Proc. Nati. Acad. Sci. USA Vol. 89, pp. 7462-7466, August 1992 Biophysics
Rapid adaptation of single mechanosensitive channels in Xenopus oocytes OWEN P. HAMILL AND DON W. MCBRIDE, JR. Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853
Communicated by A. J. Hudspeth, May 13, 1992 (received for review March 30, 1992)
ABSTRACT Mechanosensitive (MS) channels are expressed in a wide range of cell types and have been implicated in diverse functions, including osmoregulation and mechanoreception. The majority of previous studies on single MS channels have been carried out on nonsensory cells and have dealt with the steady-state properties of the channel. Here we measure the dynamic or nonstationary properties of the MS channel in Xenopus laevis oocytes. MS channels open transiently in response to a step change in suction applied to the membrane patch. This adaptive behavior occurs because of a reduction in open channel probability rather than a decrease in channel conductance. Double-step suction protocols indicate that adapted MS channels can be reactivated by application of stronger stimulation, consistent with a change in gating sensitivity rather than channel inactivation. Adaptation is highly voltage dependent, being most evident at resting or hyperpolarized potentials and absent at strongly positive potentials. Neither adaptation nor its voltage sensitivity requires the presence of extracellular Ca2+. Adaptation is fragile, dependent on patch history, and can be irreversibly abolished by moderate suction applied to the patch while MS channel activity is retained. Further suction can abolish MS channel activity without compromising the seal. We propose that the selective loss of adaptation and MS channel activity is due to different stages of membrane-cytoskeleton decoupling caused by the mechanical stresses associated with patch clamp recording.
less, there are reports that indicate MS channel activity in nonsensory cells displays nonstationary behavior and adapts in response to maintained stimulation (28-31). The mechanism and functional significance of this adaptation remain unknown. In this study we apply steps of suction of controlled amplitude and duration to membrane patches to better characterize the adaptive properties of MS channel activity in Xenopus oocytes.
MATERIALS AND METHODS Mature female frogs (Xenopus laevis) were obtained from Xenopus I (Ann Arbor, MI) and anesthetized by being placed for approximately 20 min in a beaker containing 300 mg of ethyl 3-aminobenzoate (methanesulfonic acid salt; Aldrich) in 200 ml of distilled water. After surgical removal, type V and type VI oocytes were sorted and stored overnight in Barth's medium [in mM: NaCl 88, KC1 1, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41, NaHCO3 2.4, Tris HCl 5 at pH 7.4; osmolarity 200 mOsm] containing gentamicin sulfate at 75 pg/ml at 170C. MS channel currents were activated by using a custommade stimulus system that could be used to apply suction steps to the patch pipette. The applied suction was monitored with a pressure transducer and held constant with negative feedback control using a piezoelectric valve (Maxtek; Torrance, CA). Computer control of the pressure/suction level enabled application of suction pulses of defined amplitude and duration to the patch pipette interior. Where indicated, the computer also controlled the voltage clamp steps. The method for removal of the vitelline layer for patch clamp studies was the same as that described in ref. 32. Standard patch clamp techniques (33) were used to record MS channel currents from cell-attached patches of oocytes. A "gentle" seal was essential for observing initial MS channel adaptive behavior. The suction used during sealing was monitored continuously, in gentle sealing did not exceed 5 mmHg (1 mmHg = 133 Pa), and was usually applied for less than 10 s. Except where stated, patch pipettes had a tip diameter of 1-2 sam. The pipette solution usually contained (in mM): 100 KCl, 10 EGTA (KOH), 10 Hepes (KOH) at pH 7.2. The bath solution contained Ringer's solution (in mM): 115 NaCl, 1 KCl, 1.8 CaCl2, 10 Hepes (NaOH) at pH 7.2. All experiments were carried out at room temperature (18-200C). Oocyte resting potential was estimated by extrapolation from the reversal potential of the MS current, which is 0 mV. MS current records were filtered at 500 Hz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized at 1 kHz on an IBM PC clone using the FastLab acquisition package from Indec (Sunnyvale, CA). Analysis and illustrations were also made with FastLab routines.
Mechanosensitive (MS) channels are expressed in a wide variety of cell types, including both sensory (1, 2) and nonsensory cells (3-5). While in sensory cells their role in mechanotransduction is evident, in nonsensory cells one proposed role involves volume regulation during osmotic swelling (6-8). From an evolutionary point of view this basic homeostatic mechanism may have been adapted in sensory cells for mechanotransduction. In fact, basic similarities exist between MS channels in sensory and nonsensory cells in their ion selectivity (9-11), single channel conductance (1214), and pharmacological properties (15, 16). A widespread feature of mechanotransduction in sensory receptors is rapid adaptation in response to maintained stimulation (17). Although in some cases this adaptation occurs because structures accessory to the transducer modify the mechanical stimulus (18) or because of a refractoriness in the encoding of the action potentials (19-21), there are also examples, most notably in the hair cell, in which adaptation involves an apparent change in the sensitivity of the mechanotransducer itself (22-27). Studies of single MS channels in sensory cells have proven difficult, and little data exist on the single channel mechanisms that may underlie the latter form of adaptation (27). On the other hand, most studies of single MS channels in nonsensory cells have focused on, or assumed, stationary properties of the channel (3-5). NevertheThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: MS, mechanosensitive.
7462
Biophysics: Hamill and McBride
Proc. Natl. Acad. Sci. USA 89 (1992)
RESULTS MS channels open transiently in response to a rapid step change in suction. Fig. 1A illustrates the rapidly adapting MS current in response to a single 2.5-s suction step of 10 mmHg recorded at a patch potential of -100 mV. After an initial rapid (
Rapid adaptation of single mechanosensitive channels in Xenopus oocytes OWEN P. HAMILL AND DON W. MCBRIDE, JR. Section of Neurobiology and Behavior, Cornell University, Ithaca, NY 14853
Communicated by A. J. Hudspeth, May 13, 1992 (received for review March 30, 1992)
ABSTRACT Mechanosensitive (MS) channels are expressed in a wide range of cell types and have been implicated in diverse functions, including osmoregulation and mechanoreception. The majority of previous studies on single MS channels have been carried out on nonsensory cells and have dealt with the steady-state properties of the channel. Here we measure the dynamic or nonstationary properties of the MS channel in Xenopus laevis oocytes. MS channels open transiently in response to a step change in suction applied to the membrane patch. This adaptive behavior occurs because of a reduction in open channel probability rather than a decrease in channel conductance. Double-step suction protocols indicate that adapted MS channels can be reactivated by application of stronger stimulation, consistent with a change in gating sensitivity rather than channel inactivation. Adaptation is highly voltage dependent, being most evident at resting or hyperpolarized potentials and absent at strongly positive potentials. Neither adaptation nor its voltage sensitivity requires the presence of extracellular Ca2+. Adaptation is fragile, dependent on patch history, and can be irreversibly abolished by moderate suction applied to the patch while MS channel activity is retained. Further suction can abolish MS channel activity without compromising the seal. We propose that the selective loss of adaptation and MS channel activity is due to different stages of membrane-cytoskeleton decoupling caused by the mechanical stresses associated with patch clamp recording.
less, there are reports that indicate MS channel activity in nonsensory cells displays nonstationary behavior and adapts in response to maintained stimulation (28-31). The mechanism and functional significance of this adaptation remain unknown. In this study we apply steps of suction of controlled amplitude and duration to membrane patches to better characterize the adaptive properties of MS channel activity in Xenopus oocytes.
MATERIALS AND METHODS Mature female frogs (Xenopus laevis) were obtained from Xenopus I (Ann Arbor, MI) and anesthetized by being placed for approximately 20 min in a beaker containing 300 mg of ethyl 3-aminobenzoate (methanesulfonic acid salt; Aldrich) in 200 ml of distilled water. After surgical removal, type V and type VI oocytes were sorted and stored overnight in Barth's medium [in mM: NaCl 88, KC1 1, MgSO4 0.82, Ca(NO3)2 0.33, CaCl2 0.41, NaHCO3 2.4, Tris HCl 5 at pH 7.4; osmolarity 200 mOsm] containing gentamicin sulfate at 75 pg/ml at 170C. MS channel currents were activated by using a custommade stimulus system that could be used to apply suction steps to the patch pipette. The applied suction was monitored with a pressure transducer and held constant with negative feedback control using a piezoelectric valve (Maxtek; Torrance, CA). Computer control of the pressure/suction level enabled application of suction pulses of defined amplitude and duration to the patch pipette interior. Where indicated, the computer also controlled the voltage clamp steps. The method for removal of the vitelline layer for patch clamp studies was the same as that described in ref. 32. Standard patch clamp techniques (33) were used to record MS channel currents from cell-attached patches of oocytes. A "gentle" seal was essential for observing initial MS channel adaptive behavior. The suction used during sealing was monitored continuously, in gentle sealing did not exceed 5 mmHg (1 mmHg = 133 Pa), and was usually applied for less than 10 s. Except where stated, patch pipettes had a tip diameter of 1-2 sam. The pipette solution usually contained (in mM): 100 KCl, 10 EGTA (KOH), 10 Hepes (KOH) at pH 7.2. The bath solution contained Ringer's solution (in mM): 115 NaCl, 1 KCl, 1.8 CaCl2, 10 Hepes (NaOH) at pH 7.2. All experiments were carried out at room temperature (18-200C). Oocyte resting potential was estimated by extrapolation from the reversal potential of the MS current, which is 0 mV. MS current records were filtered at 500 Hz with an eight-pole Bessel filter (Frequency Devices, Haverhill, MA) and digitized at 1 kHz on an IBM PC clone using the FastLab acquisition package from Indec (Sunnyvale, CA). Analysis and illustrations were also made with FastLab routines.
Mechanosensitive (MS) channels are expressed in a wide variety of cell types, including both sensory (1, 2) and nonsensory cells (3-5). While in sensory cells their role in mechanotransduction is evident, in nonsensory cells one proposed role involves volume regulation during osmotic swelling (6-8). From an evolutionary point of view this basic homeostatic mechanism may have been adapted in sensory cells for mechanotransduction. In fact, basic similarities exist between MS channels in sensory and nonsensory cells in their ion selectivity (9-11), single channel conductance (1214), and pharmacological properties (15, 16). A widespread feature of mechanotransduction in sensory receptors is rapid adaptation in response to maintained stimulation (17). Although in some cases this adaptation occurs because structures accessory to the transducer modify the mechanical stimulus (18) or because of a refractoriness in the encoding of the action potentials (19-21), there are also examples, most notably in the hair cell, in which adaptation involves an apparent change in the sensitivity of the mechanotransducer itself (22-27). Studies of single MS channels in sensory cells have proven difficult, and little data exist on the single channel mechanisms that may underlie the latter form of adaptation (27). On the other hand, most studies of single MS channels in nonsensory cells have focused on, or assumed, stationary properties of the channel (3-5). NevertheThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Abbreviation: MS, mechanosensitive.
7462
Biophysics: Hamill and McBride
Proc. Natl. Acad. Sci. USA 89 (1992)
RESULTS MS channels open transiently in response to a rapid step change in suction. Fig. 1A illustrates the rapidly adapting MS current in response to a single 2.5-s suction step of 10 mmHg recorded at a patch potential of -100 mV. After an initial rapid (
