Abstract
Changes in resting membrane potential (RMP) regulate membrane excitability. K+ conductance(s) are one of the main factors in regulating RMP. The functional role of K+ conductances has not been studied the in human gastric corpus smooth muscles (HGCS). To examine the role of K+ channels in regulation of RMP in HGCS we employed microelectrode recordings, patch-clamp, and molecular approaches. Tetraethylammonium and charybdotoxin did not affect the RMP, suggesting that BK channels are not involved in regulating RMP. Apamin, a selective small conductance Ca2+-activated K+ channel (SK) blocker, did not show a significant effect on the membrane excitability. 4-Aminopyridine, a Kv channel blocker, caused depolarization and increased the duration of slow wave potentials. 4-Aminopyridine also inhibited a delayed rectifying K+ current in isolated smooth muscle cells. End-product RT-PCR gel detected Kv1.2 and Kv1.5 in human gastric corpus muscles. Glibenclamide, an ATP-sensitive K+ channel (KATP) blocker, did not induce depolarization, but nicorandil, a KATP opener, hyperpolarized HGCS, suggesting that KATP are expressed but not basally activated. Kir6.2 transcript, a pore-forming subunit of KATP was expressed in HGCS. A low concentration of Ba2+, a Kir blocker, induced strong depolarization. Interestingly, Ba2+-sensitive currents were minimally expressed in isolated smooth muscle cells under whole-cell patch configuration. KCNJ2 (Kir2.1) transcript was expressed in HGCS. Unique K+ conductances regulate the RMP in HGCS. Delayed and inwardly rectifying K+ channels are the main candidates in regulating membrane excitability in HGCS. With the development of cell dispersion techniques of interstitial cells, the cell-specific functional significance will require further analysis.
Highlights
IN GASTRIC CORPUS AND ANTRUM, many studies have focused on the mechanisms of spontaneous electrical activity [4, 7, 9, 10, 25, 29, 33]
It has been shown that BK channel blockers [tetraethylammonium chloride (TEA) or charybdotoxin (ChTX)] did not affect the resting membrane potentials (RMP) in colonic smooth muscles. 4-Aminopyridine (4-AP, a Kv channel blocker) induces depolarization in colonic smooth muscle, suggesting that activation or inhibition of Kv channels can affect the changes in RMP [17, 18]
Electrical activity with use of intracellular microelectrodes was reported in human gastric smooth muscles [4, 7, 25], there is no report about the role of Kϩ conductances on membrane excitability in human gastric corpus smooth muscle (HGCS)
Summary
Dispersed smooth muscle cells were prepared from HGCS strips with use of Ca2ϩ-free. Dissected muscle strips were incubated for 40 –50 min at 37°C in a Ca2ϩ-free solution (2 ml) containing collagenase Dispersed smooth muscle cells were stored at 4°C in Ca2ϩ-free solution. The tissue chamber housing muscles was constantly perfused with oxygenated KRB solution of the following composition To measure net outward currents, smooth muscle cells were bathed in a Ca2ϩcontaining physiological salt solution (CaPSS) containing (in mmol/l) 135 NaCl, 5 KCl, 2 CaCl2, 1.2 MgCl2, 10 glucose, 10 HEPES adjusted to pH 7.4 with Tris. Cells were dialyzed with solution containing (in mmol/l) 135 KCl, 5 EGTA, 2 CaCl2, 0.1 Na2GTP, 3 MgATP, 10 glucose, 2.5 creatine phosphate disodium, and 10 HEPES and was adjusted to pH 7.2 with Tris. KCl, 10 BAPTA, 0.1 Na2GTP, 3 MgATP, 10 glucose, 2.5 creatine phosphate disodium, and 10 HEPES and was adjusted to pH 7.2
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