Jingyi Shi

Calcium-sensitive potassium channelopathy in human epilepsy and paroxysmal movement disorder

The large conductance calcium-sensitive potassium (BK) channel is widely expressed in many organs and tissues, but its in vivo physiological functions have not been fully defined. Here we report a genetic locus associated with a human syndrome of coexistent generalized epilepsy and paroxysmal dyskinesia on chromosome 10q22 and show that a mutation of the α subunit of the BK channel causes this syndrome. The mutant BK channel had a markedly greater macroscopic current. Single-channel recordings showed an increase in open-channel probability due to a three- to fivefold increase in Ca2+ sensitivity. We propose that enhancement of BK channels in vivo leads to increased excitability by inducing rapid repolarization of action potentials, resulting in generalized epilepsy and paroxysmal dyskinesia by allowing neurons to fire at a faster rate. These results identify a gene that is mutated in generalized epilepsy and paroxysmal dyskinesia and have implications for the pathogenesis of human epilepsy, the neurophysiology of paroxysmal movement disorders and the role of BK channels in neurological disease.

Mechanism of magnesium activation of calcium-activated potassium channels

Large-conductance (BK type) Ca2+-dependent K+ channels are essential for modulating muscle contraction and neuronal activities such as synaptic transmission and hearing. BK channels are activated by membrane depolarization and intracellular Ca2+ and Mg2+ (refs 6–10). The energy provided by voltage, Ca2+ and Mg2+ binding are additive in activating the channel, suggesting that these signals open the activation gate through independent pathways. Here we report a molecular investigation of a Mg2+-dependent activation mechanism. Using a combined site-directed mutagenesis and structural analysis, we demonstrate that a structurally new Mg2+-binding site in the RCK/Rossman fold domain—an intracellular structural motif that immediately follows the activation gate S6 helix—is responsible for Mg2+-dependent activation. Mutations that impair or abolish Mg2+ sensitivity do not affect Ca2+ sensitivity, and vice versa. These results indicate distinct structural pathways for Mg2+- and Ca2+-dependent activation and suggest a possible mechanism for the coupling between Mg2+ binding and channel opening.

Assembly of a Ca2+‐dependent BK channel signaling complex by binding to β2 adrenergic receptor

Large‐conductance voltage and Ca2+‐activated potassium channels (BKCa) play a critical role in modulating contractile tone of smooth muscle, and neuronal processes. In most mammalian tissues, activation of β‐adrenergic receptors and protein kinase A (PKAc) increases BKCa channel activity, contributing to sympathetic nervous system/hormonal regulation of membrane excitability. Here we report the requirement of an association of the β2‐adrenergic receptor (β2AR) with the pore forming α subunit of BKCa and an A‐kinase‐anchoring protein (AKAP79/150) for β2 agonist regulation. β2AR can simultaneously interact with both BKCa and L‐type Ca2+ channels (Cav1.2) in vivo, which enables the assembly of a unique, highly localized signal transduction complex to mediate Ca2+‐ and phosphorylation‐dependent modulation of BKCa current. Our findings reveal a novel function for G protein‐coupled receptors as a scaffold to couple two families of ion channels into a physical and functional signaling complex to modulate β‐adrenergic regulation of membrane excitability.

Kv7.1 ion channels require a lipid to couple voltage sensing to pore opening

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 (PIP2). We found that voltage-sensing domain activation failed to open the pore in the absence of PIP2. This result is due to loss of coupling because PIP2 was also required for pore opening to affect voltage-sensing domain activation. We identified a critical site for PIP2-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.

Activation of Slo1 BK channels by Mg2+ coordinated between the voltage sensor and RCK1 domains

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.

KCNE1 enhances phosphatidylinositol 4,5-bisphosphate (PIP2) sensitivity of IKs to modulate channel activity

Phosphatidylinositol 4,5-bisphosphate (PIP2) is necessary for the function of various ion channels. The potassium channel, IKs, is important for cardiac repolarization and requires PIP2 to activate. Here we show that the auxiliary subunit of IKs, KCNE1, increases PIP2 sensitivity 100-fold over channels formed by the pore-forming KCNQ1 subunits alone, which effectively amplifies current because native PIP2 levels in the membrane are insufficient to activate all KCNQ1 channels. A juxtamembranous site in the KCNE1 C terminus is a key structural determinant of PIP2 sensitivity. Long QT syndrome associated mutations of this site lower PIP2 affinity, resulting in reduced current. Application of exogenous PIP2 to these mutants restores wild-type channel activity. These results reveal a vital role of PIP2 for KCNE1 modulation of IKs channels that may represent a common mechanism of auxiliary subunit modulation of many ion channels.

An Epilepsy/Dyskinesia-Associated Mutation Enhances BK Channel Activation by Potentiating Ca2+ Sensing

Ca(2+)-activated BK channels modulate neuronal activities, including spike frequency adaptation and synaptic transmission. Previous studies found that Ca(2+)-binding sites and the activation gate are spatially separated in the channel protein, but the mechanism by which Ca(2+) 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 Ca(2+) binding and channel opening. The GEPD mutation inside the AC region increases BK channel activity by enhancing this allosteric coupling. We found that Ca(2+) 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 Ca(2+) binding to channel opening.

Participation of the S4 voltage sensor in the Mg2+-dependent activation of large conductance (BK) K+ channels

The S4 transmembrane segment is the primary voltage sensor in voltage-dependent ion channels. Its movement in response to changes in membrane potential leads to the opening of the activation gate, which is formed by a separate structural component, the S6 segment. Here we show in voltage-, Ca2+-, and Mg2+-dependent, large conductance K+ channels that the S4 segment participates not only in voltage- but also Mg2+-dependent activation. Mutations in S4 and the S4-S5 linker alter voltage-dependent activation and have little or no effect on activation by micromolar Ca2+. However, a subset of these mutations in the C-terminal half of S4 and in the S4-S5 linker either reduce or abolish the Mg2+ sensitivity of channel gating. Cysteine residues substituted into positions R210 and R213, marking the boundary between S4 mutations that alter Mg2+ sensitivity and those that do not, are accessible to a modifying reagent [sodium (2-sulfonatoethyl)methane-thiosulfonate] (MTSES) from the extracellular and intracellular side of the membrane, respectively, at -80 mV. This implies that interactions between S4 and a cytoplasmic domain may be involved in Mg2+-dependent activation. These results indicate that the voltage sensor is critical for Mg2+-dependent activation and the coupling between the voltage sensor and channel gate is a converging point for voltage- and Mg2+-dependent activation pathways.

Ion sensing in the RCK1 domain of BK channels

BK-type K+ channels are activated by voltage and intracellular Ca2+, which is important in modulating muscle contraction, neural transmission, and circadian pacemaker output. Previous studies suggest that the cytosolic domain of BK channels contains two different Ca2+ binding sites, but the molecular composition of one of the sites is not completely known. Here we report, by systematic mutagenesis studies, the identification of E535 as part of this Ca2+ binding site. This site is specific for binding to Ca2+ but not Cd2+. Experimental results and molecular modeling based on the X-ray crystallographic structures of the BK channel cytosolic domain suggest that the binding of Ca2+ by the side chains of E535 and the previously identified D367 changes the conformation around the binding site and turns the side chain of M513 into a hydrophobic core, providing a basis to understand how Ca2+ binding at this site opens the activation gate of the channel that is remotely located in the membrane.

Mg2+ mediates interaction between the voltage sensor and cytosolic domain to activate BK 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 Mg2+ and the open state of the channel pore. Mg2+ 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 Mg2+-dependent activation in BK channels and suggesting a general pathway by which intracellular factors can modulate the function of voltage-dependent proteins.