Biophysical and pharmacological characterization of stretch-activated K(+) channels.

Mechanosensitive ion channels were studied in various preparations (Lymnaea stagnalis cultured neurons, and ventricular heart cells, Aplysia californica mechanosensitive neurons, and Xenopus oocytes) using cell-attached and excised inside-out patch-clamp techniques to characterize their biophysical and pharmacological properties. It has been demonstrated that a well studied K-selective channel which plays an important role in "cellular learning" in Aplysia mechanosensory neurons, is stretch-activated. Using multi-channel patch analysis I have provided further evidence that this channel is indeed a stretch-activated (SA) K$\sp+$ channel similar to those found in virtually all molluscan neurons. I demonstrated that the number of SAK$\sp+$ channels in a patch is finite and the response saturable. Kinetic stationarity was tested. In patches which passed the stationarity tests and in which channels were found to be independent and identical, the kinetics were determined. The kinetic analysis yielded results consistent with the hypothesis that S-channels (those which responded to FMRF-amide and to 5-HT) were identical to SA K$\sp+$ channels (those which were stretch-activatable). I then tested the hypothesis that all molluscan SA K$\sp+$ channels were S-like in that they were modulated by neurotransmitters via second messengers. I found that the inherent variability of normal channel activity prohibited adequate testing of this hypothesis even when a homogeneous population of cells containing similar SA K$\sp+$ channels was examined. Dynamics. When care was taken to obtain gigaseals in a gentle fashion (10 mmHG suction) and disruption to the patch integrity was minimized, SA K$\sp+$ channels in Lymnaca neurons exhibited dynamic characteristics. These characteristics included a delayed response to rapid suction steps. The delay lessened with suction steps of increasing magnitude. The delay phenomenon was fragile in nature in that the delay was lost with repeated suction stimuli of the same magnitude of by non-gentle seal formation. The delay was voltage independent. Cells left in culture longer (4, 5, and 6 days as opposed to 1 and 2 days), responded to similar stimuli with longer delays. I found SA K$\sp+$ channels to be blocked by extracellular TEA (IC$\sb{50} = {\sim}50$mM), but not by intracellular TEA, extracellular diltiazem or Gd$\sp{3+}$. Extracellular amiloride and quinidine blocked SA K$\sp+$ channels with IC$\sb{50}$s of $\sim$2.15 mM and $\sim$0.75 mM respectively. Ethanol (3%) had no apparent effect on SA K$\sp+$ channels yet reduced the efficacy of quinidine block. Permeation. Given that Lymnaea SA K$\sp+$ channels seem to be K-selective channels which are specialized to avoid sensing membrane tension under normal conditions, I characterized the pore properties with the intent to make comparisons to other SA channels and K$\sp+$ channels whose structure/function relations are fairly well understood. I found that the pore of SA K$\sp+$ channels has an affinity for inward and outward moving K$\sp+$ currents of 28.1 $\pm$ 5.0 mM and 91.9 $\pm$ 6.7 mM. SA K$\sp+$ channels exhibited an anomalous mole fraction effect with mixtures of Rb$\sp+$ indicative of a multi-ion pore. (Abstract shortened by UMI.