Mark A. Zaydman

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.

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.

State-dependent electrostatic interactions of S4 arginines with E1 in S2 during Kv7.1 activation

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.

PIP2 regulation of KCNQ channels: biophysical and molecular mechanisms for lipid modulation of voltage-dependent gating

Voltage-gated potassium (Kv) channels contain voltage-sensing (VSD) and pore-gate (PGD) structural domains. During voltage-dependent gating, conformational changes in the two domains are coupled giving rise to voltage-dependent opening of the channel. In addition to membrane voltage, KCNQ (Kv7) channel opening requires the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2). Recent studies suggest that PIP2 serves as a cofactor to mediate VSD-PGD coupling in KCNQ1 channels. In this review, we put these findings in the context of the current understanding of voltage-dependent gating, lipid modulation of Kv channel activation, and PIP2-regulation of KCNQ channels. We suggest that lipid-mediated coupling of functional domains is a common mechanism among KCNQ channels that may be applicable to other Kv channels and membrane proteins.

Regulation of Voltage-Activated K+ Channel Gating by Transmembrane β Subunits

Voltage-activated K+ (KV) channels are important for shaping action potentials and maintaining resting membrane potential in excitable cells. KV channels contain a central pore-gate domain (PGD) surrounded by four voltage-sensing domains (VSDs). The VSDs will change conformation in response to alterations of the membrane potential thereby inducing the opening of the PGD. Many KV channels are heteromeric protein complexes containing auxiliary β subunits. These β subunits modulate channel expression and activity to increase functional diversity and render tissue specific phenotypes. This review focuses on the KV β subunits that contain transmembrane (TM) segments including the KCNE family and the β subunits of large conductance, Ca2+- and voltage-activated K+ (BK) channels. These TM β subunits affect the voltage-dependent activation of KV α subunits. Experimental and computational studies have described the structural location of these β subunits in the channel complexes and the biophysical effects on VSD activation, PGD opening, and VSD–PGD coupling. These results reveal some common characteristics and mechanistic insights into KV channel modulation by TM β subunits.

Domain–domain interactions determine the gating, permeation, pharmacology, and subunit modulation of the IKs ion channel

Voltage-gated ion channels generate electrical currents that control muscle contraction, encode neuronal information, and trigger hormonal release. Tissue-specific expression of accessory (β) subunits causes these channels to generate currents with distinct properties. In the heart, KCNQ1 voltage-gated potassium channels coassemble with KCNE1 β-subunits to generate the IKs current (Barhanin et al., 1996; Sanguinetti et al., 1996), an important current for maintenance of stable heart rhythms. KCNE1 significantly modulates the gating, permeation, and pharmacology of KCNQ1 (Wrobel et al., 2012; Sun et al., 2012; Abbott, 2014). These changes are essential for the physiological role of IKs (Silva and Rudy, 2005); however, after 18 years of study, no coherent mechanism explaining how KCNE1 affects KCNQ1 has emerged. Here we provide evidence of such a mechanism, whereby, KCNE1 alters the state-dependent interactions that functionally couple the voltage-sensing domains (VSDs) to the pore. DOI: http://dx.doi.org/10.7554/eLife.03606.001

Ion channel associated diseases: overview of molecular mechanisms.

Mechanisms Mark A. Zaydman,†,‡,§ Jonathan R. Silva,†,‡,§ and Jianmin Cui*,†,‡,§ †Department of Biomedical Engineering, Washington University, Saint Louis, Missouri 63130, United States ‡Center for the Investigation of Membrane Excitability Disorders, Washington University, Saint Louis, Missouri 63130, United States Cardiac Bioelectricity and Arrhythmia Center, Washington University, Saint Louis, Missouri 63130, United States

Direct Measurement of Cardiac Na+ Channel Conformations Reveals Molecular Pathologies of Inherited Mutations.

Background—Dysregulation of voltage-gated cardiac Na+ channels (NaV1.5) by inherited mutations, disease-linked remodeling, and drugs causes arrhythmias. The molecular mechanisms whereby the NaV1.5 voltage-sensing domains (VSDs) are perturbed to pathologically or therapeutically modulate Na+ current (INa) have not been specified. Our aim was to correlate INa kinetics with conformational changes within the 4 (DI–DIV) VSDs to define molecular mechanisms of NaV1.5 modulation. Method and Results—Four NaV1.5 constructs were created to track the voltage-dependent kinetics of conformational changes within each VSD, using voltage-clamp fluorometry. Each VSD displayed unique kinetics, consistent with distinct roles in determining INa. In particular, DIII-VSD deactivation kinetics were modulated by depolarizing pulses with durations in the intermediate time domain that modulates late INa. We then used the DII-VSD construct to probe the molecular pathology of 2 Brugada syndrome mutations (A735V and G752R). A735V shifted DII-VSD voltage dependence to depolarized potentials, whereas G752R significantly slowed DII-VSD kinetics. Both mutations slowed INa activation, although DII-VSD activation occurred at higher potentials (A735V) or at later times (G752R) than ionic current activation, indicating that the DII-VSD allosterically regulates the rate of INa activation and myocyte excitability. Conclusions—Our results reveal novel mechanisms whereby the NaV1.5 VSDs regulate channel activation and inactivation. The ability to distinguish distinct molecular mechanisms of proximal Brugada syndrome mutations demonstrates the potential of these methods to reveal how inherited mutations, post-translational modifications, and antiarrhythmic drugs alter NaV1.5 at the molecular level.

Diagnosis of red meat allergy with antigen-specific IgE tests in serum

Red meat allergy is a tick-associated hypersensitivity reaction to galactose-α-1,3-galactose (α-gal) and is characterized by anaphylaxis, angioedema, urticaria and/or gastrointestinal symptoms occurring 3–6 hours after ingesting red meat such as beef, pork, or lamb. Diagnosis of red meat allergy is challenging due to the unusually long delay in symptom onset and poor sensitivity of skin prick tests with commercial meat extracts. The primary diagnostic tools available for this disease are quantification of α-gal-, beef-, pork-, and/or lamb-specific IgE in serum, however the diagnostic performance of these tests has not been reported. Using patient data for n=135 patients with red meat allergy and n=37 controls, we found that measurement of α-gal-specific IgE using the bovine thyroglobulin (bTG) ImmunoCAP method had the best overall sensitivity (100%) and specificity (92.3%) for diagnosis of red meat allergy. Measuring α-gal-specific IgE using the streptavidin (SA)-CAP technique or beef- or pork-specific IgE using ImmunoCAP were also effective tests with high sensitivities (89–92%) and variable specificities (65–82%). Lamb-specific IgE and total IgE had essentially no diagnostic value for red meat allergy. Positive and negative predictive values mirrored these trends. Taken together, these findings indicate that the α-gal-specific IgE test by bTG ImmunoCAP is the most useful for establishing a diagnosis of red meat allergy, although α-gal-specific IgE by SA-CAP and beef- and pork-specific IgE by ImmunoCAP are also effective tests.