Determination of the Stoichiometry between α- and γ1 Subunits of the BK Channel Using LRET.

Two families of accessory proteins, ß and γ, modulate BK channel gating and pharmacology. Notably, in the absence of internal Ca2+, the γ1 subunit promotes a large shift of the BK conductance-voltage curve to more negative potentials. However, very little is known about how α- and γ1 subunits interact. In particular, the association stoichiometry between both subunits is unknown. Here, we propose a method to answer this question using lanthanide resonance energy transfer. The method assumes that the kinetics of lanthanide resonance energy transfer-sensitized emission of the donor double-labeled α/γ1 complex is the linear combination of the kinetics of the sensitized emission in single-labeled complexes. We used a lanthanide binding tag engineered either into the α- or the γ1 subunits to bind Tb+3 as the donor. The acceptor (BODIPY) was attached to the BK pore-blocker iberiotoxin. We determined that γ1 associates with the α-subunit with a maximal 1:1 stoichiometry. This method could be applied to determine the stoichiometry of association between proteins within heteromultimeric complexes.

A BK (Slo1) channel journey from molecule to physiology.

Calcium and voltage-activated potassium (BK) channels are key actors in cell physiology, both in neuronal and non-neuronal cells and tissues. Through negative feedback between intracellular Ca (2+) and membrane voltage, BK channels provide a damping mechanism for excitatory signals. Molecular modulation of these channels by alternative splicing, auxiliary subunits and post-translational modifications showed that these channels are subjected to many mechanisms that add diversity to the BK channel α subunit gene. This complexity of interactions modulates BK channel gating, modifying the energetic barrier of voltage sensor domain activation and channel opening. Regions for voltage as well as Ca (2+) sensitivity have been identified, and the crystal structure generated by the 2 RCK domains contained in the C-terminal of the channel has been described. The linkage of these channels to many intracellular metabolites and pathways, as well as their modulation by extracellular natural agents, has been found to be relevant in many physiological processes. This review includes the hallmarks of BK channel biophysics and its physiological impact on specific cells and tissues, highlighting its relationship with auxiliary subunit expression.

The first transmembrane domain (TM1) of ß2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels.

The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary ß-subunit that modulates the voltage- and Ca(2+)-dependent activation of the channel. Structural components present in ß-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the ß2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein-protein interactions demonstrated for the first time that TM1 of the ß2-subunit physically binds to the transmembrane S1 domain of the α-subunit.

Allosteric interactions and the modular nature of the voltage- and Ca2+-activated (BK) channel.

The high conductance voltage- and Ca(2+)-activated K(+) channel is one of the most broadly expressed channels in mammals. This channel is named BK for 'big K' because of its single-channel conductance that can be as large as 250 pS in 100 mm symmetrical K(+). BK channels increase their activity by membrane depolarization or an increase in cytosolic Ca(2+). One of the key features that defines the behaviour of BK channels is that neither Ca(2+) nor voltage is strictly necessary for channel activation. This and several other observations led to the idea that both Ca(2+) and voltage increase the open probability by an allosteric mechanism. In this type of mechanism, the processes of voltage sensor displacement, Ca(2+) binding and pore opening are independent equilibria that interact allosterically with each other. These allosteric interactions in BK channels reside in the structural characteristics of the BK channel in the sense that voltage and Ca(2+) sensors and the pore need to be contained in different structures or 'modules'. Through electrophysiological, mutagenesis, biochemical and fluorescence studies these modules have been identified and, more important, some of the interactions between them have been unveiled. In this review, we have covered the main advances achieved during the last few years in the elucidation of the structure of the BK channel and how this is related with its function as an allosteric protein.

Intrinsic electrostatic potential in the BK channel pore: role in determining single channel conductance and block.

The internal vestibule of large-conductance Ca(2+) voltage-activated K(+) (BK) channels contains a ring of eight negative charges not present in K(+) channels of lower conductance (Glu386 and Glu389 in hSlo) that modulates channel conductance through an electrostatic mechanism (Brelidze, T.I., X. Niu, and K.L. Magleby. 2003. Proc. Natl. Acad. Sci. USA. 100:9017-9022). In BK channels there are also two acidic amino acid residues in an extracellular loop (Asp326 and Glu329 in hSlo). To determine the electrostatic influence of these charges on channel conductance, we expressed wild-type BK channels and mutants E386N/E389N, D326N, E329Q, and D326N/E329Q channels on Xenopus laevis oocytes, and measured the expressed currents under patch clamp. Contribution of E329 to the conductance is negligible and single channel conductance of D326N/E329Q channels measured at 0 mV in symmetrical 110 mM K(+) was 18% lower than the control. Current-voltage curves displayed weak outward rectification for D326N and the double mutant. The conductance differences between the mutants and wild-type BK were caused by an electrostatic effect since they were enhanced at low K(+) (30 mM) and vanished at high K(+) (1 M K(+)). We determine the electrostatic potential change, Deltaphi, caused by the charge neutralization using TEA(+) block for the extracellular charges and Ba(2+) for intracellular charges. We measured 13 +/- 2 mV for Deltaphi at the TEA(+) site when turning off the extracellular charges, and 17 +/- 2 mV for the Deltaphi at the Ba(2+) site when the intracellular charges were turned off. To understand the electrostatic effect of charge neutralizations, we determined Deltaphi using a BK channel molecular model embedded in a lipid bilayer and solving the Poisson-Boltzmann equation. The model explains the experimental results adequately and, in particular, gives an economical explanation to the differential effect on the conductance of the neutralization of charges D326 and E329.

A marriage of convenience: beta-subunits and voltage-dependent K+ channels.

The movement of ions across cell membranes is essential for a wide variety of fundamental physiological processes, including secretion, muscle contraction, and neuronal excitation. This movement is possible because of the presence in the cell membrane of a class of integral membrane proteins dubbed ion channels. Ion channels, thanks to the presence of aqueous pores in their structure, catalyze the passage of ions across the otherwise ion-impermeable lipid bilayer. Ion conduction across ion channels is highly regulated, and in the case of voltage-dependent K(+) channels, the molecular foundations of the voltage-dependent conformational changes leading to the their open (conducting) configuration have provided most of the driving force for research in ion channel biophysics since the pioneering work of Hodgkin and Huxley (Hodgkin, A. L., and Huxley, A. F. (1952) J. Physiol. 117, 500-544). The voltage-dependent K(+) channels are the prototypical voltage-gated channels and govern the resting membrane potential. They are responsible for returning the membrane potential to its resting state at the termination of each action potential in excitable membranes. The pore-forming subunits (alpha) of many voltage-dependent K(+) channels and modulatory beta-subunits exist in the membrane as one component of macromolecular complexes, able to integrate a myriad of cellular signals that regulate ion channel behavior. In this review, we have focused on the modulatory effects of beta-subunits on the voltage-dependent K(+) (Kv) channel and on the large conductance Ca(2+)- and voltage-dependent (BK(Ca)) channel.