Kinetic states and modes of single large-conductance calcium-activated potassium channels in cultured rat skeletal muscle (original) (raw)
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The Journal of physiology, 1983
The stochastic properties of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle cells were studied using the patch-clamp technique. The distribution of all open intervals was described by the sum of two exponential distributions of short and long mean open time, suggesting at least two major open-channel states. Increasing the concentration of Ca at the inner membrane surface, [Ca]i, increased the mean duration of the long open distribution, while having little effect on the mean duration of the short open distribution. The frequency of openings to each distribution increased with [Ca]i. The rate of increase was a much steeper function of [Ca]i for openings in the long open distribution than for openings in the short open distribution; about 80% of the openings were to the long open distribution with 0.1 microM-Cai, increasing to 97% with 1 microM-Cai (+ 30 mV). These results suggest that openings in both open distributions are Ca-dependen...
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...
Burst kinetics of single calcium-activated potassium channels in cultured rat muscle
The Journal of Physiology, 1983
Burst kinetics of single Ca-activated K channels in excised patches of surface membrane from cultured rat muscle were studied using the patch-clamp technique. 2. Channel activity was separated into bursts using a calculated gap derived from the distribution of shut intervals. Shut intervals greater than the calculated gap were taken as gaps between bursts. 3. The distribution of burst duration was described as the sum of two exponential with mean durations of about 0-8 and 24 msec (1 /SM-Ca1, + 20 mV), suggesting two classes of bursts (short and long). 4. The composition of short and long bursts was determined from comparisons of the distributions of open intervals, unit bursts (bursts of single openings), and openings/burst. 5. Short bursts consisted mainly of single openings to the open channel state of short mean lifetime. Long bursts consisted ofone or more openings to the (compound) open-channel state of long mean lifetime, plus, in fewer than 70 % of the long bursts, one or more openings to the short open-channel state. 6. The frequency of occurrence of bursts from each class first increased and then decreased with increasing [Ca]i, with the number of long bursts increasing at a greater rate than the number of short bursts. 7. The number of openings/short burst was relatively independent of [Ca]i, while the number of openings/long burst increased, often more than linearly, with increasing [Ca]j. This increase arose almost entirely from an increase in openings to the long open state. 8. These results suggest that openings to the long open state typically require the binding of three or more Ca ions, and openings to the short open state typically require the binding of at least one Ca ion. This is the case whether the openings occur in isolation as bursts of single openings or in bursts composed of both types of openings. An obvious burst of channel activity would occur when the channel opens and closes several times without losing all its bound Ca.
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+ ...
Potassium channels from normal and denervated mouse skeletal muscle fibers
Muscle & Nerve, 1993
The properties of singles K+ channels in normal and denervated muscles were compared using the "patch-clamp" technique. Single channels were recorded from vesicles obtained by stretching bundles of normal and denervated extensor digitorium longus (EDL) muscles. The most frequently observed channel in normal muscles was a high conductance (266 pS) Ca+ +activated K+ channel. Although channel density, as estimated by patch recording, showed a significant decrease in denervated muscles, no differences were found in conductance and gating properties. Another voltage-dependent K+ channel (81 pS) was only recorded from normal muscles, but never from denervated ones. In addition, a 35 pS conductance was recorded from both normal and denervated fibers. This channel displayed neither voltage dependence nor sensitivity to tetraethylammonium (TEA). In contrast, another TEAinsensitive (16 pS) channel was recorded only from denervated muscles. We conclude that denervation induces significant changes in the distribution and expression of K+ channels in mammalian skeletal muscles. 0 1993 John Wiley & Sons, Inc.
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 ].
Kinetic properties of calcium channels of twitch muscle fibres of the frog
The Journal of physiology, 1983
Calcium currents (ICa) were recorded in frog skeletal muscle fibres using the three-micro-electrode voltage-clamp technique. The sartorius muscle was bathed in TEA methanesulphonate saline with 350 mM-sucrose. 5 mM-3,4-diaminopyridine was added to the saline to minimize K+ currents. The I-V relationship for peak Ca2+ currents showed that ICa was detected at -40 mV and reached a maximum value at ca. -10 mV. No net inward current was recorded at potentials positive to ca. +40 mV. Remaining K+ currents (IK) were recorded by replacing 10 mM-Ca2+ with 5.5 mM-Co2+. They were not noticeably time-dependent up to +20 mV and would tend to diminish the amplitude of ICa without greatly affecting its time course. ICa tail currents could be separated from non-linear capacity currents. Tail currents were measured 5 msec after repolarization and extrapolated to the end of the pulse. ICa tail-current amplitudes at EK were measured with pulses of different durations. The envelope of tail-current ampl...
Methods in Enzymology, 1999
High-conductance Ca2+-activated K+ (maxi-K+) channels are activated by both membrane depolarization and binding of Ca 2+ to sites at the intracellular face of the channel. Maxi-K+ channels are present in both electrically excitable and nonexcitable cells, and display high conductance and selectivity for K+. These channels are involved in regulation of the excitation-contraction coupling process in smooth muscle, as well as in control of transmitter release from neuroendocrine tissues. The pharmacology of maxi-K+ channels has been developed during the last few years and efforts are continuing to identify novel and selective modulators of this channel family. This chapter discusses procedures developed to purify maxi-K+ channels from smooth muscle tissues, as well as the way purified preparation can be reconstituted into liposomes for determination of channel activity. The chapter also discusses ways of obtaining protein sequence information from components of the maxi-K+ channel preparation that are useful for obtaining full-length cDNA clones of these proteins.