Localization of the K+ Lock-In and the Ba2+ Binding Sites in a Voltage-gated Calcium-modulated Channel: Implications for Survival of K+ Permeability (original) (raw)
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Localization of the K Lock-in and the Ba Binding Sites in a Voltage-Gated Calcium-Modulated Channel
The Journal of General Physiology
Using Ba2+ as a probe, we performed a detailed characterization of an external K+ binding site located in the pore of a large conductance Ca2+-activated K+ (BKCa) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba2+ blocks BKCa channels and decreasing external K+ using a K+ chelator, (+)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba2+-blocked events. Average Ba2+ dwell time changes from 10 s at 10 mM external K+ to 100 ms in the limit of very low [K+]. Using a model where external K+ binds to a site hindering the exit of Ba2+ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 mircoM for K) at this lock-in site. We also found that BK(Ca) channels enter into a long-lasting nonconductive state when the external [K+] is reduced below 4 microM using the crown ether. Channel activity can be recovered by adding K+, Rb+, Cs+, or NH4+ to the externa...
The Journal of general …, 1999
Using Ba 2 ϩ as a probe, we performed a detailed characterization of an external K ϩ binding site located in the pore of a large conductance Ca 2 ϩ-activated K ϩ (BK Ca) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba 2 ϩ blocks BK Ca channels and decreasing external K ϩ using a K ϩ chelator, (ϩ)-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba 2 ϩ-blocked events. Average Ba 2 ϩ dwell time changes from 10 s at 10 mM external K ϩ to 100 ms in the limit of very low [K ϩ ]. Using a model where external K ϩ binds to a site hindering the exit of Ba 2 ϩ toward the external side (Neyton, J., and C. Miller. 1988. J. Gen. Physiol. 92:549-568), we calculated a dissociation constant of 2.7 M for K ϩ at this lock-in site. We also found that BK Ca channels enter into a long-lasting nonconductive state when the external [K ϩ ] is reduced below 4 M using the crown ether. Channel activity can be recovered by adding K ϩ , Rb ϩ , Cs ϩ , or NH 4 ϩ to the external solution. These results suggest that the BK Ca channel stability in solutions of very low [K ϩ ] is due to K ϩ binding to a site having a very high affinity. Occupancy of this site by K ϩ avoids the channel conductance collapse and the exit of Ba 2 ϩ toward the external side. External tetraethylammonium also reduced the Ba 2 ϩ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K ϩ. It is explained in terms of a model in which the conduction pore contains Ba 2 ϩ , K ϩ , and tetraethylammonium simultaneously, with the K ϩ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (
Voltage-gated Calcium-modulated Channel Implications for Survival of K 1 Permeability
Using Ba 2 ϩ as a probe, we performed a detailed characterization of an external K ϩ binding site located in the pore of a large conductance Ca 2 ϩ -activated K ϩ (BK Ca ) channel from skeletal muscle incorporated into planar lipid bilayers. Internal Ba 2 ϩ blocks BK Ca channels and decreasing external K ϩ using a K ϩ chelator, ( ϩ )-18-Crown-6-tetracarboxylic acid, dramatically reduces the duration of the Ba 2 ϩ -blocked events. Average Ba 2 ϩ dwell time changes from 10 s at 10 mM external K ϩ to 100 ms in the limit of very low [K ϩ ]. Using a model where external K ϩ binds to a site hindering the exit of Ba 2 ϩ toward the external side (Neyton, J., and C. Miller. 1988. J . Gen . Physiol . 92:549-568), we calculated a dissociation constant of 2.7 M for K ϩ at this lock-in site. We also found that BK Ca channels enter into a long-lasting nonconductive state when the external [K ϩ ] is reduced below 4 M using the crown ether. Channel activity can be recovered by adding K ϩ , Rb ϩ , Cs ϩ , or NH 4 ϩ to the external solution. These results suggest that the BK Ca channel stability in solutions of very low [K ϩ ] is due to K ϩ binding to a site having a very high affinity. Occupancy of this site by K ϩ avoids the channel conductance collapse and the exit of Ba 2 ϩ toward the external side. External tetraethylammonium also reduced the Ba 2 ϩ off rate and impeded the channel from entering into the long-lasting nonconductive state. This effect requires the presence of external K ϩ . It is explained in terms of a model in which the conduction pore contains Ba 2 ϩ , K ϩ , and tetraethylammonium simultaneously, with the K ϩ binding site located internal to the tetraethylammonium site. Altogether, these results and the known potassium channel structure (Doyle, D.A., J.M. Cabral, R.A. Pfuetzner, A. Kuo, J.M. Gulbis, S.L. Cohen, B.T. Chait, and R. MacKinnon. 1998. Science. 280:69-77) imply that the lock-in site and the Ba 2 ϩ sites are the external and internal ion sites of the selectivity filter, respectively. key words: K Ca channel • multiple occupancy • barium block • tetraethylammonium • lipid bilayer
Interaction of internal Ba2+ with a cloned Ca(2+)-dependent K+ (hslo) channel from smooth muscle
Journal of General Physiology, 1996
We have studied potassium currents through a cloned Ca2+-dependent K § channel (hs/o) from human myometrium, Currents were recorded in inside-out macropatches from membranes of Xenopus laevis oocytes. In particular, the, inactivation-like, process that these channels show at high positive potentials was assessed in order tcr explore its molecular nature. This current inhibition conferred a bell shape to the current-voltage curves. The kinetic and voltage dependence of this process suggested the possibility of a Ba 2+ block. There were the following similarities between the'inactivation process observed at zero-added Ba 2+ and the internal Ba 2 § block of hslo channels: (a) in the steady state, the voltage dependence of the current inhibition observed at zero-added Ba 2+ was the same as the voltage dependence of the Ba 2+ block; (b) the time constant fdr recovery from current decay at zero-added Ba 2+ was the same as the time constant for current recovery from Ba 2+ blockade; and (c) current decay was largely suppressed in both cases by adding a Ba 2+ chelator [ (+)-18-crown-6-tetracarboxylic acid] to the internal solution. In our experimental conditions, we determined that the~Ka, for the complex chelator-Ba 2+ is 1.6 • 10 -l~ M. We conclude that the current decay observed at zero-added Ba 2+ to the internal solution is due to contaminant Ba 2+ present in our solutions (N70 nM) and not to an intrinsic gating process. The Ba 2+ blocking reaction in hslo channels is bimolecular. Ba 2+ binds to a site (K~ = 0.36 + 0.05 mM at zero applied voltage) that senses 92 + 25% of the potential drop from the internal membrane surface.
The Journal of general physiology, 1983
The interaction of Cat' and Ba t+ with a Ca 2 '-activated K + channel from rabbit skeletal muscle membranes is studied in planar lipid bilayers. At [Ca 21] 2_100 AM in the cis side (the side to which the vesicles are added) and at positive voltages, the channel kinetics consisted of bursts of activity interrupted by long periods of quiescence. We found that the reciprocal of the mean burst time increases linearly with [Ca"], whereas the mean time for the quiescent (closed) periods is independent of [Ca 2+]. The number of quiescent periods is reduced by increasing [K +]. Micromolar amounts of cis Ba t + do not activate the channel, but induce similar "slow" closings. Also, in this case, the mean burst time is inversely proportional to the [Ba 2+] and the mean closed time is independent of [Ba 2+]. Raising [K +] either symmetrically or only in the trans side relieved the Ba t+ effect. trans Bat' also induces changes in the slow kinetics, but in millimolar amounts. These results suggest that the quiescent periods correspond to a channel blocked by a Ba ion. The voltage dependence of the cis blockade indicates that the Ba t+ binding site is past the middle of the membrane field. The similarities in the slow kinetics induced by Ca2' and Ba t + suggest that Ca 2+ blocks the channel by binding to the same site. However, binding of Ca 2+ to the site is 10 5-fold weaker .
Mechanisms of Cs+ blockade in a Ca2+-activated K+ channel from smooth muscle
Biophysical Journal, 1987
Large unitary conductance Ca2+-activated K+ channels from smooth muscle membrane were incorporated into phospholipid planar bilayers, and the blockade induced by internally and externally applied Cs+ was characterized. Internal Cs+ blockade is voltage dependent and can be explained on the basis of a Cs' binding to a site that senses 54% of the applied voltage, with an apparent dissociation constant, Kd(O), of 70 mM. On the other hand, external Cs+ blocks the channel in micromolar amounts, and the voltage dependence of blockade is a function of Cs+ concentration. The fractional electrical distance can be as large as 1.4 at 10 mM Cs+. This last result suggests that the channel behaves as a multi-ion pore. At large negative voltages the I-V relationships in the presence of external Cs+ show an upturn, indicating relief of Cs+ block. External Cs+ blockade is relieved by increasing the internal K+ concentration, but can be enhanced by increasing the external K+. All the characteristics of external Cs' block can be explained by a model that incorporates a "knock-on" of Cs+ by K+.
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+ ...
Proceedings of the National Academy of Sciences, 1982
Addition of membrane vesicles prepared from transverse tubule (T-tubule) membranes of rabbit skeletal muscle to the aqueous phase of a planar lipid bilayer induces a stepwise increase in conductance. This conductance is both voltage and Ca2' dependent. At 1 mM Ca2+, the steady-state conductance is maximal at voltages higher than +20 mV and decreases for more negative voltages. (Voltages refer to the side to which the vesicles are added, cia.) Decreasing the Ca2+ concentration reversibly shifts the conductance-voltage curve toward the right along the voltage axis. Furthermore, Ca2+ can activate the conductance only if added to the cis compartment. Neither Mg2+, Ba2+, nor Cd2+ can activate the conductance induced by T-tubule vesicles. Addition of 5 mM tetraethylammonium ion to the trans, but not the cis, side abolishes the T-tubule-induced conductance. The Ca2+_ dependent conductance appears as a consequence ofionic channel formation. Single-channel activity appears in bursts followed by periods of time in which the channel remains "silent". The conductance of the oven channel averages 226 pS in 0.1 M KCl and is voltage and Ca + independent. However, the fraction of time that the channel remains in the open state is voltage and Ca2+ dependent in a manner that parallels the voltage and Ca2+ dependence ofthe multichannel membrane. The channel is 6.6 times more permeable to K+ than to Na+ and is impermeable to C1-.
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 ].