Characterization of calcium-activated potassium channels in single smooth muscle cells using the patch-clamp technique

Abstract

Single-channel currents were recorded with the patch-clamp technique from freshly dissociated vertebrate smooth muscle cells from the stomach of Bufo marinus. Of the variety of channels observed, one displayed a large linear conductance of 250 pS (in symmetric 130 mM KCl) which in excised patches was shown to be highly K+ selective. The probability of the channel being open (Po) increased when [Ca2+]i was elevated and/or when the membrane potential was made more positive. Thus, the features of this channel resemble the large-conductance Ca2+-activated K+ channel found in a wide variety of cell types. The voltage sensitivity of the channel was studied in detail. For patches containing a single large-conductance channel a plot of Po versus membrane potential followed the Boltzmann relationship. Increasing [Ca2+]i shifted this plot to the left along the voltage axis to more negative potentials. Both the mean closed time and mean open time varied with potential as a single exponential with almost all of the voltage sensitivity of Po residing in the mean closed time. These results were verified with a series of experiments carried out at low Po (less than 0.1) in patches containing multiple (N) large-conductance channels. Here the ln (NPo) was a linear function of potential with an inverse slope of 9 mV. Almost all of the potential sensitivity lay in the mean closed time the natural log of which was also a linear function of potential with an inverse slope 11 mV in magnitude. The characteristics of this channel as well as the appearance of several of them in almost every patch suggest that they underlie the large peak outward macroscopic current found with whole-cell voltage-clamp studies.

Ionic channels in smooth muscle studied with patch-clamp methods.

“…shortening of the closed state and partially to prolong the open state. The effect of depolarization was found to be the same as that of increasing [Ca]; in the toad stomach cells [53].…”

“…The presence of K channels controlled by intracellular Ca (Ca-activated K channels) has been demonstrated in several smooth muscles by recording currents flowing through a single channel with the patch-clamp method (toad and frog stomach [10,52,53,63]; rabbit portal vein [28,29]; rabbit jejunum and guinea-pig mesenteric artery [7,8]; canine trachea [37]). From the effect of altering ionic composition of the media, it has been shown that these channels are highly selective to K ions, activated by a low concentration of cytoplasmic Ca (0.01-1 µM), and have a very high conductance (200-270 pS at a symmetrical high K concentration).…”

“…The high conductance is likely to indicate that the channel has a large pathway, but the high ionic selectivity probably results from the presence of a narrow point in the pathway [35]. In the cells of the toad stomach, the density of Ca-activated K channel was estimated to be approximately one channel/pmt [53]. This channel's physiological role is to participate in the refractory period and possibly also in the generation of spontaneous activity [61].…”

Visualization of Ca2+entry through single stretch-activated cation channels

“…Therefore, over the range of patch membrane potentials that occurred during our experiments, the fraction of the SAC current that is carried by Ca 2ϩ appears to be relatively independent of this parameter. Ca 2ϩ entering the cell through SACs is sufficient to cause a substantial local increase in the free [Ca 2ϩ ] (see discussion), as indicated by large-conductance Ca 2ϩ -activated K ϩ (BK Ca ) channel (19) openings in the same patch (Fig. 5).…”

Natural Bile Acids and Synthetic Analogues Modulate Large Conductance Ca2+-activated K+ (BKCa) Channel Activity in Smooth Muscle Cells

“…Over the range of potentials used here, the voltage dependence of activation can be described by a Boltzmann relationship such that a plot of ln NP o as a function of membrane potential is linear at low values of P o . The reciprocal of the slope ("slope factor"), a measure of the voltage dependence of channel gating, is the potential required to produce an e-fold change in P o (Singer and Walsh, 1987). Data from an inside-out patch (Fig.…”