Hypotonic stretch has been reported in other cell types to stretch the cell membrane.
28 29 38 This procedure increases cell membrane tension caused by cell swelling. Whole-cell experiments were performed to evaluate outward currents in response to hypotonic shock in LC cells.
Figure 2Ashows typical traces of whole-cell currents recorded from an LC cell under isotonic stretch (left panel), hypotonic stretch (middle panel), and after washout (right panel). Cells displayed outward current rectification at positive membrane potentials. The summary of whole-cell outward currents obtained from 23 cells is represented as the current-voltage (I–V) relationship seen in
Figure 2B . The reversal potential was approximately 0 mV, which is close to the theoretical Nernst potential for the K
+-selective channel in our experimental conditions. Whole-cell mean conductance measured at +100 mV was increased from 114 ± 5 pS under isotonic (control) conditions to 193 ± 6 pS under hypotonic conditions (
n = 23;
P < 0.05). Mean maximal current density measured at Vp = +100 mV was 13.3 ± 11 pA/pF before and 99 ± 8.5 pA/pF (at Vp = +100 mV) after hypotonic stretch, and the difference was statistically different (
n = 23;
P < 0.05). The current increase was reversed on return to isotonic conditions (washout) 28.6 ± 8.7 pA/pF (at Vp = +100 mV;
n = 23). These current profiles resembled maxi-K
+ currents
23 26 ; therefore, we used the potassium channel blocker BaCl
2 to test whether the outward currents observed in LC cells were K
+ currents and found that they were. Tetraethylammonium (TEA) was then used to test whether the K
+ currents were Ca
2+-dependent, and iberiotoxin (Ibtx) was used to test the maxi-K
+ signature. Comparable studies have been reported by Fernández-Fernández et al.
23 in human bronchial epithelial cells. Adding K
+ channel blockers, TEA (5 mM;
n = 8;
P < 0.02) and Ba
2+(5 mM;
n = 8;
P < 0.02) or Ibtx (100 nM;
n = 12;
P < 0.02) resulted in a large inhibition of the outward current
(Fig. 3) , indicating that most of the outward current in LC cells occurs through K
+ channels.