Independence and Cooperativity in Rearrangements of a Potassium Channel Voltage Sensor Revealed by Single Subunit Fluorescence

BK channels (Slo1) are widely distributed K+ channels that control Ca2+-dependent processes and cellular excitability. Their activation by intracellular Ca2+ (Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}) is highly cooperative, with Hill coefficients of typically 2–5. To investigate the cooperativity contributed by each of the four α subunits that form the BK channel, we studied single channels comprised of mixtures of functional subunits and subunits with a mutation to disrupt a key site (Ca-bowl) required for activation by low concentrations of Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document}. As the number of functional subunits increased, we found a stepwise increase in the Hill coefficient of 0.3–0.8 per functional subunit and a stepwise decrease in the Ca\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}_{i}^{2+}\end{equation*}\end{document} required for half activation (Kd). These results show directly that BK channels can open with 0, 1, 2, 3, or 4 functional Ca-bowls, and that each subunit with a functional Ca-bowl contributes a stepwise increase to both the cooperativity of activation and the apparent Ca2+ affinity. A model with 0–4 high-affinity allosteric activators and four low-affinity allosteric activators was examined. In this model, Ca2+ bindings were independent of one another and the cooperativity arose from the joint action of the allosteric activators on the open–closed equilibrium. Although this model described well the major features of the experimental data, some differences between the observed and predicted results indicated that additional factors not included in the model also contribute to the cooperativity

Section: Discussionmentioning

confidence: 93%

Stepwise contribution of each subunit to the cooperative activation of BK channels by Ca ²⁺

Niu

Magleby

2002

“…The assertion of concerted pore opening implies that no intermediates exist along the pathway leading to the last channel opening transition and that all subunits switch from the final closed to the open state in an all-or-none fashion. Earlier evidence by others, using a tandem tetrameric channel construct, offers support for such sequential voltage-sensor gating transitions (15,16). No direct evidence, however, supporting a late concerted pore-opening transition has, thus far, been provided.…”

mentioning

confidence: 97%

“…channel inactivation and assembly (12)(13)(14). In only a few cases have such tandem-linked subunit constructs been used to ascertain the contribution of individual subunits to cooperativity in the function of the oligomeric protein (15)(16)(17). These studies, however, did not exploit the full potential of such constructs to decipher the contributions of intersubunit interactions to cooperativity in protein function.…”

mentioning

confidence: 99%

Direct analysis of cooperativity in multisubunit allosteric proteins

Zandany

Ovadia

Orr

et al. 2008

Allosteric regulation of protein function is a fundamental phenomenon of major importance in many cellular processes. Such regulation is often achieved by ligand-induced conformational changes in multimeric proteins that may give rise to cooperativity in protein function. At the heart of allosteric mechanisms offered to account for such phenomenon, involving either concerted or sequential conformational transitions, lie changes in intersubunit interactions along the ligation pathway of the protein. However, structurefunction analysis of such homooligomeric proteins by means of mutagenesis, although it provides valuable indirect information regarding (allosteric) mechanisms of action, it does not define the contribution of individual subunits nor interactions thereof to cooperativity in protein function, because any point mutation introduced into homooligomeric proteins will be present in all subunits. Here, we present a general strategy for the direct analysis of cooperativity in multisubunit proteins that combines measurement of the effects on protein function of all possible combinations of mutated subunits with analysis of the hierarchy of intersubunit interactions, assessed by using high-order double-mutant cyclecoupling analysis. We show that the pattern of high-order intersubunit coupling can serve as a discriminative criterion for defining concerted versus sequential conformational transitions underlying protein function. This strategy was applied to the particular case of the voltage-activated potassium channel protein (Kv) to provide compelling evidence for a concerted all-or-none activation gate opening of the Kv channel pore domain. An direct and detailed analysis of the contribution of high-order intersubunit interactions to cooperativity in the function of an allosteric protein has not previously been presented. activation gate ͉ allosteric enzymes ͉ double-mutant cycles ͉ voltage-activated potassium channels ͉ non-additivity A llosteric regulation of protein function is often achieved by changes in protein conformation induced by the binding of substrate or other ligand molecules (1). In multisubunit proteins, such conformational changes may give rise to cooperativity in ligand binding. Several mechanistic models, in particular the Monod-Wymann-Changeux (MWC) model (2) and the Koshland-Némethy-Filmer (KNF) model (3), were developed in the 1960s to describe cooperativity in ligand binding. In both models, cooperativity is explained by binding-induced conformational changes in the subunits of the protein that may be concerted (MWC), sequential (KNF), or a combination of both (4). No matter the type of conformational change, cooperativity in ligand binding in all allosteric models is manifested by changes in intersubunit interactions along the ligation pathway of the protein (1).Despite the general acceptance of intersubunit interactions playing a fundamental role in determining the magnitude of cooperativity in ligand binding by an allosteric enzyme, structure-function analysis of such proteins throu...

“…Recordings were carried out in the W434F mutation, which locks the channel in the nonconducting P-type inactivated state, but permits free motion of S4 (16,17). A single cysteine mutation was also introduced in the extracellular portion of the Shaker S4 segment (A359C) to allow for site-specific conjugation with tetramethylrhodammine-6-maleimide, an environmentally sensitive fluorophore, and monitor the movement of the voltage-sensing domain (18,19). Ligand 4 (200 M) was found to slow the rate of S4 motion at the top of the voltage-dependence curve where channels open (Fig.…”

Section: Inhibition Of Kv1 Channels By Calix[4]arene Compoundsmentioning

confidence: 99%

Calix[4]arene-based conical-shaped ligands for voltage-dependent potassium channels

Martos

Bell

Santos

et al. 2009

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