Inward rectifier potassium conductance regulates membrane potential of canine colonic smooth muscle

Abstract

1. The membrane potential of gastrointestinal smooth muscles determines the open probability of ion channels involved in rhythmic electrical activity. The role of Ba2+-sensitive K+ conductances in the maintenance of membrane potential was examined in canine proximal colon circular muscle. 2. Application of Ba2+ (1-100 microM) to strips of tunica muscularis produced depolarization of cells along the submucosal surface of the circular muscle layer. Significantly higher concentrations of Ba2+ were needed to depolarize preparations from which the submucosal and myenteric pacemaker regions were removed. 3. Elevation of extracellular [K+]o (from 5.9 to 12 mM) brought membrane potentials closer to EK (the Nernst potential for K+ ions), suggesting activation of a K+ conductance. This occurred at potentials much more negative than the activation range for delayed rectifier channels (Kv). 4. Forskolin (1 microM) caused hyperpolarization and a leftward shift in the dose-response relationship for Ba2+, suggesting that forskolin may activate a Ba2+-sensitive conductance. 5. Patch-clamp recordings from interstitial cells of Cajal (ICC) revealed the presence of a Ba2+-sensitive inward rectifier potassium conductance. Far less of this conductance was present in smooth muscle cells. 6. Kir2.1 was expressed in the circular muscle layer of the canine proximal colon, duodenum, jejunum and ileum. Kir2.1 mRNA was expressed in greater abundance along the submucosal surface of the circular muscle layer in the colon. 7. These results demonstrate that ICC express a Ba2+-sensitive conductance (possibly encoded by Kir2.1). This conductance contributes to the generation and maintenance of negative membrane potentials between slow waves.

Figure 1. Schematic drawing showing preparations used

Strips of canine proximal colon were cut parallel to the circular muscle fibres. Full-thickness preparations consisted of a strip of the entire tunica muscularis. Pinning these strips in a cross-sectional orientation allowed selective impalement of smooth muscle cells near the submucosal surface of the circular muscle layer. Further dissections were performed to produce submucosal circular muscle strips (SCM) and isolated circular muscle strips (ICM). SCM strips contained the submucosal pacemaker region and a thin strip of adjacent circular muscle. ICM strips were devoid of the submucosal pacemaker region, the myenteric region and the longitudinal muscle layer. ICM strips could be pinned with either the inner or outer aspect of the muscle strip facing upward, thus permitting selective impalements of either population of cells.

Figure 2. Effects of Ba²⁺ on electrical activity

Membrane potentials of cells near the submucosal surface of the circular muscle typically had negative membrane potentials between slow waves (A; see text for details). Ba²⁺ caused concentration-dependent depolarization (B-D) and reduction of (B and C) or block of slow waves (D).

Figure 3. Effects of Ba²⁺ on slow wave parameters

The graphs show summaries of the effects of Ba²⁺ on resting potential (A), amplitude (B) and frequency (C) in experiments on 5 muscles from 5 dogs. C here and in others figures, indicates control value. Data are means ±s.e.m.; * P < 0.05.

Figure 4. Comparison of the effects of Ba²⁺ on full-thickness muscle strips (•, data reproduced from Fig. 3) and ICM preparations (▪)

Resting membrane potential depolarized in ICM preparations when the submucosal pacemaker region and myenteric regions were removed. Ba²⁺ (1-100 μM) had no significant effect on membrane potential in ICM preparations. RMP was significantly decreased by 250-1000 μM Ba²⁺ (*P < 0.05). Data are means ±s.e.m. from 14 muscles from 14 dogs.

Figure 5. Summary of the effects of elevated external K⁺ ([K⁺]_o) on resting membrane potential (RMP)

Full-thickness muscle strips were exposed to three concentrations of [K⁺]_o (5.9-12 mM). A-C, the effects of changes in [K⁺]_o on membrane potential and slow wave activity. D, elevated [K⁺]_o caused significant depolarization (• *P < 0.05) and brought membrane potential vs.[K⁺]_o closer to the slope defined by the Nernst relationship (▾). Data are means ±s.e.m. from 5 preparations from 5 dogs.

Figure 6. Sensitivity to Ba²⁺ increases in ICM after application of forskolin

Isolated circular muscle preparations were exposed to forskolin (1 μM). Forskolin caused hyperpolarization of RMP (see text). For grapical comparison, the data were plotted as the degree of depolarization caused by Ba²⁺ before (▪) and after forskolin (•). In the presence of forskolin, Ba²⁺ depolarized ICM at lower concentrations, suggesting activation of a Ba²⁺-sensitive conductance. Data are means ±s.e.m. from 4-14 preparations from 4-14 dogs. * Significance compared with the membrane potentials before the addition of Ba²⁺ (P < 0.05).

Figure 7. Effects of ouabain and Ba²⁺ on RMP of colonic muscles

Cells near the submucosal surface of full-thickness muscle preparations were depolarized by ouabain (1 μM). Addition of Ba²⁺ (100 μM) caused further depolarization. Data are means ±s.e.m. from 5 muscle strips. In another series of 5 experiments the order of application of ouabain and Ba²⁺ was reversed. Initial application of Ba²⁺ caused depolarization, and further application of ouabain produced a small depolarization that did not reach statistical significance. Significance between data groups denoted by brackets; * P < 0.05; ** P < 0.01; n.s., not significant).

Figure 8. Effects of Ba²⁺ on whole-cell currents of voltage-clamped ICC and smooth muscle cells

The top trace shows the voltage-ramp protocol used. Traces in A and C are averaged current responses from 15 episodes in physiological K⁺ gradients (5/140 mM). A, current responses recorded from ICC before (○) and after (•) application of Ba²⁺ (50 μM). Prominent inward rectification was observed at potentials negative to E_K (-88 mV). Ba²⁺ inhibited the inward current. B, difference current describing the Ba²⁺-sensitive currents in ICC. C, a similar experiment conducted on a circular smooth muscle cell. Much less inward current was observed in these cells at negative potentials (•), and Ba²⁺ (50 μM; ○) had very little effect. D, Ba²⁺ difference currents obtained by subtraction from control.

Figure 9. Quantification of Kir2.1 cDNA in the canine gastrointestinal tract

A representative gel of Q-PCR for Kir2.1 in the submucosal layer of canine colon is shown in A; 2-fold serial dilutions of mimic DNA were included in the PCR reactions while Kir2.1 cDNA remained constant. The concentration of Kir2.1 cRNA in different regions of the GI tract expressed relative to β-actin cDNA is illustrated in B. The amount of Kir2.1 cDNA in SCM, ICM and MyCM preparations is depicted in C. Results are expressed as means ±s.e.m.; * significant difference in the level of Kir2.1 transcript in ICM and MyCM compared with SCM preparations (P < 0.05).