Voltage and Ca2+ Activation of Single Large-Conductance Ca2+-activated K+ Channels Described by a Two-Tiered Allosteric Gating Mechanism (original) (raw)
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The Journal of physiology, 1991
1. The Ca(2+)-dependent kinetics of large-conductance Ca(2+)-activated K+ channels from cultured rat skeletal muscle were studied with the patch clamp technique. Data were collected in the absence of Na+ and Mg2+, which can alter the kinetics. About 2 x 10(5) open and shut intervals were analysed from each of five different excised membrane patches containing a single active channel. Analysis was restricted to activity in the normal mode, which includes 96% of the intervals. 2. The open probability (Popen) and dwell-time distributions of open and shut intervals were obtained at three to four different [Ca2+]i for each of the channels. Popen data were also obtained from some multichannel patches. 3. Increasing [Ca2+]i increased Popen. At a pH of 7.0 the Hill coefficient was 3.7 +/- 0.8 (range of 3.0-5.0) and a Popen of 0.5 occurred at 14 +/- 7 microM [Ca2+]i (K0.5) for data obtained at +30 mV (n = 6). At a pH of 7.2 the Hill coefficient was 3.0 +/- 0.5 (range of 2.2-3.7) and K0.5 was...
The Journal of physiology, 1988
1. Kinetic states and modes of a large-conductance Ca2+-activated K+ channel in excised patches of membrane from cultured rat skeletal muscle were studied with the patch clamp technique. Up to 10(6) open and shut intervals were analysed from each of seven different excised membrane patches containing a single channel. 2. Plots of the mean durations of consecutive groups of ten to fifty open and shut intervals were made to assess kinetic stability of the channel. Occasional abrupt decreases in the mean open interval duration from normal to different distinct levels, which were maintained for hundreds to thousands of consecutive intervals, indicated entry of the channel into different modes. 3. Four different kinetic modes were identified: normal mode, which included 96% of the intervals; intermediate open mode with 3.2% of the intervals; brief open mode with 0.5% of the intervals; and buzz mode with 0.1% of the intervals. The mean open interval durations were 61% of normal during the...
Journal of General Physiology, 1983
The gating kinetics of a Ca2+-activated K+ channel from adult rat muscle plasma membrane are studied in artificial planar bilayers. Analysis of single-channel fluctuations distinguishes two Ca2+- and voltage-dependent processes: (a) short-lived channel closure (less than 1 ms) events appearing in a bursting pattern; (b) opening and closing events ranging from one to several hundred milliseconds in duration. The latter process is studied independently of the first and is denoted as the primary gating mode. At constant voltage, the mean open time of the primary gating mode is a linear function of the [Ca2+], whereas the mean closed time is a linear function of the reciprocal [Ca2+]. In the limits of zero and infinite [Ca2+], the mean open and the mean closed times are, respectively, independent of voltage. These results are predicted by a kinetic scheme consisting of the following reaction steps: (a) binding of Ca2+ to a closed state; (b) channel opening; (c) binding of a second Ca2+ ...
The Journal of General Physiology, 1999
The Ca 2 ϩ -dependent gating mechanism of large-conductance calcium-activated K ϩ (BK) channels from cultured rat skeletal muscle was examined from low (4 M) to high (1,024 M) intracellular concentrations of calcium (Ca 2 ϩ i ) using single-channel recording. Open probability ( P o ) increased with increasing Ca 2 ϩ i ( K 0.5 11.2 Ϯ 0.3 M at ϩ 30 mV, Hill coefficient of 3.5 Ϯ 0.3), reaching a maximum of ف 0.97 for Ca 2 ϩ i ف 100 M. Increasing Ca 2 ϩ i further to 1,024 M had little additional effect on either P o or the single-channel kinetics. The channels gated among at least three to four open and four to five closed states at high levels of Ca 2 ϩ i ( Ͼ 100 M), compared with three to four open and five to seven closed states at lower Ca 2 ϩ i . The ability of kinetic schemes to account for the single-channel kinetics was examined with simultaneous maximum likelihood fitting of twodimensional (2-D) dwell-time distributions obtained from low to high Ca 2 ϩ i . Kinetic schemes drawn from the 10state Monod-Wyman-Changeux model could not describe the dwell-time distributions from low to high Ca 2 ϩ i . Kinetic schemes drawn from Eigen's general model for a ligand-activated tetrameric protein could approximate the dwell-time distributions but not the dependency (correlations) between adjacent intervals at high Ca 2 ϩ i . However, models drawn from a general 50 state two-tiered scheme, in which there were 25 closed states on the upper tier and 25 open states on the lower tier, could approximate both the dwell-time distributions and the dependency from low to high Ca 2 ϩ i . In the two-tiered model, the BK channel can open directly from each closed state, and a minimum of five open and five closed states are available for gating at any given Ca 2 ϩ i . A model that assumed that the apparent Ca 2 ϩ -binding steps can reach a maximum rate at high Ca 2 ϩ i could also approximate the gating from low to high Ca 2 ϩ i . The considered models can serve as working hypotheses for the gating of BK channels. key words: BK channel • K Ca channel • Monod-Wyman-Changeux • Eigen • Markov
The Journal of General Physiology, 1998
Mechanisms for the Ca2+-dependent gating of single large-conductance Ca2+-activated K+channels from cultured rat skeletal muscle were developed using two-dimensional analysis of single-channel currents recorded with the patch clamp technique. To extract and display the essential kinetic information, the kinetic structure, from the single channel currents, adjacent open and closed intervals were binned as pairs and plotted as two-dimensional dwell-time distributions, and the excesses and deficits of the interval pairs over that expected for independent pairing were plotted as dependency plots. The basic features of the kinetic structure were generally the same among single large-conductance Ca2+-activated K+channels, but channel-specific differences were readily apparent, suggesting heterogeneities in the gating. Simple gating schemes drawn from the Monod- Wyman-Changeux (MWC) model for allosteric proteins could approximate the basic features of the Ca2+dependence of the kinetic stru...
Allosteric Gating of a Large Conductance Ca-activated K+ Channel
The Journal of General Physiology, 1997
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 channelmslo, we have recently proposed a general kinetic scheme to describe the interaction between voltage and Ca2+in the gating of themslochannel (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 mimicm...
The Journal of General Physiology, 2000
Over the past few years, it has become clear that an important mechanism by which large-conductance Ca2+-activated K+channel (BKCa) activity is regulated is the tissue-specific expression of auxiliary β subunits. The first of these to be identified, β1, is expressed predominately in smooth muscle and causes dramatic effects, increasing the apparent affinity of the channel for Ca2+10-fold at 0 mV, and shifting the range of voltages over which the channel activates −80 mV at 9.1 μM Ca2+. With this study, we address the question: which aspects of BKCagating are altered by β1 to bring about these effects: Ca2+binding, voltage sensing, or the intrinsic energetics of channel opening? The approach we have taken is to express the β1 subunit together with the BKCaα subunit inXenopusoocytes, and then to compare β1's steady state effects over a wide range of Ca2+concentrations and membrane voltages to those predicted by allosteric models whose parameters have been altered to mimic changes ...
Large conductance Ca 2-activated K (BK) channel: Activation by Ca 2 and voltage
Biol. Res, 2006
Large conductance Ca 2+ -activated K + (BK) channels belong to the S4 superfamily of K + channels that include voltage-dependent K + (Kv) channels characterized by having six (S1-S6) transmembrane domains and a positively charged S4 domain. As Kv channels, BK channels contain a S4 domain, but they have an extra (S0) transmembrane domain that leads to an external NH 2 -terminus. The BK channel is activated by internal Ca 2+ , and using chimeric channels and mutagenesis, three distinct Ca 2+ -dependent regulatory mechanisms with different divalent cation selectivity have been identified in its large COOH-terminus. Two of these putative Ca 2+ -binding domains activate the BK channel when cytoplasmic Ca 2+ reaches micromolar concentrations, and a low Ca 2+ affinity mechanism may be involved in the physiological regulation by Mg 2+ . The presence in the BK channel of multiple Ca 2+ -binding sites explains the huge Ca 2+ concentration range (0.1 μM-100 μM) in which the divalent cation influences channel gating. BK channels are also voltage-dependent, and all the experimental evidence points toward the S4 domain as the domain in charge of sensing the voltage. Calcium can open BK channels when all the voltage sensors are in their resting configuration, and voltage is able to activate channels in the complete absence of Ca 2+ . Therefore, Ca 2+ and voltage act independently to enhance channel opening, and this behavior can be explained using a two-tiered allosteric gating mechanism.
Biophysical Journal, 1998
The Ca 2ϩ sensitivity of large conductance Ca 2ϩ -and voltage-activated K ϩ channels (BK V,Ca ) has been determined in situ in freshly isolated myocytes from the guinea pig urinary bladder. In this study, in situ denotes that BK V,Ca channel activity was recorded without removing the channels from the cell. By combining patch clamp recording in the cell-attached configuration and microfluorometry of fura-2, we were able to correlate BK V,Ca channel activity with changes in cytoplasmic intracellular [Ca 2ϩ ] ([Ca 2ϩ ] i ). The latter were induced by ionomycin, an electroneutral Ca 2ϩ ionophore. At 0 mV, the Hill coefficient (n H ) and the [Ca 2ϩ ] i to attain half of the maximal BK V,Ca channel activity (Ca 50 ) were 8 and 1 M, respectively. The data suggest that this large Hill number was not a consequence of the difference between the nearmembrane [Ca 2ϩ ] ([Ca 2ϩ ] s ) and the bulk [Ca 2ϩ ] i , indicated by fura-2. High Hill numbers in the activation by Ca 2ϩ of BK V,Ca channels have been seen by different groups (e.g., filled squares in of Silberberg, S. D., A. Lagrutta, J. P. Adelman, and K. L. . Biophys. J. 70:2640 -2651. However, such high n H has always been considered a peculiarity rather than the rule. This work shows that a high Ca 2ϩ cooperativity is the normal situation for BK V,Ca channels in myocytes from guinea pig urinary bladder. Furthermore, the Ca 50 did not display any significant variation among different channels or cells. It was also evident that BK V,Ca channel activity could decrease in elevated [Ca 2ϩ ] i , either partially or completely. This work implies that the complete activation of BK V,Ca channels occurs with a smaller increment in [Ca 2ϩ ] s than previously expected from in vitro characterization of the Ca 2ϩ sensitivity of these channels. Additionally, it appears that the activity of BK V,Ca channels in situ does not strictly follow changes in near-membrane [Ca 2ϩ ].
The Journal of General Physiology, 1999
Coexpression of the β subunit (KV,Caβ) with the α subunit of mammalian large conductance Ca2+- activated K+(BK) channels greatly increases the apparent Ca2+sensitivity of the channel. Using single-channel analysis to investigate the mechanism for this increase, we found that the β subunit increased open probability (Po) by increasing burst duration 20–100-fold, while having little effect on the durations of the gaps (closed intervals) between bursts or on the numbers of detected open and closed states entered during gating. The effect of the β subunit was not equivalent to raising intracellular Ca2+in the absence of the beta subunit, suggesting that the β subunit does not act by increasing all the Ca2+binding rates proportionally. The β subunit also inhibited transitions to subconductance levels. It is the retention of the BK channel in the bursting states by the β subunit that increases the apparent Ca2+sensitivity of the channel. In the presence of the β subunit, each burst of ope...