XB-ART-15108
J Gen Physiol
1998 Apr 01;1114:565-81. doi: 10.1085/jgp.111.4.565.
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Gating of recombinant small-conductance Ca-activated K+ channels by calcium.
Hirschberg B, Maylie J, Adelman JP, Marrion NV.
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Small-conductance Ca-activated K+ channels play an important role in modulating excitability in many cell types. These channels are activated by submicromolar concentrations of intracellular Ca2+, but little is known about the gating kinetics upon activation by Ca2+. In this study, single channel currents were recorded from Xenopus oocytes expressing the apamin-sensitive clone rSK2. Channel activity was detectable in 0.2 micro M Ca2+ and was maximal above 2 micro M Ca2+. Analysis of stationary currents revealed two open times and three closed times, with only the longest closed time being Ca dependent, decreasing with increasing Ca2+ concentrations. In addition, elevated Ca2+ concentrations resulted in a larger percentage of long openings and short closures. Membrane voltage did not have significant effects on either open or closed times. The open probability was approximately 0.6 in 1 micro M free Ca2+. A lower open probability of approximately 0.05 in 1 micro M Ca2+ was also observed, and channels switched spontaneously between behaviors. The occurrence of these switches and the amount of time channels spent displaying high open probability behavior was Ca2+ dependent. The two behaviors shared many features including the open times and the short and intermediate closed times, but the low open probability behavior was characterized by a different, long Ca2+-dependent closed time in the range of hundreds of milliseconds to seconds. Small-conductance Ca- activated K+ channel gating was modeled by a gating scheme consisting of four closed and two open states. This model yielded a close representation of the single channel data and predicted a macroscopic activation time course similar to that observed upon fast application of Ca2+ to excised inside-out patches.
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Species referenced: Xenopus
Genes referenced: kcnn2 rps6ka3
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Figure 3. Voltage independence of kinetic parameters. (A) Open probability is shown as a function of voltage for three patches individually. Values differed between patches, due to different intracellular Ca2+ concentrations (▴, ♦, 1 μM; ▪, 0.8 μM) and different types of channel behavior (high and low open probability behavior, see later), but for all patches open probability was essentially voltage independent. The line was drawn for display purposes only and has no physical meaning. (B) Average short and long open times for the patches in A. Data are shown as mean ± SD, and the lines represent the best fit of the data to single exponential functions. (C) Relative amplitude of the short open-time component as a function of voltage shown individually for each patch in A. The line was drawn for display purposes only. Data from each patch are represented by the same symbol in A, C, E, and F. (D) Short and intermediate closed times for the patches in A. Data are shown as mean ± SD and the lines represent the best fit of the data to exponential functions. (E) Long closed times shown for two patches individually and fit to a single exponential (solid lines). In the patch with the highest open probability, too few events were associated with the longest time constant to reliably fit this component. (F) Relative amplitudes of the short (closed symbols) and intermediate (open symbols) closed-time components plotted individually for the patches in A. |
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Figure 1. Calcium dependence of SK channel activity. (A) Recordings from an inside-out patch in different calcium concentrations at a holding potential of −80 mV. Traces show 2-s periods of the single channel recordings used to calculate the open probabilities in B. Channel openings are shown as downward deflections and calculated free intracellular [Ca2+] is noted on the left. Channel current was slightly smaller in the trace recorded in 5 μM Ca2+ due to an off-set of ∼11 mV that occurred upon switching to the EGTA-free solution (see methods). (B) Open probability as a function of intracellular calcium concentration. 30–60-s steady state recordings in 0.6, 0.8, 1.0, and 5.0 μM Ca2+ were used to calculate the open probability. Least squares fitting of the data to the Hill equation of the form Po = Po,max · [Ca2+]n/(EC50n+ [Ca2+]n) (where Po,max is the maximal open probability in saturating [Ca2+], and other terms have their usual meanings) yielded Po,max = 81%, EC50 = 0.74 μM, and n = 2.2. |
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Figure 10. Simulation of SK channel activity using a sequential gating scheme. Traces were obtained from simulations of the gating scheme presented here. In this scheme, forward transitions between closed states are calcium dependent and are expressed as absolute rate constants in units of seconds−1 per micromolar−1, all other rates are in units of seconds−1. The rates for transitions between open and closed states were derived from the measured open and closed times, whereas the rates for transitions between closed states reflect the longest closed time as well as the relative contributions of different time constants. Simulations used the same acquisition rate and filter frequency as was used in single channel recordings, and simulated data were analyzed in the same manner as recorded data. Resulting open-duration histograms are shown on the left, closed-duration histograms on the right. Relative amplitudes of open-duration components were in 1.0 μM Ca2+: 1.2 ms, 29%; 12.6 ms, 71%; in 0.4 μM Ca2+: 1.0 ms, 51%; 12.2 ms, 49%. Relative amplitudes of closed-duration components were in 1.0 μM Ca2+: 0.8 ms, 71%; 6.3 ms, 21%; 18.8 ms, 8%; in 0.4 μM Ca2+: 0.8 ms, 49%; 4.4 ms, 33%; 53.9 ms, 18%. |
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Figure 11. Simulation of low open probability behavior. Traces were obtained from simulations of the modified gating scheme shown in this figure. Calcium-dependent rates are lower by a factor of 6.7 compared with the gating scheme in Fig. 7 and are expressed as absolute rate constants in units of seconds−1 per micromolar−1, whereas all other rates are in units of seconds−1. Open-duration histograms are shown on the left, closed-duration histograms on the right. Relative amplitudes of open-duration components were in 1.0 μM Ca2+: 1.0 ms, 73%; 11.4 ms, 27%; in 0.4 μM Ca2+: 1.0 ms, 89%; 13.4 ms, 11%. Relative amplitudes of closed-duration components were in 1.0 μM Ca2+: 0.8 ms, 32%; 5.4 ms, 44%; 234 ms, 24%; in 0.4 μM Ca2+: 1.2 ms, 19%; 5.2 ms, 48%; 1,410 ms, 33%. |
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Figure 7. Calcium dependence of low and high open probability behavior. (A) Absolute recording time (in seconds) from all single channel patches combined during which low (dark gray) and high (light gray) open probability was observed. An open probability of <10% and the appearance of a closed-time constant >100 ms were taken as indicators of low open probability (see Fig. 8). The inset shows the fraction of the total recording time during which high open probability was observed and a fit of the data to the Hill equation yielding an EC50 of 0.52 μM and a Hill coefficient of 4.1. (B, •) The single channel open probability for a representative patch containing a channel displaying only high activity (Po(high)). (□) The “effective open probability” obtained by multiplying the single channel open probability by the relative time channels spent in high open probability behavior (Po(high) · f(high)) (Fig. 7 A, inset). Lines represent fits to the Hill equation, yielding an EC50 of 0.43 μM and a Hill coefficient of 3.3 for the single channel open probability, and an EC50 of 0.63 μM and a Hill coefficient of 3.6 for the “effective open probability.” |
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Figure 8. Low open probability SK channel behavior in different concentrations of calcium. Traces shown are from a single channel patch exposed to 1.0, 0.6, and 0.4 μM intracellular Ca2+. Scaling is the same for all traces, and open probabilities are noted above each trace. Open-duration histograms are displayed on the left and closed-duration histograms on the right. Solid lines and time constants represent maximum likelihood fits to the sum of two or three exponential components (see Fig. 3). Relative amplitudes of open- duration components were in 1.0 μM Ca2+: 0.9 ms, 71%; 9.8 ms, 29%; in 0.6 μM Ca2+: 0.8 ms, 80%; 8.3 ms, 20%; in 0.4 μM Ca2+: 0.9 ms, 83%; 11.6 ms, 17%. Relative amplitudes of closed-duration components were in 1.0 μM Ca2+: 0.7 ms, 41%; 6.2 ms, 27%; 293 ms, 32%; in 0.6 μM Ca2+: 0.9 ms, 39%; 6.5 ms, 27%; 489 ms, 34%; in 0.4 μM Ca2+: 0.9 ms, 31%; 7.5 ms, 26%; 1,050 ms, 43%. |
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Figure 4. SK channel activity in different concentrations of Ca2+. Data shown are from a single channel patch voltage clamped at −80 mV. Scaling is the same for all traces and open probabilities are noted above each trace. Open-duration histograms are displayed on the left and closed-duration histograms on the right underneath the corresponding 2-s traces. Solid lines and time constants represent maximum likelihood fits to the sum of two or three exponential components shown for each component separately as well as for the sum. Relative amplitudes of open-duration components were in 1.0 μM Ca2+: 1.1 ms, 34%; 11.9 ms, 66%; in 0.6 μM Ca2+: 1.0 ms, 43%; 10.1 ms, 57%; in 0.4 μM Ca2+: 1.1 ms, 51%; 13.5 ms, 49%. Relative amplitudes of closed-duration components were in 1.0 μM Ca2+: 0.8 ms, 71%; 4.2 ms, 19%; 20.0 ms, 10%; in 0.6 μM Ca2+: 0.7 ms, 67%; 4.0 ms, 23%; 32.6 ms, 10%; in 0.4 μM Ca2+: 0.8 ms, 63%; 4.8 ms, 25%; 60.2 ms, 12%. |
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Figure 5. Calcium dependence of channel open and closed durations. Data were from five excised patches, each containing a single channel displaying high open probability behavior (see text). Except in C, lines were drawn to illustrate the Ca2+ independence of the kinetic parameters and are not intended to have physical meaning. (A) Average short and long open times were independent of intracellular Ca2+ concentration. (B) Average short and intermediate closed-times were independent of Ca2+ concentration. (C) Ca2+ dependence of the average long closed times. Linear regression analysis of the means yielded a slope of −66 ms μM−1. (D) The relative amplitude of the short open-time component decreased as a function of Ca2+ concentration. (E) The relative amplitude of the short closed-time component increased with increasing Ca2+ concentration. (F) Increasing Ca2+ concentrations lead to a decrease in the relative amplitude of the long closed-time component. |
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Figure 6. Low and high open probability behavior. The traces in A show 20 s of a recording from a single channel patch voltage clamped at −100 mV starting 11 s after excision into 1.0 μM free Ca2+. The parallel lines mark a 32-s break in the continuously acquired recording. Approximately 19 s after patch excision (arrow), channel open probability decreased from ∼60% to <10%. During the same continuous recording, ∼63 s after patch excision (second arrow), channel open probability spontaneously increased to its starting level. B shows the open probability during 1-s intervals as a function of time after patch excision with the arrows indicating the switches in channel activity (same as A). |
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Figure 9. Calcium dependence of kinetic parameters describing low open probability behavior. Data are from three single channel patches except in 0.2 μM Ca2+ (one patch) and in 0.6 μM Ca2+ (two patches) during periods of low open probability activity. (A) Average short and long open times were independent of intracellular Ca2+ concentration. Linear regression analysis of the data is shown as the solid lines and illustrates the Ca2+ independence of the open-time constants. (B) Average short and intermediate closed times were independent of Ca2+ concentration. Solid lines represent linear regression analysis. (C) The long closed times during low open probability activity was Ca2+ dependent, similar to what was observed during high channel open probability (compare Fig. 5). Linear regression analysis yielded a slope of −2,460 ms μM−1. (D) The relative amplitude of the short open-time component decreased as a function of Ca2+ concentration. Data are shown on a semi-logarithmic scale for comparison with Fig. 5 (high open probability behavior). The line in D–F is included for display purposes only. (E) The relative amplitude of the short closed-time component increased with increasing Ca2+ concentration. (F) Increasing Ca2+ concentrations lead to a decrease in the relative amplitude of the long closed-time component. |
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Figure 12. Correlations between successive openings. (A) Open-duration histograms for the patch in Fig. 4 exposed to 0.4 μM Ca2+ including all openings (left), openings after openings shorter than 1 ms (middle), and openings after openings longer than 10 ms (right). The lifetimes and relative amplitudes of the two components were 1.1 ms, 51%, and 13.5 ms for all openings; 1.1 ms, 80%, and 15.8 ms after short openings; and 1.3 ms, 26%, and 14.0 ms after long openings. Insets show corresponding histograms for simulations of the model in Fig. 10. The short and long components had lifetimes of 1.0 ms, 51%, and 12.2 ms for all openings; 1.0 ms, 77%, and 12.3 ms after short openings; and 1.1 ms, 19%, and 12.8 ms after long openings. (B) Open-duration histograms for the channel in Fig. 7 displaying low open probability behavior in 1.0 μM Ca2+. Open times were fit to two components with lifetimes of 0.9 ms, 71%, and 9.8 ms (left). Openings after openings shorter than 1 ms (middle) had lifetimes of 0.7 ms, 92%, and 8.8 ms. Openings after openings longer than 10 ms (right) were predominantly long but could not be fit due to the small number of events. Insets show corresponding histograms for the model in Fig. 11. The two components had lifetimes of 1.0 ms, 73%, and 11.4 ms for all openings; 1.0 ms, 91%, and 9.1 ms after short openings; and 1.2 ms, 24%, and 13.3 ms after long openings. |
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Figure 13. Time dependence of the macroscopic current activation. (A) An inside-out macropatch excised from an oocyte expressing SK2 was positioned near a solution interface formed by a continuous flow through a θ tube of 0 and 9.5 μM Ca2+. At a holding potential of −80 mV, Ca2+ was pulsed on and off by rapidly moving the solution interface across the patch pipette tip using a bimorph translator (Morgan Matroc Inc., Bedford, OH) attached to the θ tube. The rising and falling phase of activation and deactivation, respectively, were well described by single exponentials, yielding time constants of 4.9 and 58 ms. Overlaid on the data is a simulation of the model in Fig. 10. (B) Computer simulations of the gating scheme in Fig. 10 for 400 ms Ca2+-jumps from 0 to the Ca2+ concentration indicated above each trace. For each trace, the rising and falling phase of the activation and deactivation time course, respectively, were fit to single exponential functions with the fitted lines coinciding with the traces. The deactivation time course was independent of the Ca2+ concentration during the jump and gave a time constant of 76 ms. (C) Ca2+ dependence of the activation time course. Activation time-constants obtained from fitting the macroscopic current activation (○) and the simulated traces in B (•) were plotted as a function of Ca2+concentration. Over the range of 0.2 to 10 μM Ca2+, the Ca2+ dependence of the simulated data appeared linear and regression analysis yielded an apparent Ca2+-binding rate of 47 μM−1 s−1. |
References [+] :
Adelman,
Calcium-activated potassium channels expressed from cloned complementary DNAs.
1992, Pubmed,
Xenbase
Adelman, Calcium-activated potassium channels expressed from cloned complementary DNAs. 1992, Pubmed , Xenbase
Auerbach, Heterogeneous kinetic properties of acetylcholine receptor channels in Xenopus myocytes. 1986, Pubmed , Xenbase
Blatz, Single apamin-blocked Ca-activated K+ channels of small conductance in cultured rat skeletal muscle. , Pubmed
DiChiara, Distinct effects of Ca2+ and voltage on the activation and deactivation of cloned Ca(2+)-activated K+ channels. 1995, Pubmed , Xenbase
Donnelly, Single-channel currents from diethylpyrocarbonate-modified NMDA receptors in cultured rat brain cortical neurons. 1995, Pubmed
Goh, Pharmacological and physiological properties of the after-hyperpolarization current of bullfrog ganglion neurones. 1987, Pubmed
Grissmer, Ca(2+)-activated K+ channels in human leukemic T cells. 1992, Pubmed
Gurney, Activation of a potassium current by rapid photochemically generated step increases of intracellular calcium in rat sympathetic neurons. 1987, Pubmed
Hamill, Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. 1981, Pubmed
Hess, Different modes of Ca channel gating behaviour favoured by dihydropyridine Ca agonists and antagonists. , Pubmed
Horn, Estimating kinetic constants from single channel data. 1983, Pubmed
Ishii, A human intermediate conductance calcium-activated potassium channel. 1997, Pubmed , Xenbase
Köhler, Small-conductance, calcium-activated potassium channels from mammalian brain. 1996, Pubmed , Xenbase
Lancaster, Calcium-dependent current generating the afterhyperpolarization of hippocampal neurons. 1986, Pubmed
Lancaster, Photolytic manipulation of Ca2+ and the time course of slow, Ca(2+)-activated K+ current in rat hippocampal neurones. 1994, Pubmed
Lancaster, Potassium currents evoked by brief depolarizations in bull-frog sympathetic ganglion cells. 1987, Pubmed
Lancaster, Calcium activates two types of potassium channels in rat hippocampal neurons in culture. 1991, Pubmed
Lang, Large and small conductance calcium-activated potassium channels in the GH3 anterior pituitary cell line. 1987, Pubmed
Lang, Tetraethylammonium blockade of apamin-sensitive and insensitive Ca2(+)-activated K+ channels in a pituitary cell line. 1990, Pubmed
Latorre, Varieties of calcium-activated potassium channels. 1989, Pubmed
Madison, Control of the repetitive discharge of rat CA 1 pyramidal neurones in vitro. 1984, Pubmed
Magleby, Calcium dependence of open and shut interval distributions from calcium-activated potassium channels in cultured rat muscle. 1983, Pubmed
Marrion, Selective reduction of one mode of M-channel gating by muscarine in sympathetic neurons. 1993, Pubmed
McManus, Accounting for the Ca(2+)-dependent kinetics of single large-conductance Ca(2+)-activated K+ channels in rat skeletal muscle. 1991, Pubmed
McManus, Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle. 1988, Pubmed
Moczydlowski, Gating kinetics of Ca2+-activated K+ channels from rat muscle incorporated into planar lipid bilayers. Evidence for two voltage-dependent Ca2+ binding reactions. 1983, Pubmed
Neely, Two components of calcium-activated potassium current in rat adrenal chromaffin cells. 1992, Pubmed
Park, Ion selectivity and gating of small conductance Ca(2+)-activated K+ channels in cultured rat adrenal chromaffin cells. 1994, Pubmed
Patlak, Single glutamate-activated channels in locust muscle. 1979, Pubmed
Patlak, Opentime heterogeneity during bursting of sodium channels in frog skeletal muscle. 1986, Pubmed
Pennefather, Two distinct Ca-dependent K currents in bullfrog sympathetic ganglion cells. 1985, Pubmed
Rothberg, High Ca2+ concentrations induce a low activity mode and reveal Ca2(+)-independent long shut intervals in BK channels from rat muscle. 1996, Pubmed
Sah, Ca(2+)-activated K+ currents in neurones: types, physiological roles and modulation. 1996, Pubmed
Sah, Kinetic properties of a slow apamin-insensitive Ca(2+)-activated K+ current in guinea pig vagal neurons. 1993, Pubmed
Sah, Ca(2+)-activated K+ currents underlying the afterhyperpolarization in guinea pig vagal neurons: a role for Ca(2+)-activated Ca2+ release. 1991, Pubmed
Sah, Properties of channels mediating the apamin-insensitive afterhyperpolarization in vagal motoneurons. 1995, Pubmed
Sah, Channels underlying the slow afterhyperpolarization in hippocampal pyramidal neurons: neurotransmitters modulate the open probability. 1995, Pubmed
Schwindt, Calcium-dependent potassium currents in neurons from cat sensorimotor cortex. 1992, Pubmed
Silberberg, Wanderlust kinetics and variable Ca(2+)-sensitivity of Drosophila, a large conductance Ca(2+)-activated K+ channel, expressed in oocytes. 1996, Pubmed , Xenbase
Storm, Action potential repolarization and a fast after-hyperpolarization in rat hippocampal pyramidal cells. 1987, Pubmed
Valiante, Analysis of current fluctuations during after-hyperpolarization current in dentate granule neurones of the rat hippocampus. 1997, Pubmed
Wakamatsu, Transition of localization of the N-Myc protein from nucleus to cytoplasm in differentiating neurons. 1993, Pubmed
Wilson, Mode-switching of a voltage-gated cation channel is mediated by a protein kinase A-regulated tyrosine phosphatase. 1993, Pubmed
Yarom, Ionic currents and firing patterns of mammalian vagal motoneurons in vitro. 1985, Pubmed