Two-pore-domain potassium channels in smooth muscles: new components of myogenic regulation

Abstract

Gastrointestinal (GI) smooth muscles are influenced by many levels of regulation, including those provided by enteric motor neurones, hormones and paracrine substances. The integrated contractile responses to these regulatory mechanisms depend heavily on the state of excitability of smooth muscle cells. Resting ionic conductances and myogenic responses to agonists and physical parameters, such as stretch, are important in establishing basal excitability. This review discusses the role of 2-pore-domain K+ channels in contributing to background conductances and in mediating responses of GI muscles to enteric inhibitory nerve stimulation and stretch. Murine GI muscles express TREK-1 channels and display a stretch-dependent K+ (SDK) conductance that is also activated by nitric oxide via a cGMP-dependent mechanism. Cloning and expression of mTREK-1 produced an SDK conductance that was activated by cGMP-dependent phosphorylation at serine-351. GI muscle cells also express TASK-1 and TASK-2 channels that are inhibited by lidocaine and external acidification. These conductances appear to provide significant background K+ permeability that contributes to the negative resting potentials of GI muscles.

Figure 1. Structural and functional subclasses of two-pore-domain K⁺ (K2P) channels

A phylogenic tree of the K2P channel family is shown with different nomenclature indicated (IUPHAR, HUGO). Six functional subfamilies (TWIK, TREK, TASK, TASK-2, THIK and TRESK) of the K2P channel can be classified based on contained functional domain. The TREK subfamily (TREK-1, TREK-2 and TRAAK, white oval) is activated by arachidonic acid, unsaturated fatty acids and mechanical stretch. Two subfamilies of extracellular pH-dependent K2P channels are identified. The TASK subfamily (TASK-1 and TASK-3, black oval) are inhibited by extracellular acidic pH. In contrast the TASK-2 family (TASK-2, TALK-1 and TALK-2, grey oval) are activated by alkaline pH. TASK-2 can also be inhibited by acidosis. Volatile anaesthetics (e.g. halothane, isoflurane) inhibit the THIK subfamily but activate TREK-1, TREK-2, TASK-1, TASK-2, TALK-1 and TRESK (dotted lines). Several K2P channels are not functionally expressed (thin lines).

Figure 2. Current–voltage relationship of SDK channels in excised patch

A, representative traces of SDK channels showing channel activity recorded from holding potentials between −60 and +20 mV in an excised patch under asymmetrical K⁺ gradients. B, relationship between current amplitude and voltage in asymmetrical K⁺ (5 mm/140 mm) gradient was fitted by the GHK equation (•). Similar experiments were also performed in symmetrical K⁺ (140 mm/140 mm) gradients (○). The conductance of SDK channels under these conditions was 95 pS.

Figure 3. Mechanosensitivity of SDK channels

A, the effect of negative pressure on open probability of SDK channels. A negative pressure of −20 cmH₂O had little effect on channel activity. However, greater negative pressures (−40 cmH₂O) applied to the same patch increased NP_o (i.e. channel number × open probability) to 6.2. Further negative pressure (−60 and −80 cmH₂O) increased NP_o to the maximal level. After removal of negative pressure in each step, the open probability returned to near zero. After application of pressure pulses, the patch was excised. This caused maximal activation of channels in the patch. B, activation of stretch-dependent K⁺ (SDK) channels via cell elongation in murine colonic myocytes. Ba, two patch pipettes were sealed to the same cell. Single channel currents were measured via one pipette, and the other pipette was used to stretch the cell. Bb, after confirming that negative patch pressure (−60 cmH₂O) activated SDK channels in this patch, the cells were elongated (in this example by 8 μm). Cell elongation caused activation of channels with the same properties as negative pressure.

Figure 4. Effects of extracellular acidic pH on outward currents in the presence of 4-aminopyridine and TEA in colonic myocytes (A) and effects of lidocaine (LDC) on membrane potential of murine ileum (B)

Aa, control traces from colonic myocytes are shown in the presence of 4-aminopyridine (5 mm) and TEA (10 mm). Ab, exposure to pH 6.5 solution decreased outward current. Ac, difference currents obtained by subtracting currents obtained at pH 6.5 (Ab) from control currents (Aa). Inset shows exponential fits (continuous lines) of outward currents generated by steps to −10, 0 and +10 mV to obtain time constants of activation. Ba, lidocaine (10⁻⁴m) depolarized murine ileal tissue. Bb, exposure of ileal muscles to pH 6.0 caused depolarization. Application of lidocaine (1 mm) after pH 6.0 solution did not induce further depolarization. Bc, in reverse order, lidocaine caused depolarization, but addition of acidic pH (6.0) in the continued presence of lidocaine had no further effect on membrane potential.