Pairing E1R with individual charge reversal mutations of arginines in S4 (R1–R4) can restore current, demonstrating that R1–R4 interact with E1. After mutating E1 to cysteine, we probed E1C with charged methanethiosulfonate (MTS) reagents. MTS reagents could not modify E1C in the absence of KCNE1. With KCNE1, (2-sulfonatoethyl) MTS (MTSES)− could modify E1C, but [2-(trimethylammonium)ethyl] MTS (MTSET)+ could not, confirming the presence of a positively charged environment around E1C that allows approach by MTSES− but repels MTSET+. We could change the local electrostatic environment of E1C by making charge reversal and/or neutralization mutations of R1 and R4, such that MTSET+ modified these constructs depending on activation states of the voltage sensor. Our results confirm the interaction between E1 and the fourth arginine in S4 (R4) predicted from open-state crystal structures of Kv channels and reveal an E1–R1 interaction in the resting state. Thus, E1 engages in electrostatic interactions with arginines in S4 sequentially during the gating movement of S4. These electrostatic interactions contribute energetically to voltage-dependent gating and are important in setting the limits for S4 movement.

The KCNE1 auxiliary subunit coassembles with the Kv7.1 channel and modulates its properties to generate the cardiac I(Ks) current. Recent biophysical evidence suggests that KCNE1 interacts with the voltage-sensing domain (VSD) of Kv7.1. To investigate the mechanism of how KCNE1 affects the VSD to alter the voltage dependence of channel activation, we perturbed the VSD of Kv7.1 by mutagenesis and chemical modification in the absence and presence of KCNE1. Mutagenesis of S4 in Kv7.1 indicates that basic residues in the N-terminal half (S4-N) and C-terminal half (S4-C) of S4 are important for stabilizing the resting and activated states of the channel, respectively. KCNE1 disrupts electrostatic interactions involving S4-C, specifically with the lower conserved glutamate in S2 (Glu(170) or E2). Likewise, Trp scanning of S4 shows that mutations to a cluster of residues in S4-C eliminate current in the presence of KCNE1. In addition, KCNE1 affects S4-N by enhancing MTS accessibility to the top of the VSD. Consistent with the structure of Kv channels and previous studies on the KCNE1-Kv7.1 interaction, these results suggest that KCNE1 alters the interactions of S4 residues with the surrounding protein environment, possibly by changing the protein packing around S4, thereby affecting the voltage dependence of Kv7.1.

Large conductance Ca(2+)- and voltage-activated K(+) (BK) channels, composed of pore-forming alpha-subunits and auxiliary beta-subunits, play important roles in diverse physiological processes. The differences in BK channel phenotypes are primarily due to the tissue-specific expression of beta-subunits (beta1-beta4) that modulate channel function differently. Yet, the molecular basis of the subunit-specific regulation is not clear. In our study, we demonstrate that perturbation of the voltage sensor in BK channels by mutations selectively disrupts the ability of the beta1-subunit--but not that of the beta2-subunit--to enhance apparent Ca(2+) sensitivity. These mutations change the number of equivalent gating charges, the voltage dependence of voltage sensor movements, the open-close equilibrium of the channel, and the allosteric coupling between voltage sensor movements and channel opening to various degrees, indicating that they alter the conformation and movements of the voltage sensor and the activation gate. Similarly, the ability of the beta1-subunit to enhance apparent Ca(2+) sensitivity is diminished to various degrees, correlating quantitatively with the shift of voltage dependence of voltage sensor movements. In contrast, none of these mutations significantly reduces the ability of the beta2-subunit to enhance Ca(2+) sensitivity. These results suggest that the beta1-subunit enhances Ca(2+) sensitivity by altering the conformation and movements of the voltage sensor, whereas the similar function of the beta2-subunit is governed by a distinct mechanism.

Large-conductance, voltage-, and Ca 2ϩ -dependent K ϩ (BK) channels are broadly expressed in various tissues to modulate neuronal activity, smooth muscle contraction, and secretion. BK channel activation depends on the interactions among the voltage sensing domain (VSD), the cytosolic domain (CTD), and the pore gate domain (PGD) of the Slo1 ␣-subunit, and is further regulated by accessory ␤ subunits (␤1-␤4). How ␤ subunits fine-tune BK channel activation is critical to understand the tissue-specific functions of BK channels. Multiple sites in both Slo1 and the ␤ subunits have been identified to contribute to the interaction between Slo1 and the ␤ subunits. However, it is unclear whether and how the interdomain interactions among the VSD, CTD, and PGD are altered by the ␤ subunits to affect channel activation. Here we show that human ␤1 and ␤2 subunits alter interactions between bound Mg 2ϩ and gating charge R213 and disrupt the disulfide bond formation at the VSD-CTD interface of mouse Slo1, indicating that the ␤ subunits alter the VSD-CTD interface. Reciprocally, mutations in the Slo1 that alter the VSD-CTD interaction can specifically change the effects of the ␤1 subunit on the Ca 2ϩ activation and of the ␤2 subunit on the voltage activation. Together, our data suggest a novel mechanism by which the ␤ subunits modulated BK channel activation such that a ␤ subunit may interact with the VSD or the CTD and alter the VSD-CTD interface of the Slo1, which enables the ␤ subunit to have effects broadly on both voltage and Ca 2ϩ -dependent activation.