1Department of Physiology and Biophysics, University of Miami School of Medicine, Miami, Florida 33136; and 2Physiology and Biophysical Sciences, Center for Single Molecule Biophysics, State University of New York, Buffalo, New York 14214
Submitted 5 May 2004 ; accepted in final form 13 July 2004
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ABSTRACT |
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mechanical stress; lens
Other connexins, including Cx43 and Cx38, have also been reported to form open hemichannels (10, 25). However, these were only observed under special experimental conditions, typically with nonphysiological micromolar extracellular Ca2+. Recently, a series of papers have invoked connexins in the release of cellular ATP and NAD+ to the extracellular space through nonjunctional hemichannels (5, 8, 24, 41). These agents can act as extracellular second messengers (21). Ca2+ waves propagate through many cell networks (4, 7, 36, 37, 42), and connexins are known to play a crucial role in the intracellular propagation process (44). The intracellular mode utilizes gap junctions to pass inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] from cell to cell (6, 35). The extracellular mode involves release of ATP and its binding to purinergic receptors (14, 37). Connexin hemichannels may be involved in this mode because they are permeant to ATP (24, 41).
Mechanical stress can initiate Ca2+ waves (40), and it is well documented that mechanical stress can result in ATP release (16, 47). The simplest scenario in which connexin hemichannels could initiate mechanosensitive Ca2+ waves would be if they were mechanosensitive and permeant to ATP.
In the present study, we tested the sensitivity of hemichannels to mechanical stress. We chose Cx46 hemichannels because they are well characterized at both the macroscopic and single-channel levels (11, 31, 32, 45). Cx46 is present in the ocular lens, and hemichannels might have a physiological role permitting rapid fluid equilibration under the mechanical stresses associated with accommodation.
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METHODS |
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In vitro transcription of mRNAs. Cx46 cloned into the expression vector pSP64T was obtained from Dr. D. L. Paul (Harvard University, Boston, MA) (31). mRNA was transcribed by Sp6 RNA polymerase from 10 µg of EcoRI-linearized plasmid with the mMessage mMachine kit (Ambion). mRNA was quantified by absorbance (260 nm), and the proportion of full-length transcripts was checked by agarose gel electrophoresis. Twenty nanoliters of mRNA (50 ng/µl) was injected into oocytes. The injected oocytes were then transferred into fresh OR2 medium with elevated Ca2+ (5 mM) to keep the gap junction hemichannels closed and incubated at 18°C for 1824 h. For electrophysiological recordings, oocytes were transferred to regular OR2.
Solutions. OR2 solution contained (in mM) 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 CaCl2, 1.0 Na2HPO4, and 5.0 HEPES (pH 7.5) with antibiotics (penicillin 10,000 U/ml, streptomycin 10 mg/ml). Hypotonic solution contained (in mM) 2.5 KCl, 1.0 MgCl2, 1.0 CaCl2, 1.0 Na2HPO4, and 5.0 HEPES (pH 7.5). Hypertonic solution contained (in mM) 82.5 NaCl, 2.5 KCl, 1.0 MgCl2, 1.0 CaCl2, 1.0 Na2HPO4, 5.0 HEPES (pH 7.5), and 100 sucrose.
Voltage clamp. Whole cell voltage-clamp recording was performed with two intracellular electrodes as described previously (9).
Patch-clamp technique. Single Cx46 hemichannels were studied by the patch-clamp technique (19) with a WPC-100 amplifier (E.S.F. Electronic, Goettingen, Germany). Currents were filtered at 5 kHz, digitized with a VR-10B digital data recorder, and stored on videotape. The recordings were transferred to a Power Macintosh (Apple) computer with an ITC-18 computer interface (Instrutech) and analyzed. Acquisition and analysis were done with the Acquire and TAC programs (both from Bruxton).
The vitelline membrane was removed, and the oocyte was washed once before it was transferred to a new dish containing potassium gluconate (KGlu) solution (in mM: 140 KGlu, 10 KCl, 5.0 TES, pH 7.5). Patch pipettes were made from borosilicate glass tubing (1.50.86 mm; no. GC150F-15; Warner Instrument), drawn with a Flaming-Brown micropipette puller (model P-97; Sutter Instrument), and polished with a microforge (Narishige Scientific Instruments) to an inside diameter of 0.51 µm with a resistance of 1020 M in KGlu solution. The pipette and bath usually contained the KGlu solution.
Negative pressure was applied to the membrane patch pneumatically through the pipette holder. The pressure, measured with a water manometer, was established first in a reservoir with a syringe. Step changes of pressure were then applied to the patch by connecting the pipette to the reservoir or atmospheric pressure with a valve.
Statistics. Channel kinetics were analyzed only for patches containing single channels. Student's paired t-tests were used to quantitate the effects of stress on single channels and whole oocyte conductance.
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RESULTS |
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Figure 1 shows the activity of a Cx46 hemichannel at 50 mV after a jump back from a positive holding potential. The frequency of channel openings declined over the first 20 s, but application of negative pressure reactivated the channel with a latency of 1 s. Activity was maintained as long as the pressure was applied. With the release of pressure, the channels closed after passing through substates. Figure 2 shows a multichannel record with two negative pressure jumps that induced intense channel activity. The release of suction ended activity abruptly, but that was followed by a slow (
1 s) return to steady-state activity. Such rebound behavior has been reported for mechanosensitive K+ channels (probably TREK-1) in rat heart (30) and snail heart (29). The rebound was postulated to represent retensioning of the cytoskeleton on release of stress and was highly variable. Because the stimulus rise time was <100 ms, the latency was not instrument based.
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An obvious question is whether we are observing endogenous SACs or connexins. The result is quite clear: they are two different channels (Fig. 4). SACs are abundant in all oocytes, whereas the large-conductance (280 pS) channel can only be observed in CX46-injected oocytes (32, 45). This channel is directly gated by pH (32, 46). The strongest argument that this channel must be ascribed to Cx46 is the observation that channel properties change with mutation of the CX46 sequence (22, 23, 32). The conductance and the selectivity are different between Cx46 (45) and endogenous SACs (20, 52). The voltage dependence of mechanosensitivity also differs (20, 52). The endogenous channels are activated by mechanical stress at all potentials, and only inactivation is voltage dependent (Fig. 5).
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When the membrane potential of oocytes expressing Cx46 was clamped at 50 mV the hemichannels were closed and the membrane conductance was 1 µS, a value similar to that of uninjected oocytes (11, 31, 32). Hypotonic solutions caused the oocytes to swell and increased the membrane conductance (Fig. 6). On switching back to normal Ringer solution, the conductance transiently increased and then returned to resting values within
10 min. The transient increase in conductance is due to the reintroduction of the higher-conductivity normal Ringer solution. Currents in hypotonic medium are carried mainly by cytoplasmic anions, such as Cl, and the remaining extracellular salts (10 mM). Thus the "instantaneous" current obtained just after restoration of normal Ringer solution represents the osmotically activated change in conductance.
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DISCUSSION |
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Because Cx46 is expressed at high levels exclusively in the lens, the mechanosensitivity of Cx46 hemichannels may play a physiological role. Under resting conditions, the membrane potential keeps the channels closed. During visual accommodation, the mechanical stress imposed on the cells to change the shape of the lens could open the hemichannels and allow a more rapid volume flow to relieve stress. This process would be self-limiting, because depolarization rapidly closes the channels, preserving cell viability. Such a scenario is consistent with the findings that in normal resting lens fiber cells there is no significant contribution of Cx46 hemichannels to membrane currents (27). However, such currents become visible under certain experimental conditions, such as low Ca2+ (12).
Although oocytes endogenously express mechanosensitive cation channels (20, 28, 48), they cannot be reliably activated by volume changes. This inability to evoke whole cell currents corresponding to the single-channel currents has been ascribed to several potential causes: a large "membrane reserve" (53), limited water permeability (39), or the presence of a stiff cytoskeleton that is disrupted during patch formation. The bipolar effect of potential on the kinetics of mechanosensitive ion channels is reminiscent of Shaker K+ channels. In that case it was postulated that the intermediate states of the channel, those between fully closed and fully open, are physically larger and hence occupancy is favored by mechanical stress (17).
Nearly all cells release ATP with mechanical stress (13, 16, 38, 49, 51), and connexins have been suggested to mediate at least part of the process. Stress-induced ATP fluxes have been measured in normal oocytes, and the activation of endogenous mechanosensitive channels was invoked as a possible sensory mechanism (3). The present study establishes that, in principle, connexins can be mechanosensitive and can mediate both sensing and ATP release. Cx46 hemichannels are sufficiently large to permit the flux of ATP. For example, when Cx46 hemichannels are active, extracellular application of cAMP results in a CFTR-mediated Cl current that depends on elevation of intracellular cAMP (33).
It has been suggested in the literature that connexins, such as Cx43, could form open hemichannels that mediate the physiological stress-induced release of ATP from cells (24, 25, 41). However, the Cx43 hemichannel conductance only occurs in nonphysiological, low extracellular Ca2+. Complicating the simple view of the Cx43 hemichannel as an ATP transporter/transducer is evidence that ATP release may be vesicular (1, 26). Also, in astrocytes from Cx43 knockout mice, intercellular Ca2+ waves propagate in these cells mainly through an extracellular pathway (42) with the same velocity as in wild-type astrocytes (37).
Whether other connexins, or gap junction channels, are mechanosensitive is not yet known. Although Cx43 hemichannel activity has only been found under nonphysiological bath conditions, it is possible that second messengers may modify the gating of the channel to function in vivo.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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