Stretch-dependent potassium channels in murine colonic smooth muscle cells

Abstract

Gastrointestinal muscles are able to maintain negative resting membrane potentials in spite of stretch. We investigated whether stretch-dependent K+ channels might contribute to myogenic regulation of smooth muscle cells from the mouse colon. Negative pressure applied to on-cell membrane patches activated K+ channels that were voltage independent and had a slope conductance of 95 pS in symmetrical K+ gradients. The effects of negative pressure on open probability were graded as a function of pressure and reversible when atmospheric pressure was restored. Cell elongation activated K+ channels with the same properties as those activated by negative pressure, suggesting that the channels were stretch-dependent K+ (SDK) channels. Channels with the same properties were maximally activated by patch excision, suggesting that either an intracellular messenger or interactions with the cytoskeleton regulate open probability. Internal 4-aminopyridine, Ca2+ (10(-8) to 10(-6) M), and tetraethylammonium (internal or external) were without effect on SDK channels. Nitric oxide donors (and cell-permeant cGMP analogues) activated SDK channels, suggesting that these channels may mediate a portion of the enteric inhibitory neural response in colonic muscles. In summary, SDK channels are an important conductance expressed by colonic muscle cells. SDK channels may stabilize membrane potential during dynamic changes in cell length and mediate responses to enteric neurotransmitters.

Figure 4. Activation of stretch-dependent K⁺ (SDK) channels via cell elongation in murine colonic myocytes

A, 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. B and C, 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 1. Activation of stretch-dependent K⁺ (SDK) channels by negative pipette pressure

A, application of negative pressure (-40 cmH₂O) to patch pipettes increased channel activity in cell-attached patches of murine colonic myocytes. In order to remove contaminating currents from non-selective cation channels, cells were held at 0 mV in asymmetrical K⁺ (5/140 mm). B, NP_o plotted as a function of time in response to negative pipette pressure. Data points tabulated every 2 s. C, negative pipette pressure increased the open probability of channels with the same properties in canine colonic circular myocytes studied under identical conditions.

Figure 2. Relationship between pressure and open probability of channels activated by negative pressure in murine colonic myocytes

A, 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 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, the graph summarizes the relationship between pressure and NP_o in patches from 5 cells. I-O denotes inside-out patches.

Figure 3. I-V relationship before recovery from negative pressure in murine colonic myocytes

A, representative traces of current-voltage relationship before recovery from negative pressure. B, current voltage relationship in asymmetrical K⁺ (5/140 mm) gradient was fitted by the GHK equation. C, 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. D, relationship between current amplitude and voltage in asymmetrical K⁺ (5/140 mm) gradient was fitted by the GHK equation (•). Similar experiments were also performed in symmetrical K⁺ (140/140 mm) gradients (○). The conductance of SDK channels under these conditions was 95 pS (○).

Figure 5. Effects of SNP on SDK channels in murine colonic myocytes

A, in on-cell patches, the open probability of SDK channels was close to 0 at a holding potential of 0 mV. Application of SNP (10⁻⁶m) resulted in openings of potassium channels, as previously documented (Koh et al. 1995). SNP (10⁻⁵m) increased the open probability of channels with the same properties as SDK channels. The patch used for the illustration also contained a small (4 pS) channel that was also activated by SNP. B and C, corresponding amplitude histograms before and after SNP (10⁻⁵m). D, in the example shown negative pressure (-30 cmH₂O) activated SDK channels. Addition of SNP (10⁻⁵m) in the presence of negative pressure further increased the NP_o of SDK channels.

Figure 6. Effects of 8-Br-cGMP on SDK channels in murine colonic myocytes

A, application of 8-Br-cGMP (10⁻⁵m) to the bathing solution dramatically increased the activity of SDK channels in on-cell patches at a holding potential of 0 mV. B and C, corresponding amplitude histograms before and after 8-Br-cGMP (10⁻⁵m) from the boxed area of trace in A.