Tuning the mechanosensitivity of a BK channel by changing the linker length

Some large-conductance Ca(2+) and voltage-activated K(+)(BK) channels are activated by membrane stretch. However, the mechanism of mechano-gating of the BK channels is still not well understood. Previous studies have led to the proposal that the linker-gating ring complex functions as a passive spring, transducing the force generated by intracellular Ca(2+) to the gate to open the channel. This raises the question as to whether membrane stretch is also transmitted to the gate of mechanosensitive (MS) BK channels via the linker-gating complex. To study this, we changed the linker length in the stretch-activated BK channel (SAKCaC), and examined the effect of membrane stretch on the gating of the resultant mutant channels. Shortening the linker increased, whereas extending the linker reduced, the channel mechanosensitivity both in the presence and in the absence of intracellular Ca(2+). However, the voltage and Ca(2+) sensitivities were not significantly altered by membrane stretch. Furthermore, the SAKCaC became less sensitive to membrane stretch at relatively high intracellular Ca(2+) concentrations or membrane depolarization. These observations suggest that once the channel is in the open-state conformation, tension on the spring is partially released and membrane stretch is less effective. Our results are consistent with the idea that membrane stretch is transferred to the gate via the linker-gating ring complex of the MS BK channels.     

BK通道的生物物理特性与门控及其最新研究进展

BK_(Ca)通道是细胞膜上受Ca~(2+)和膜电位双重调控的离子通道,其与细胞信号系统偶联并发挥着重要作用,该通道高度表达于高等动物的多种组织.最近的研究证实,在心肌细胞膜上存在力敏感BK通道并参与了心脏收缩与舒张的调控.本文将介绍BK通道与L-型钙通道功能上的耦合,心肌细胞质膜力敏感BK通道门控和功能的研究,以及对基底刚度的响应.这有助于更好地理解力敏感离子通道相关心脏疾病的病理和生理学基础.     

Functional coupling of ion channels in cellular mechanotransduction

The major players in the processes of cellular mechanotransduction are considered to be mechanosensitive (MS) or mechano-gated ion channels. Non-selective Ca²⁺-permeable channels, whose activity is directly controlled by membrane stretch (stretch-activated channels, SACs) are ubiquitously present in mammalian cells of different origin. Ca²⁺ entry mediated by SACs presumably has a significant impact on various Ca²⁺-dependent intracellular and membrane processes. It was proposed that SACs could play a crucial role in the different cellular reactions and pathologies, including oncotransformation, increased metastatic activity and invasion of malignant cells. In the present work, coupling of ion channels in transformed fibroblasts in course of stretch activation was explored with the use of patch-clamp technique. The combination of cell-attached and inside-out single-current experiments showed that Ca²⁺ influx via SACs triggered the activity of Ca²⁺-sensitive K⁺ channels indicating functional compartmentalization of different channel types in plasma membrane. Importantly, the analysis of single channel behavior demonstrated that K⁺ currents could be activated by the rise of intracellular calcium but displayed no direct mechanosensitivity. Taken together, our data imply that local changes in Ca²⁺ concentration due to SAC activity may provide a functional link between various Ca²⁺-dependent molecules in the processes of cellular mechanotransduction.     

大电导钾离子通道(BK)结构与功能的研究进展

大电导钙离子激活钾通道(BK)是细胞膜上唯一接受细胞内Ca2+和膜电位双重调控的离子通道.最新发表的关于BK通道电镜结构及其胞质功能域的晶体结构的文章,第一次展示了BK通道各亚基的组装,并证实通道各功能域在通道门控机制中存在紧密的相互作用.近年来,针对BK通道的功能调节及其门控动力学模拟的研究取得较多进展,有助于更好地理解BK通道发挥生理功能的门控机制,并揭示BK通道相关疾病的病理生理学基础.     

Ca2+-activated K channels in parotid acinar cells: The functional basis for the hyperpolarized activation of BK channels

Fluid secretion relies on a close interplay between Ca²⁺-activated Cl and K channels. Salivary acinar cells contain both large conductance, BK, and intermediate conductance, IK1, K channels. Physiological fluid secretion occurs with only modest (<500 nM) increases in intracellular Ca²⁺ levels but BK channels in many cell types and in heterologous expression systems require very high concentrations for significant activation. We report here our efforts to understand this apparent contradiction. We determined the Ca²⁺ dependence of IK1 and BK channels in mouse parotid acinar cells. IK1 channels activated with an apparent Ca²⁺ affinity of about 350 nM and a hill coefficient near 3. Native parotid BK channels activated at similar Ca²⁺ levels unlike the BK channels in other cell types. Since the parotid BK channel is encoded by an uncommon splice variant, we examined this clone in a heterologous expression system. In contrast to the native parotid channel, activation of this expressed “parslo” channel required very high levels of Ca²⁺. In order to understand the functional basis for the special properties of the native channels, we analyzed the parotid BK channel in the context of the horrigan-Aldrich model of BK channel gating. We found that the shifted activation of parotid BK channels resulted from a hyperpolarizing shift of the voltage dependence of voltage sensor activation and channel opening and included a large change in the coupling of these two processes.Key words: ion channels, Ca²⁺-activated K channels, maxi-K channels, IK1 channels     

Effects of mechanical stretch on the functions of BK and L-type Ca2+ channels in vascular smooth muscle cells

It is well recognized that pathologically increased mechanical stretch plays a critical role in vascular remodeling during hypertension. However, how the stretch modulates the functions of ion channels of vascular smooth muscle cells (VSMCs) remains to be elucidated. Here, we demonstrated the effects of mechanical stretch on the activity of large conductance calcium, voltage-activated potassium (BK) and L-type Ca²⁺ channels. In comparison with 5% stretch (physiological), 15% stretch (pathological) upregulated the current density of L-type Ca²⁺ and BK channels as well as the frequency and amplitude of calcium oscillation in VSMCs. 15% stretch also increased the open probability and mean open time of the BK channel compared with 5% stretch. BK and L-type Ca²⁺ channels participated in the mechanical stretch-modulated calcium oscillation. Our results suggested that during hypertension, pathological stretch altered the activity of BK and L-type Ca²⁺ channels and manipulated the calcium oscillation of VSMCs.     

Impaired Ca2+-dependent activation of large-conductance Ca2+-activated K+ channels in the coronary artery smooth muscle cells of Zucker Diabetic Fatty rats

The large-conductance Ca²⁺-activated K⁺ (BK) channels play an important role in the regulation of cellular excitability in response to changes in intracellular metabolic state and Ca²⁺ homeostasis. In vascular smooth muscle, BK channels are key determinants of vasoreactivity and vital-organ perfusion. Vascular BK channel functions are impaired in diabetes mellitus, but the mechanisms underlying such changes have not been examined in detail. We examined and compared the activities and kinetics of BK channels in coronary arterial smooth muscle cells from Lean control and Zucker Diabetic Fatty (ZDF) rats, using single-channel recording techniques. We found that BK channels in ZDF rats have impaired Ca²⁺ sensitivity, including an increased free Ca²⁺ concentration at half-maximal effect on channel activation, a reduced steepness of Ca²⁺ dose-dependent curve, altered Ca²⁺-dependent gating properties with decreased maximal open probability, and a shortened mean open-time and prolonged mean closed-time durations. In addition, the BK channel β-subunit-mediated activation by dehydrosoyasaponin-1 (DHS-1) was lost in cells from ZDF rats. Immunoblotting analysis confirmed a 2.1-fold decrease in BK channel β₁-subunit expression in ZDF rats, compared with that of Lean rats. These abnormalities in BK channel gating lead to an increase in the energy barrier for channel activation, and may contribute to the development of vascular dysfunction and complications in type 2 diabetes mellitus.     

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 Ca²⁺ 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 Ca²⁺ 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.     

Mechanosensitive Properties of BK Channels from Embryonic Rat Neuroepithelium

The mechanosensitive properties of large-conductance Ca²⁺-activated K⁺ (BK) channels from embryonic rat neuroepithelium were investigated with the cell-attached and inside-out configurations of the patch-clamp technique. The channels were activated in both recording configurations by negative pressures applied to the patch electrode, but reversal of the effect was total and immediate in inside-out patches whereas it was incomplete and delayed in on-cell patches. This mechanosensitivity was not mediated by Ca²⁺ ions or fatty acids, suggesting that it is an intrinsic property of these channels. Cytochalasin B did not affect mechanosensitivity in on-cell patches but increased it in inside-out patches. Kinetic studies showed that stretch increased the mean open time of the channels and decreased the slowest time constant of their closed-time distributions. The present as well as previous results suggest complex interactions between embryonic BK channels and their membranous and submembranous environment. Received: 1 February 1996/Revised: 25 March 1996     

Structural Determinants of Phosphatidylinositol 4,5-Bisphosphate (PIP2) Regulation of BK Channel Activity through the RCK1 Ca2+ Coordination Site

Big or high conductance potassium (BK) channels are activated by voltage and intracellular calcium (Ca²⁺). Phosphatidylinositol 4,5-bisphosphate (PIP₂), a ubiquitous modulator of ion channel activity, has been reported to enhance Ca²⁺-driven gating of BK channels, but a molecular understanding of this interplay or even of the PIP₂ regulation of this channel''s activity remains elusive. Here, we identify structural determinants in the KDRDD loop (which follows the αA helix in the RCK1 domain) to be responsible for the coupling between Ca²⁺ and PIP₂ in regulating BK channel activity. In the absence of Ca²⁺, RCK1 structural elements limit channel activation through a decrease in the channel''s PIP₂ apparent affinity. This inhibitory influence of BK channel activation can be relieved by mutation of residues that (a) connect either the RCK1 Ca²⁺ coordination site (Asp³⁶⁷ or its flanking basic residues in the KDRDD loop) to the PIP₂-interacting residues (Lys³⁹² and Arg³⁹³) found in the αB helix or (b) are involved in hydrophobic interactions between the αA and αB helix of the RCK1 domain. In the presence of Ca²⁺, the RCK1-inhibitory influence of channel-PIP₂ interactions and channel activity is relieved by Ca²⁺ engaging Asp³⁶⁷. Our results demonstrate that, along with Ca²⁺ and voltage, PIP₂ is a third factor critical to the integral control of BK channel activity.     

Interaction between residues in the Mg2+-binding site regulates BK channel activation

As a unique member of the voltage-gated potassium channel family, a large conductance, voltage- and Ca²⁺-activated K⁺ (BK) channel has a large cytosolic domain that serves as the Ca²⁺ sensor, in addition to a membrane-spanning domain that contains the voltage-sensing (VSD) and pore-gate domains. The conformational changes of the cytosolic domain induced by Ca²⁺ binding and the conformational changes of the VSD induced by membrane voltage changes trigger the opening of the pore-gate domain. Although some structural information of these individual functional domains is available, how the interactions among these domains, especially the noncovalent interactions, control the dynamic gating process of BK channels is still not clear. Previous studies discovered that intracellular Mg²⁺ binds to an interdomain binding site consisting of D99 and N172 from the membrane-spanning domain and E374 and E399 from the cytosolic domain. The bound Mg²⁺ at this narrow interdomain interface activates the BK channel through an electrostatic interaction with a positively charged residue in the VSD. In this study, we investigated the potential interdomain interactions between the Mg²⁺-coordination residues and their effects on channel gating. By introducing different charges to these residues, we discovered a native interdomain interaction between D99 and E374 that can affect BK channel activation. To understand the underlying mechanism of the interdomain interactions between the Mg²⁺-coordination residues, we introduced artificial electrostatic interactions between residues 172 and 399 from two different domains. We found that the interdomain interactions between these two positions not only alter the local conformations near the Mg²⁺-binding site but also change distant conformations including the pore-gate domain, thereby affecting the voltage- and Ca²⁺-dependent activation of the BK channel. These results illustrate the importance of interdomain interactions to the allosteric gating mechanisms of BK channels.     

Arachidonic acid effect on the allosteric gating mechanism of BK (Slo1) channels associated with the β1 subunit

Arachidonic acid (AA) is a fatty acid involved in the modulation of several ion channels. Previously, we reported that AA activates the high conductance Ca²⁺- and voltage-dependent K⁺ channel (BK) in vascular smooth muscle depending on the expression of the auxiliary β1 subunit. Here, using the patch-clamp technique on BK channel co-expressed with β1 subunit in a heterologous cell expression system, we analyzed whether AA modifies the three functional modules involved in the channel gating: the voltage sensor domain (VSD), the pore domain (PD), and the intracellular calcium sensor domain (CSD). We present evidence that AA activates BK channel in a direct way, inducing VSD stabilization on its active configuration observed as a significant left shift in the Q-V curve obtained from gating currents recordings. Moreover, AA facilitates the channel opening transitions when VSD are at rest, and the CSD are unoccupied. Furthermore, the activation was independent of the intracellular Ca²⁺ concentration and reduced when the BK channel was co-expressed with the Y74A mutant of the β1 subunit. These results allow us to present new insigths in the mechanism by which AA modulates BK channels co-expressed with its auxiliary β1 subunit.     

Allosteric Gating of a Large Conductance Ca-activated K+ Channel

Large-conductance Ca-activated potassium channels (BK channels) are uniquely sensitive to both membrane potential and intracellular Ca²⁺. Recent work has demonstrated that in the gating of these channels there are voltage-sensitive steps that are separate from Ca²⁺ binding steps. Based on this result and the macroscopic steady state and kinetic properties of the cloned BK channel mslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca²⁺ in the gating of the mslo channel (Cui, J., D.H. Cox, and R.W. Aldrich. 1997. J. Gen. Physiol. In press.). This scheme supposes that the channel exists in two main conformations, closed and open. The conformational change between closed and open is voltage dependent. Ca²⁺ binds to both the closed and open conformations, but on average binds more tightly to the open conformation and thereby promotes channel opening. Here we describe the basic properties of models of this form and test their ability to mimic mslo macroscopic steady state and kinetic behavior. The simplest form of this scheme corresponds to a voltage-dependent version of the Monod-Wyman-Changeux (MWC) model of allosteric proteins. The success of voltage-dependent MWC models in describing many aspects of mslo gating suggests that these channels may share a common molecular mechanism with other allosteric proteins whose behaviors have been modeled using the MWC formalism. We also demonstrate how this scheme can arise as a simplification of a more complex scheme that is based on the premise that the channel is a homotetramer with a single Ca²⁺ binding site and a single voltage sensor in each subunit. Aspects of the mslo data not well fitted by the simplified scheme will likely be better accounted for by this more general scheme. The kinetic schemes discussed in this paper may be useful in interpreting the effects of BK channel modifications or mutations.     

Involvement of TRP channels in the signal transduction of bradykinin in human osteoblasts

Bradykinin (BK), a mediator of pain and inflammation, is involved in bone metabolism. We have previously reported that BK increased the synthesis of interleukin-6 and prostaglandin E₂ via phosphorylation of ERK1/2 in human osteoblasts, SaM-1. In the present study, we investigated the signal transduction pathway of BK focusing on intracellular Ca²⁺ kinetics in SaM-1 cells. Bath-applied BK increased intracellular Ca²⁺ concentration through the activation of B₂ receptors. Removal of extracellular Ca²⁺ attenuated the effects of BK. Additionally, thapsigargin, endoplasmic reticulum Ca²⁺ pump inhibitor, completely inhibited BK-induced increase of intracellular Ca²⁺. These results suggested that bath-applied BK activated store-operated Ca²⁺ channels (SOCCs) following Ca²⁺ store depletion via B₂ receptor. Although the molecular components of SOCCs have yet to be conclusively identified in all cell types, recent studies demonstrated that transient receptor potential canonical (TRPC) channels are candidates for them. TRPC1, TRPC3, TRPC4 and TRPC6 were expressed in SaM-1 cells and inhibitors of TRP channel, 2-aminoethoxydiphenyl borate, GdCl₃, LaCl₃ and flufenamic acid, inhibited the effects of BK. These findings suggested that BK activated SOCCs and induced Ca²⁺ influx via B₂ receptor in human osteoblasts. Molecular components of the SOCCs are suggested to be TRPC channels.     

IL-1β modulate the Ca2+-activated big-conductance K channels (BK) via reactive oxygen species in cultured rat aorta smooth muscle cells

The large conductance Ca²⁺-activated K⁺ (BK) channel, abundantly expressed in vascular smooth muscle cells, plays a critical role in controlling vascular tone. Activation of BK channels leads to membrane hyperpolarization and promotes vasorelaxation. BK channels are activated either by elevation of the intracellular Ca²⁺ concentration or by membrane depolarization. It is also regulated by a diversity of vasodilators and vasoconstrictors. Interleukin-1β (IL-1β) is one of the cytokines that play important roles in the development and progression of a variety of cardiovascular diseases. The effects of IL-1β on vascular reactivity are controversial, and little is known about the modulation of BK channel function by IL-1β. In this study, we investigated how IL-1β modulates BK channel function in cultured arterial smooth muscle cells (ASMCs), and examined the role of H₂O₂ in the process. We demonstrated that IL-1β had biphasic effects on BK channel function and membrane potential of ASMCs, that is both concentration and time dependent. IL-1β increased BK channel-dependent K⁺ current and hyperpolarized ASMCs when applied for 30 min. While long-term (24–48 h) treatment of IL-1β resulted in decreased expression of α-subunit of BK channel, suppressed BK channel activity, decreased BK channel-dependent K⁺ current and depolarization of the cells. H₂O₂ scavenger catalase completely abolished the early effect of IL-1β, while it only partly diminished the long-term effect of IL-1β. These results may provide important molecular mechanisms for therapeutic strategies targeting BK channel in inflammation-related diseases.     

Four Ca2+ Ions Activate TRPM2 Channels by Binding in Deep Crevices near the Pore but Intracellularly of the Gate

TRPM2 is a tetrameric Ca²⁺-permeable channel involved in immunocyte respiratory burst and in postischaemic neuronal death. In whole cells, TRPM2 activity requires intracellular ADP ribose (ADPR) and intra- or extracellular Ca²⁺, but the mechanism and the binding sites for Ca²⁺ activation remain unknown. Here we study TRPM2 gating in inside-out patches while directly controlling intracellular ligand concentrations. Concentration jump experiments at various voltages and Ca²⁺ dependence of steady-state single-channel gating kinetics provide unprecedented insight into the molecular mechanism of Ca²⁺ activation. In patches excised from Xenopus laevis oocytes expressing human TRPM2, coapplication of intracellular ADPR and Ca²⁺ activated ∼50-pS nonselective cation channels; K_1/2 for ADPR was ∼1 µM at saturating Ca²⁺. Intracellular Ca²⁺ dependence of TRPM2 steady-state opening and closing rates (at saturating [ADPR] and low extracellular Ca²⁺) reveals that Ca²⁺ activation is a consequence of tighter binding of Ca²⁺ in the open rather than in the closed channel conformation. Four Ca²⁺ ions activate TRPM2 with a Monod-Wymann-Changeux mechanism: each binding event increases the open-closed equilibrium constant ∼33-fold, producing altogether 10⁶-fold activation. Experiments in the presence of 1 mM of free Ca²⁺ on the extracellular side clearly show that closed channels do not sense extracellular Ca²⁺, but once channels have opened Ca²⁺ entering passively through the pore slows channel closure by keeping the “activating sites” saturated, despite rapid continuous Ca²⁺-free wash of the intracellular channel surface. This effect of extracellular Ca²⁺ on gating is gradually lost at progressively depolarized membrane potentials, where the driving force for Ca²⁺ influx is diminished. Thus, the activating sites lie intracellularly from the gate, but in a shielded crevice near the pore entrance. Our results suggest that in intact cells that contain micromolar ADPR a single brief puff of Ca²⁺ likely triggers prolonged, self-sustained TRPM2 activity.     

State-dependent inhibition of BK channels by the opioid agonist loperamide

Large-conductance Ca²⁺-activated K⁺ (BK) channels control a range of physiological functions, and their dysfunction is linked to human disease. We have found that the widely used drug loperamide (LOP) can inhibit activity of BK channels composed of either α-subunits (BKα channels) or α-subunits plus the auxiliary γ1-subunit (BKα/γ1 channels), and here we analyze the molecular mechanism of LOP action. LOP applied at the cytosolic side of the membrane rapidly and reversibly inhibited BK current, an effect that appeared as a decay in voltage-activated BK currents. The apparent affinity for LOP decreased with hyperpolarization in a manner consistent with LOP behaving as an inhibitor of open, activated channels. Increasing LOP concentration reduced the half-maximal activation voltage, consistent with relative stabilization of the LOP-inhibited open state. Single-channel recordings revealed that LOP did not reduce unitary BK channel current, but instead decreased BK channel open probability and mean open times. LOP elicited use-dependent inhibition, in which trains of brief depolarizing steps lead to accumulated reduction of BK current, whereas single brief depolarizing steps do not. The principal effects of LOP on BK channel gating are described by a mechanism in which LOP acts as a state-dependent pore blocker. Our results suggest that therapeutic doses of LOP may act in part by inhibiting K⁺ efflux through intestinal BK channels.     

Emerging Role of the Calcium-Activated,Small Conductance,SK3 K+ Channel in Distal Tubule Function: Regulation by TRPV4

The Ca²⁺-activated, maxi-K (BK) K⁺ channel, with low Ca²⁺-binding affinity, is expressed in the distal tubule of the nephron and contributes to flow-dependent K⁺ secretion. In the present study we demonstrate that the Ca²⁺-activated, SK3 (K_Ca2.3) K⁺ channel, with high Ca²⁺-binding affinity, is also expressed in the mouse kidney (RT-PCR, immunoblots). Immunohistochemical evaluations using tubule specific markers demonstrate significant expression of SK3 in the distal tubule and the entire collecting duct system, including the connecting tubule (CNT) and cortical collecting duct (CCD). In CNT and CCD, main sites for K⁺ secretion, the highest levels of expression were along the apical (luminal) cell membranes, including for both principal cells (PCs) and intercalated cells (ICs), posturing the channel for Ca²⁺-dependent K⁺ secretion. Fluorescent assessment of cell membrane potential in native, split-opened CCD, demonstrated that selective activation of the Ca²⁺-permeable TRPV4 channel, thereby inducing Ca²⁺ influx and elevating intracellular Ca²⁺ levels, activated both the SK3 channel and the BK channel leading to hyperpolarization of the cell membrane. The hyperpolarization response was decreased to a similar extent by either inhibition of SK3 channel with the selective SK antagonist, apamin, or by inhibition of the BK channel with the selective antagonist, iberiotoxin (IbTX). Addition of both inhibitors produced a further depolarization, indicating cooperative effects of the two channels on Vm. It is concluded that SK3 is functionally expressed in the distal nephron and collecting ducts where induction of TRPV4-mediated Ca²⁺ influx, leading to elevated intracellular Ca²⁺ levels, activates this high Ca²⁺-affinity K⁺ channel. Further, with sites of expression localized to the apical cell membrane, especially in the CNT and CCD, SK3 is poised to be a key pathway for Ca²⁺-dependent regulation of membrane potential and K⁺ secretion.     

Identification of Changes in the Functional Profile of the Cardiac Ryanodine Receptor Caused by the Coupled Gating Phenomenon

The objective of this work was to identify and further characterize potential changes in the functional profile of the cardiac ryanodine receptor (RyR2) channel caused by the coupled gating phenomenon. By reconstituting an ion channel into a planar lipid membrane, we showed that coupled RyR2 channels were activated by cytosolic Ca²⁺ with similar efficacy and potency as reported for the single RyR2 channel. In contrast, all examined parameters of gating kinetics were affected by the functional interaction between channels. Ignoring brief closings during main open events, the average open and closed times were considerably prolonged and the frequency of opening was reduced. Interestingly, when luminal Ca²⁺ was used as a charge carrier, Ca²⁺-activated coupled RyR2 channels did not exhibit a sudden switch from slow to fast gating kinetics at an open probability of 0.5 as reported for the single RyR2 channel. Regarding flicker gating, the average closed time was significantly shorter and the frequency of closing was greatly enhanced. Furthermore, in contrast to the single RyR2 channel, both parameters for coupled channels were independent of cytosolic Ca²⁺. Selected permeation properties of coupled RyR2 channels were comparable to those found for the single RyR2 channel. The Ca²⁺ current amplitude-luminal Ca²⁺ relationship displayed a simple saturation and the channel selectivity for Ba²⁺ and Ca²⁺ ions was similar. Our results suggest that the major targets influenced by coupled gating are likely the gates of individual RyR2 channels recruited into a functional complex, thus ensuring the correlation of Ca²⁺ fluxes.