Regulation of large-conductance Ca2+- and voltage-gated K+ channels by electrostatic interactions with auxiliary β subunits

Large-conductance Ca2+- and voltage-gated K+ (BK KCa1.1) channel complexes include pore-forming Slo1 α subunits and often auxiliary β subunits, latter of which noticeably modify the channel’s pharmacological and gating characteristics. In the absence of intracellular Ca2+, β1 and β4 modestly shift the overall voltage dependence of the channel to the positive direction by decreasing the probability that the ion conduction gate is open without any allosteric influence from the channel’s voltage or Ca2+ sensors. This intrinsic open probability is also critically regulated by the intracellular-facing 329RKK331 segment of human Slo1 (hSlo1) downstream of the transmembrane segment S6 in association with two negatively charged residues in S6 (E321 and E324) (Tian et al., Proc Natl Acad Sci USA, 116, 8591-8596, 2019). This study examined how β1/β4 and the RKK segment function together to control the channel gate. With select mutations in the RKK segment, inclusions of β1 or β4 can dramatically increase the intrinsic gate opening probability and shift the overall voltage dependence of the channel to the negative direction by up to 200 mV without Ca2+. This remarkable shift is mediated at least in part by electrostatic interactions between the Slo1 RKK and β N-terminal segments as suggested by the results of double-mutant cycle analysis, ionic strength experiments, and molecular modelling. With or without auxiliary β subunits, the Slo1 RKK and E321/E324 segments are thus critical determinants of the intrinsic open probability of the ion conduction gate and changes in the electrostatic environment near the RKK-EE segments are a potential mechanism of pharmacological gating modifiers.

Double Mutant Cycles as a Tool to Address Folding, Binding, and Allostery

Quantitative measurement of intramolecular and intermolecular interactions in protein structure is an elusive task, not easy to address experimentally. The phenomenon denoted ‘energetic coupling’ describes short- and long-range interactions between two residues in a protein system. A powerful method to identify and quantitatively characterize long-range interactions and allosteric networks in proteins or protein–ligand complexes is called double-mutant cycles analysis. In this review we describe the thermodynamic principles and basic equations that underlie the double mutant cycle methodology, its fields of application and latest employments, and caveats and pitfalls that the experimentalists must consider. In particular, we show how double mutant cycles can be a powerful tool to investigate allosteric mechanisms in protein binding reactions as well as elusive states in protein folding pathways.

Cross-subunit interactions that stabilize open states mediate gating in NMDA receptors

NMDA receptors are excitatory channels with critical functions in the physiology of central synapses. Their activation reaction proceeds as a series of kinetically distinguishable, reversible steps, whose structural bases are currently under investigation. Very likely, the earliest steps include glutamate binding to glycine-bound receptors and subsequent constriction of the ligand-binding domain. Later, three short linkers transduce this movement to open the gate by mechanical pulling on transmembrane helices. Here, we used molecular and kinetic simulations and double-mutant cycle analyses to show that a direct chemical interaction between GluN1-I642 (on M3 helix) and GluN2A-L550 (on L1-M1 linker) stabilizes receptors after they have opened and thus represents one of the structural changes that occur late in the activation reaction. This native interaction extends the current decay, and its absence causes deficits in charge transfer by GluN1-I642L, a pathogenic human variant.

Physical and functional interaction sites in cytoplasmic domains of KCNQ1 and KCNE1 channel subunits

The cardiac potassium IKs current is carried by a channel complex formed from α-subunits encoded by KCNQ1 and β-subunits encoded by KCNE1. Deleterious mutations in either gene are associated with hereditary long QT syndrome. Interactions between the transmembrane domains of the α- and β-subunits determine the activation kinetics of IKs. A physical and functional interaction between COOH termini of the proteins has also been identified that impacts deactivation rate and voltage dependence of activation. We sought to explore the specific physical interactions between the COOH termini of the subunits that confer such control. Hydrogen/deuterium exchange coupled to mass spectrometry narrowed down the region of interaction to KCNQ1 residues 352–374 and KCNE1 residues 70–81, and provided evidence of secondary structure within these segments. Key mutations of residues in these regions tended to shift voltage dependence of activation toward more depolarizing voltages. Double-mutant cycle analysis then revealed energetic coupling between KCNQ1-I368 and KCNE1-D76 during channel activation. Our results suggest that the proximal COOH-terminal regions of KCNQ1 and KCNE1 participate in a physical and functional interaction during channel opening that is sensitive to perturbation and may explain the clustering of long QT mutations in the region. NEW & NOTEWORTHY Interacting ion channel subunits KCNQ1 and KCNE1 have received intense investigation due to their critical importance to human cardiovascular health. This work uses physical (hydrogen/deuterium exchange with mass spectrometry) and functional (double-mutant cycle analyses) studies to elucidate precise and important areas of interaction between the two proteins in an area that has eluded structural definition of the complex. It highlights the importance of pathogenic mutations in these regions.

Supramolecular cage encapsulation as a versatile tool for the experimental quantification of aromatic stacking interactions

A Double Mutant Cycle is built up using a supramolecular cage that binds two aromatic carboxylates in a stacked geometry is used to quantify aromatic stacking interactions.

The inactivation domain of STIM1 is functionally coupled with the Orai1 pore to enable Ca2+-dependent inactivation

The inactivation domain of STIM1 (IDSTIM: amino acids 470–491) has been described as necessary for Ca2+-dependent inactivation (CDI) of Ca2+ release–activated Ca2+ (CRAC) channels, but its mechanism of action is unknown. Here we identify acidic residues within IDSTIM that control the extent of CDI and examine functional interactions of IDSTIM with Orai1 pore residues W76 and Y80. Alanine scanning revealed three IDSTIM residues (D476/D478/D479) that are critical for generating full CDI. Disabling IDSTIM by a triple alanine substitution for these three residues (“STIM1 3A”) or by truncation of the entire domain (STIM11–469) reduced CDI to the same residual level observed for the Orai1 pore mutant W76A (approximately one third of the extent seen with full-length STIM1). Results of noise analysis showed that STIM11–469 and Orai1 W76A mutants do not reduce channel open probability or unitary Ca2+ conductance, factors that determine local Ca2+ accumulation, suggesting that they diminish CDI instead by inhibiting the CDI gating mechanism. We tested for functional coupling between IDSTIM and the Orai1 pore by double-mutant cycle analysis. The effects on CDI of mutations disabling IDSTIM or W76 were not additive, demonstrating that IDSTIM and W76 are strongly coupled and act in concert to generate full-strength CDI. Interestingly, disabling IDSTIM and W76 separately gave opposite results in Orai1 Y80A channels: channels with W76 but lacking IDSTIM generated approximately two thirds of the WT extent of CDI but those with IDSTIM but lacking W76 completely failed to inactivate. Together, our results suggest that Y80 alone is sufficient to generate residual CDI, but acts as a barrier to full CDI. Although IDSTIM is not required as a Ca2+ sensor for CDI, it acts in concert with W76 to progress beyond the residual inactivated state and enable CRAC channels to reach the full extent of inactivation.

Chelate cooperativity effects on the formation of di- and trivalent pseudo[2]rotaxanes with diketopiperazine threads and tetralactam wheels

Double mutant cycle analyses of isothermal titration calorimetry data on di- and trivalent amide pseudorotaxanes provide insight into chelate cooperativity effects on multiply threaded structures.