Electrophysiology of dissociated gastrointestinal muscle cells

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Electrophysiology of dissociated gastrointestinal muscle cells

Kenton M. Sanders

10.1002/cphy.cp060104

Source: Supplement 16: Handbook of Physiology, The Gastrointestinal System, Motility and Circulation

Originally published: 1989

Published online: January 2011

Full Article on Wiley Online Library

Abstract
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References

Abstract

The sections in this article are:

1 Dispersion Techniques

1.1 Comparison of Approaches

1.2 Evaluation of Dispersed Cells

2 Rhythmicity In Cellular Preparations

3 Voltage Recordings From Isolated Cells

3.1 Agonist Effects on Voltage

3.2 Other Voltage Recordings in Isolated Cells

4 Voltage‐Clamp Studies Of Isolated Cells

4.1 Microelectrode Voltage‐Clamp Studies

4.2 Whole‐Cell Current and Voltage Clamp by Patch‐Clamp Technique

4.3 Properties of Potassium Channels

4.4 Properties of Calcium Channels

4.5 Properties of Chloride Channels

5 Effects of Physiological Agonists on Channels

5.1 Cholinergic Regulation of Potassium Channels

5.2 Cholinergic Effects on Calcium Channels

5.3 Effects of Other Agonists

6 Relating Studies of Ionic Channels to Electrical Events of the Gut

7 Implications of Single‐Channel Studies on Models of Electrical Activity

	Figure 1. Freshly dispersed circular muscle cells from canine proximal colon. Cells average 400 μm in length and have a relaxed, smooth appearance. They appear bright when visualized with phase‐contrast optics. Most cells with this appearance are Ca²⁺ tolerant, contract to a variety of agonists 42, maintain a resting membrane potential, and express Ca²⁺‐ and K⁺‐channel activities. Cells in this condition have been maintained for up to 36 h after dispersion.
	Figure 2. Spontaneous electrical activity recorded from longitudinal cells dispersed from rabbit jejunum. Cells demonstrated rhythmic depolarizations, some of which led to fast action potential transients. Under current clamp, application of hyperpolarizing currents caused cessation of spontaneous activity. Second hyperpolarization was not maintained by the constant current pulse, suggesting that membrane conductance increases during pulse, which is evidence for presence of inward rectifier channels in these cells. From Benham et al. 9
	Figure 3. Membrane potentials and currents recorded from isolated longitudinal muscle cell of rabbit jejunum using current‐ and voltage‐clamp conditions. Pipette contained K⁺ (126 mM), EGTA (0.77 mM), and no Ca²⁺. A and B, top traces: in normal physiological solution (2.5 mM Ca²⁺, 6 mM K⁺, 137 mM Na⁺), depolarizing current evoked overshooting action potentials. A and B, bottom traces: hyperpolarizing current yielded an electrotonic response. B: in Ca²⁺‐free solution that contained 20 mM Ba²⁺, amplitude and duration of action potentials were increased. Increase in input resistance was also noted; amplitude of electrotonic response increased. C, top traces: under voltage‐clamp conditions, depolarizing test pulses evoked 2 current phases: transient inward current followed by sustained outward current. C, bottom traces: in Ba²⁺‐containing solution, amplitude and duration of inward current increased and outward current was essentially blocked. Adapted from Bolton et al. 17
	Figure 4. Current‐voltage relationship for gastric muscle cell from Bufo marinus. Inset: voltage‐clamp step from holding potential (V_H) of −78 to +10 mV. Symbols, 3 current phases recognized from these traces: 1) inward (circles), 2) transient outward (triangles), and 3) steady‐state outward (squares). Amplitudes of these 3 current phases are plotted as function of a series of test pulses to several levels. Note voltages at which currents appear to activate and reversal potentials of currents. Inward current was carried by Ca²⁺ ions; outward currents were carried by K⁺ currents. Transient outward current (triangles) activates steeply in range where inward current (circles) is greatest in amplitude. Transient outward current depended on magnitude of inward Ca²⁺ current and was thought to result from opening of Ca²⁺‐activated K⁺ currents. As Ca²⁺ is removed from the cytoplasm, this current decays. Adapted from Walsh and Singer 79
	Figure 5. Schematic showing procedures for obtaining several membrane preparations that can be studied with patch‐clamp technique. Top, blunt pipette tip pressed against cell membrane. With suction a gigaseal can be formed that isolates interior of pipette and surface of membrane patch from bath solution. Therefore majority of current flowing through pipette must pass through membrane patch. In this cell‐attached configuration, single‐channel openings in membrane patch can be monitored without losing cellular regulation by second messengers. The drawback is that unless cell membrane potential is monitored independently, one cannot be sure of the potential across the isolated patch. From cell‐attached configuration, patch can be excised by withdrawing pipette from cell surface. In these inside‐out patches one can control ionic gradients and transmembrane potential precisely. By passing a small pulse of current or suction, it is also possible to break the isolated patch in the cell‐attached configuration while maintaining the gigaohm seal. This provides low‐resistance access to cell interior. Contents of pipette rapidly exchange with cellular contents (dialyze). In this configuration one has control over ionic gradients and transmembrane potential. Whole‐cell or macroscopic currents can be quantitated. Disadvantage is that important second messengers that regulate channels can be lost by dialysis. Finally it is also possible to withdraw the pipette after whole‐cell conditions have been obtained, which results in an excised patch that forms with exterior of membrane facing the bath. This configuration is particularly useful in studies of receptor‐activated channels, because exterior surface can be easily exposed to agonists and blockers. Adapted from Hamill et al. 36
	Figure 6. Voltage dependence of K⁺ channels in isolated membrane patch from gastric cell of Bufo marinus (C, closed; O, open). Pipette and bath contained equivalent concentrations of K⁺. In symmetrical K⁺ concentrations, current flowing through channel should reverse direction at 0 mV. Bath contained 10⁻⁶ M Ca²⁺; pipette was Ca²⁺ free with 2 mM EGTA. Top trace, membrane was held at +28 mV. At this potential channel was frequently open; probability of being open was 0.43. At −14 mV channel was open infrequently; probability was only 0.07. Amplitude of current is voltage dependent, and direction of current is opposite at 2 test potentials. From Singer and Walsh 69
	Figure 7. Permeability of K⁺ channels to Rb⁺ in excised patch from rabbit jejunal muscle cell. Pipette and bath contained symmetrical K⁺ concentrations. Top, current records at several test potentials. At positive potentials current was outward with respect to inner surface of membrane (current flowed from bath into pipette). At negative potentials current direction reversed, but K⁺ ions passed through the channel equally well. Data are plotted in graph (solid circles). When K⁺ was replaced in bath solution with 126 mM Rb⁺, outward current was greatly decreased. Current still passed easily out of pipette. Data are plotted in graph (crosses). Data can be interpreted as follows. Channel passed K⁺ in either direction (no rectification). Rb⁺ does not pass through channel as well as K⁺. When Rb⁺ served as current carrier (current flowing from bath into pipette), current was diminished, but at potentials favoring inward current, K⁺ in pipette served as current carrier and effects of Rb⁺ were not apparent. From Benham et al. 12
	Figure 8. Calcium dependence of K⁺ channels in isolated patch from circular muscle cell of canine gastric antrum. Data from excised inside‐out patch in symmetrical K⁺ gradient. Patch held at −70 mV. A: transition in activity when concentration of Ca²⁺ bathing inner surface of membrane was increased from 10⁻⁷ to 10⁻⁶ M. This caused a large increase in the probability of opening and revealed that the patch contained at least 6 channels. B: reduction in Ca²⁺ (from 10⁻⁶ to 10⁻⁷ M) rapidly decreased probability of opening. Changes in intracellular Ca²⁺ concentration in submicromolar range shift activation voltage for these K⁺ channels into physiological range. Data from A. Carl
	Figure 9. Voltage and Ca²⁺ dependence of K⁺ channels from mesenteric artery cell. A: channel activity in 10⁻⁶ M Ca²⁺ at several voltages. Channel is highly active at 10⁻⁶ M Ca²⁺. B: when Ca²⁺ was reduced to 10‐⁷ M, channel rarely opened at negative potentials, but opening was increased by depolarization. C and D: probability of opening of channels from mesenteric and jejunal cells, respectively; Ca²⁺ concentration given next to curves. Data describe Ca²⁺ and voltage dependence of channel. When Ca²⁺ increases from 10⁻⁷ to 10⁻⁶ M, channel becomes highly activable at potentials achieved physiologically (negative to 0 mV). During excitation‐contraction coupling in situ when cytoplasmic Ca²⁺ increases, activation range of Ca²⁺‐activated K⁺ channels can shift to more negative, physiological potentials. From Benham et al. 12
	Figure 10. Effects of cholinergic stimulation on electrical activity of isolated gastric cells of Bufo marinus. A: intracellular recording of membrane potential (top trace) in response to ionophoretic application of acetylcholine (ACh) (bottom trace). ACh depolarized membrane potential and caused spiking. Magnitude of response was related to amplitude and duration of ionophoretic pulse (pulses a‐e). B and C: current‐clamp records; top traces, application of square pulses of hyperpolarizing current; lower traces, voltage responses. Bars, muscarine was applied. Muscarine depolarized the membrane and increased amplitude of voltage responses to injected current. C: offset current was applied to hold membrane potential fairly constant during muscarine application. This further increased the amplitude of voltage responses during muscarine application. Data suggest that cholinergic stimulation decreases membrane conductance. A: extracellular [Ca²⁺] ([Ca²⁺]_o) = 50 mM, [Na⁺]_o = 39 mM, and extracellular tetraethylammonium ion ([TEA⁺]_o) = 18 mM. B and C: [Ca²⁺]₀ = 20 M, [Na⁺]_o = 97 mM, and [TEA⁺]_o = 5 mM. From Sims et al. 63
	Figure 11. Cholinergic responses of gastric muscle cell from Bufo marinus studied with voltage clamp. Traces, time (sweep speed is varied during trace, but tics denote seconds), voltage (stepped between −36 and −65 mV), and current responses. Expanded pulse at beginning of record shows control responses. Stepping to −65 mV revealed relaxation of outward current (outward tail current); stepping back to −36 mV shows development of outward current. Bar, muscarine was added to bath. This caused development of sustained inward current (departure from dashed line) and blockade of current relaxations upon hyperpolarization and development of outward current upon depolarization. Data suggest that cholinergic agonists act to block K⁺ current. V_h, holding potential; I_ss steady‐state current. From Sims et al. 63
	Figure 12. Current records under whole‐cell voltage clamp of guinea pig ileum. Pipette contained 140 mM Cs⁺ to block outward currents. Traces, voltage dependence of inward current. Graph, I‐ V relationship at peak inward current. Current activated at about −40 mV, reached a maximum at −10 mV, and reversed at approximately −40 mV. Tests showed that inward current was carried by Ca²⁺ ions. From Droogmans and Callewaert 27
	Figure 13. Steady‐state activation (solid circles) and inactivation (open circles) of inward current as function of membrane potential. Data taken from isolated cell from guinea pig ileum. Curves, fit of data with Boltzmann equation. Activation and inactivation curves overlap, indicating that at approximately −40 mV a fraction of the Ca²⁺ may be open, producing a small sustained inward current (window current). From Droogmans and Callewaert 27
	Figure 14. Effect of acetylcholine (AcCho) on voltage‐activated Ca²⁺ current in gastric muscle cell of Bufo marinus. Current response under voltage clamp from a holding potential of −78 mV to a test potential of 9 mV. Acetylcholine increased the magnitude of inward current. From Clapp et al. 23
	Figure 15. Effects of acetylcholine (ACh) on electrical activity of gastric muscle of Bufo marinus. Record, continuous intracellular recording from circular muscle cell of corpus region. Arrow, ACh (10⁻⁵ M) was applied by superfusion. This caused a significant increase in slow‐wave frequency, a small tonic depolarization, and changed the waveforms of slow waves by reducing upstroke velocity and amplitude. Insets, specific portions of record enlarged to display effects of ACh on waveform. None of these effects on electrical activity are easily reconcilable on basis of suppression of an M current (see ref. 63. Data from P. Shonnard

Figure 1.

Freshly dispersed circular muscle cells from canine proximal colon. Cells average 400 μm in length and have a relaxed, smooth appearance. They appear bright when visualized with phase‐contrast optics. Most cells with this appearance are Ca²⁺ tolerant, contract to a variety of agonists 42, maintain a resting membrane potential, and express Ca²⁺‐ and K⁺‐channel activities. Cells in this condition have been maintained for up to 36 h after dispersion.

Figure 2.

Spontaneous electrical activity recorded from longitudinal cells dispersed from rabbit jejunum. Cells demonstrated rhythmic depolarizations, some of which led to fast action potential transients. Under current clamp, application of hyperpolarizing currents caused cessation of spontaneous activity. Second hyperpolarization was not maintained by the constant current pulse, suggesting that membrane conductance increases during pulse, which is evidence for presence of inward rectifier channels in these cells.

From Benham et al. 9

Figure 3.

Membrane potentials and currents recorded from isolated longitudinal muscle cell of rabbit jejunum using current‐ and voltage‐clamp conditions. Pipette contained K⁺ (126 mM), EGTA (0.77 mM), and no Ca²⁺. A and B, top traces: in normal physiological solution (2.5 mM Ca²⁺, 6 mM K⁺, 137 mM Na⁺), depolarizing current evoked overshooting action potentials. A and B, bottom traces: hyperpolarizing current yielded an electrotonic response. B: in Ca²⁺‐free solution that contained 20 mM Ba²⁺, amplitude and duration of action potentials were increased. Increase in input resistance was also noted; amplitude of electrotonic response increased. C, top traces: under voltage‐clamp conditions, depolarizing test pulses evoked 2 current phases: transient inward current followed by sustained outward current. C, bottom traces: in Ba²⁺‐containing solution, amplitude and duration of inward current increased and outward current was essentially blocked.

Adapted from Bolton et al. 17

Figure 4.

Current‐voltage relationship for gastric muscle cell from Bufo marinus. Inset: voltage‐clamp step from holding potential (V_H) of −78 to +10 mV. Symbols, 3 current phases recognized from these traces: 1) inward (circles), 2) transient outward (triangles), and 3) steady‐state outward (squares). Amplitudes of these 3 current phases are plotted as function of a series of test pulses to several levels. Note voltages at which currents appear to activate and reversal potentials of currents. Inward current was carried by Ca²⁺ ions; outward currents were carried by K⁺ currents. Transient outward current (triangles) activates steeply in range where inward current (circles) is greatest in amplitude. Transient outward current depended on magnitude of inward Ca²⁺ current and was thought to result from opening of Ca²⁺‐activated K⁺ currents. As Ca²⁺ is removed from the cytoplasm, this current decays.

Adapted from Walsh and Singer 79

Figure 5.

Schematic showing procedures for obtaining several membrane preparations that can be studied with patch‐clamp technique. Top, blunt pipette tip pressed against cell membrane. With suction a gigaseal can be formed that isolates interior of pipette and surface of membrane patch from bath solution. Therefore majority of current flowing through pipette must pass through membrane patch. In this cell‐attached configuration, single‐channel openings in membrane patch can be monitored without losing cellular regulation by second messengers. The drawback is that unless cell membrane potential is monitored independently, one cannot be sure of the potential across the isolated patch. From cell‐attached configuration, patch can be excised by withdrawing pipette from cell surface. In these inside‐out patches one can control ionic gradients and transmembrane potential precisely. By passing a small pulse of current or suction, it is also possible to break the isolated patch in the cell‐attached configuration while maintaining the gigaohm seal. This provides low‐resistance access to cell interior. Contents of pipette rapidly exchange with cellular contents (dialyze). In this configuration one has control over ionic gradients and transmembrane potential. Whole‐cell or macroscopic currents can be quantitated. Disadvantage is that important second messengers that regulate channels can be lost by dialysis. Finally it is also possible to withdraw the pipette after whole‐cell conditions have been obtained, which results in an excised patch that forms with exterior of membrane facing the bath. This configuration is particularly useful in studies of receptor‐activated channels, because exterior surface can be easily exposed to agonists and blockers.

Adapted from Hamill et al. 36

Figure 6.

Voltage dependence of K⁺ channels in isolated membrane patch from gastric cell of Bufo marinus (C, closed; O, open). Pipette and bath contained equivalent concentrations of K⁺. In symmetrical K⁺ concentrations, current flowing through channel should reverse direction at 0 mV. Bath contained 10⁻⁶ M Ca²⁺; pipette was Ca²⁺ free with 2 mM EGTA. Top trace, membrane was held at +28 mV. At this potential channel was frequently open; probability of being open was 0.43. At −14 mV channel was open infrequently; probability was only 0.07. Amplitude of current is voltage dependent, and direction of current is opposite at 2 test potentials.

From Singer and Walsh 69

Figure 7.

Permeability of K⁺ channels to Rb⁺ in excised patch from rabbit jejunal muscle cell. Pipette and bath contained symmetrical K⁺ concentrations. Top, current records at several test potentials. At positive potentials current was outward with respect to inner surface of membrane (current flowed from bath into pipette). At negative potentials current direction reversed, but K⁺ ions passed through the channel equally well. Data are plotted in graph (solid circles). When K⁺ was replaced in bath solution with 126 mM Rb⁺, outward current was greatly decreased. Current still passed easily out of pipette. Data are plotted in graph (crosses). Data can be interpreted as follows. Channel passed K⁺ in either direction (no rectification). Rb⁺ does not pass through channel as well as K⁺. When Rb⁺ served as current carrier (current flowing from bath into pipette), current was diminished, but at potentials favoring inward current, K⁺ in pipette served as current carrier and effects of Rb⁺ were not apparent.

From Benham et al. 12

Figure 8.

Calcium dependence of K⁺ channels in isolated patch from circular muscle cell of canine gastric antrum. Data from excised inside‐out patch in symmetrical K⁺ gradient. Patch held at −70 mV. A: transition in activity when concentration of Ca²⁺ bathing inner surface of membrane was increased from 10⁻⁷ to 10⁻⁶ M. This caused a large increase in the probability of opening and revealed that the patch contained at least 6 channels. B: reduction in Ca²⁺ (from 10⁻⁶ to 10⁻⁷ M) rapidly decreased probability of opening. Changes in intracellular Ca²⁺ concentration in submicromolar range shift activation voltage for these K⁺ channels into physiological range.

Data from A. Carl

Figure 9.

Voltage and Ca²⁺ dependence of K⁺ channels from mesenteric artery cell. A: channel activity in 10⁻⁶ M Ca²⁺ at several voltages. Channel is highly active at 10⁻⁶ M Ca²⁺. B: when Ca²⁺ was reduced to 10‐⁷ M, channel rarely opened at negative potentials, but opening was increased by depolarization. C and D: probability of opening of channels from mesenteric and jejunal cells, respectively; Ca²⁺ concentration given next to curves. Data describe Ca²⁺ and voltage dependence of channel. When Ca²⁺ increases from 10⁻⁷ to 10⁻⁶ M, channel becomes highly activable at potentials achieved physiologically (negative to 0 mV). During excitation‐contraction coupling in situ when cytoplasmic Ca²⁺ increases, activation range of Ca²⁺‐activated K⁺ channels can shift to more negative, physiological potentials.

From Benham et al. 12

Figure 10.

Effects of cholinergic stimulation on electrical activity of isolated gastric cells of Bufo marinus. A: intracellular recording of membrane potential (top trace) in response to ionophoretic application of acetylcholine (ACh) (bottom trace). ACh depolarized membrane potential and caused spiking. Magnitude of response was related to amplitude and duration of ionophoretic pulse (pulses a‐e). B and C: current‐clamp records; top traces, application of square pulses of hyperpolarizing current; lower traces, voltage responses. Bars, muscarine was applied. Muscarine depolarized the membrane and increased amplitude of voltage responses to injected current. C: offset current was applied to hold membrane potential fairly constant during muscarine application. This further increased the amplitude of voltage responses during muscarine application. Data suggest that cholinergic stimulation decreases membrane conductance. A: extracellular [Ca²⁺] ([Ca²⁺]_o) = 50 mM, [Na⁺]_o = 39 mM, and extracellular tetraethylammonium ion ([TEA⁺]_o) = 18 mM. B and C: [Ca²⁺]₀ = 20 M, [Na⁺]_o = 97 mM, and [TEA⁺]_o = 5 mM.

From Sims et al. 63

Figure 11.

Cholinergic responses of gastric muscle cell from Bufo marinus studied with voltage clamp. Traces, time (sweep speed is varied during trace, but tics denote seconds), voltage (stepped between −36 and −65 mV), and current responses. Expanded pulse at beginning of record shows control responses. Stepping to −65 mV revealed relaxation of outward current (outward tail current); stepping back to −36 mV shows development of outward current. Bar, muscarine was added to bath. This caused development of sustained inward current (departure from dashed line) and blockade of current relaxations upon hyperpolarization and development of outward current upon depolarization. Data suggest that cholinergic agonists act to block K⁺ current. V_h, holding potential; I_ss steady‐state current.

From Sims et al. 63

Figure 12.

Current records under whole‐cell voltage clamp of guinea pig ileum. Pipette contained 140 mM Cs⁺ to block outward currents. Traces, voltage dependence of inward current. Graph, I‐ V relationship at peak inward current. Current activated at about −40 mV, reached a maximum at −10 mV, and reversed at approximately −40 mV. Tests showed that inward current was carried by Ca²⁺ ions.

From Droogmans and Callewaert 27

Figure 13.

Steady‐state activation (solid circles) and inactivation (open circles) of inward current as function of membrane potential. Data taken from isolated cell from guinea pig ileum. Curves, fit of data with Boltzmann equation. Activation and inactivation curves overlap, indicating that at approximately −40 mV a fraction of the Ca²⁺ may be open, producing a small sustained inward current (window current).

From Droogmans and Callewaert 27

Figure 14.

Effect of acetylcholine (AcCho) on voltage‐activated Ca²⁺ current in gastric muscle cell of Bufo marinus. Current response under voltage clamp from a holding potential of −78 mV to a test potential of 9 mV. Acetylcholine increased the magnitude of inward current.

From Clapp et al. 23

Figure 15.

Effects of acetylcholine (ACh) on electrical activity of gastric muscle of Bufo marinus. Record, continuous intracellular recording from circular muscle cell of corpus region. Arrow, ACh (10⁻⁵ M) was applied by superfusion. This caused a significant increase in slow‐wave frequency, a small tonic depolarization, and changed the waveforms of slow waves by reducing upstroke velocity and amplitude. Insets, specific portions of record enlarged to display effects of ACh on waveform. None of these effects on electrical activity are easily reconcilable on basis of suppression of an M current (see ref. 63.

Data from P. Shonnard

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Article Title:	Electrophysiology of dissociated gastrointestinal muscle cells
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Kenton M. Sanders. Electrophysiology of dissociated gastrointestinal muscle cells. Compr Physiol 2011, Supplement 16: Handbook of Physiology, The Gastrointestinal System, Motility and Circulation: 163-185. First published in print 1989. doi: 10.1002/cphy.cp060104

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