ELECTRO- AND MAGNETOBIOLOGY, 19(1), 81–89 (2000)

EVIDENCE FOR MECHANOSENSITIVETRANSMEMBRANE ION CHANNELS OF

SMALL CONDUCTANCE INMAGNETOTACTIC BACTERIA

Zoe Stewart,1 Boris Martinac,2 and Jon Dobson1,3*1Department of PhysicsBiophysics Programme

The University of Western AustraliaNedlands/Perth WA6907, Australia

2Department of PharmacologyThe University of Western AustraliaNedlands/Perth WA6907, Australia

3Centre for Science and Technology in MedicineStoke-on-Trent

United Kingdom

ABSTRACT

To determine whether or not mechanosensitive (MS) ion channels arepresent in the magnetotactic bacterium Magnetospirillum magnetotacticum,techniques for spheroplast preparation in Escherichia coli were adapted forthis bacterium. This resulted in the formation of 2–3-µm spheroplasts, whichwere used for patch clamp analysis. Ion channel activity in M. magnetotac-ticum was compared with that of the MS of small conductance (MscS) inE. coli. This comparison reveals the presence of MscS-like channels in M.magnetotacticum and, as this bacterium produces intracellular magnetite(Fe3O4) particles similar to those found in the human brain, provides a modelfor investigation of the effects of magnetic fields on MS ion channels in mag-netite-bearing cells.

INTRODUCTION

Magnetite (Fe3O4) was first discovered in living organisms as a capping materialon the major lateral teeth of the chiton (a marine mollusc) (1). Later it was found that

* To whom correspondence should be addressed, at the Centre for Science and Technologyin Medicine, Thornburrow Drive, Hartshill, Stoke-on-Trent, ST4 7QB, United Kingdom. E-mail:jdobson@chem.ufl.edu

81

Copyright 2000 by Marcel Dekker, Inc. www.dekker.com

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82 STEWART, MARTINAC, AND DOBSON

chains of single-magnetic-domain biogenic magnetite are produced in magnetotactic bac-teria and are used for navigation in the earth’s magnetic field (2,3). It has since been foundin many organisms from bacteria to vertebrates, with evidence suggesting it is used formagnetic field sensing in some cases, e.g., refs. 4–8.

Prior to the discovery of magnetite in vertebrates, it was not considered feasiblethat animals could use magnetoreception to sense the earth’s magnetic field. Generally,sensory systems have associated receptor cells specifically designed for response to anexternal stimulus. These are coupled to neurons to bring information regarding the stimu-lus to the brain. Although the earth’s magnetic field is transduced by magnetite chains inmagnetotactic bacteria, this is a purely passive system and works by the application of atorque to the entire chain of particles—and thus the entire bacterium. It is not coupled toany sensory receptor, as the whole organism acts as the receptor. Recent work by Walkeret al. (8), however, has shown that such a magnetite-based receptor system does existsin the case of at least one vertebrate—the rainbow trout (Oncorhynchus mykiss).

With the discovery and verification of the presence of magnetite and/or maghemite(γFe2O3) in the human brain (6,7,10,11), its role in the brain and the possibility of thepresence of magnetite-based receptors in humans has been a source of scientific specula-tion. In addition, the presence of magnetite in the human brain has led to magnetite-based models for the interactions of environmental and applied magnetic fields with theseparticles in a way that could influence human physiologic processes—mainly throughinterference with the normal functioning of mechanosensitive (MS) transmembrane ionchannels (12,13).

MS ion channels are activated by membrane tension and were originally thought tounderlie the mechano-electrical transduction that occurs in muscle spindles, crustaceanstretch receptors, Pacinian corpuscles, and other specialized mechanoreceptors (14–16).In 1984, MS channels were first discovered and characterized by Guharay and Sachs (17)in cultured chick muscle cells. Since then, MS channels of varying ionic selectivities havebeen reported in many organisms (14,18,19). The role of MS channels in cells other thanspecialized receptors is thought to be associated with osmolarity regulation, cell volume,and cell growth (19,20).

To evaluate magnetite-based receptor models properly in the human brain and toexamine the interaction of these particles in magnetic fields with ion channels, an in vitromodel system should be developed. As magnetotactic bacteria contain chains of biogenicmagnetite, they would make an ideal model system for studying these interactions provid-ing they contain MS transmembrane ion channels. Development of such a model systemcould also lead to methods for the remote activation of transmembrane ion channels bycoupling magnetic particles to cell membranes. This type of process could find applicationin the treatment of disease.

To examine the possibility of using magnetotactic bacteria as a model system, sphe-roplast preparation techniques were developed for the magnetotactic bacterium Magne-tospirillum magnetotacticum and patch clamp experiments were performed under appliedpressures so that the possible presence of MS transmembrane ion channels could be inves-tigated. M. magnetotacticum are gram-negative bacteria that are microaerophillic andchemoheterotrophic and possess flagella (21).

Channel activity in M. magnetotacticum was then compared with measurements ofEscherichia coli, which are known to have at least three types of MS channels—the MSchannel of large conductance (MscL), the MS channel of small conductance (MscS), andthe MS channel of miniconductance (MscM) (22).

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MS ION CHANNELS IN MAGNETOTACTIC BACTERIA 83

MATERIALS AND METHODS

Magnetospirillum magnetotacticum Cultures

Non-magnetite-producing cultures of M. magnetotacticum were grown by inoculat-ing 200 µL from frozen stock (obtained from the American Type Culture Collection,ATCC strain number 31632) into a 5-mL sterile, plastic, screw-cap sample tube filled tothe top with ATCC Culture Medium 1653 [Revised Magnetic Spirillum Growth Medium(MSGM)]. The culture was left in a 30°C water bath under aerobic conditions (oxygenlevel 5 21%) for 10 days or until the pink-colored medium became colorless and slightlycloudy. From this culture, 4.5 mL was transferred into a 45-mL sterilized, screw-cap Beck-man centrifuge tube and filled with fresh MSGM media. This culture was left for approxi-mately 5 days or until the medium became dull in color and cloudy. A 15-µL sample ofthe culture was viewed on an Olympus IMT2 phase contrast microscope to check forcontamination. This culture was then used as the inoculation culture for preparation ofspheroplasts.

The growth of M. magnetotacticum was monitored daily by taking a 15-µL sampleand observing the culture on the phase-contrast microscope. This was done to ensure thatculture samples were uncontaminated. M. magnetotacticum bacteria are spiral-like rodsand do not grow to high densities. Because of these features, when viewed on the phase-contrast microscope the M. magnetotacticum cells are distinct from common contamina-tion bacteria, which are short rods and very motile. Another distinctive feature of M.magnetotacticum is that over time culture growth discolors the medium because the cellscreate alkaline ions and reduce the oxygen level in their environment (23). If contamina-tion had occurred in the cultures it would have become apparent, as the medium wouldhave become extremely cloudy but not discolored within 24 h.

Spheroplast Preparation

To compare the ion channel properties of M. magnetotacticum with known MscSchannels in E. coli, giant round-up cells (spheroplasts) of E. coli (wild-type strain AW-737) were produced by culturing the bacteria in cephalexin, a penicillin analog, whichallows cell elongation but blocks septation (22). These multinucleated filaments were thencollapsed into spheres measuring 3–10 µm in diameter by degrading the peptidoglycanwall with lysozyme and EDTA (24).

As with E. coli, M. magnetotacticum are also gram-negative bacteria, with a cellenvelope comprising an outer membrane and an inner membrane separated by a peptido-glycan cell wall (25). M. magnetotacticum cell dimensions are approximately 0.5 µm inwidth and 3 µm in length (25). To patch these bacteria, spheroplast preparations werealso required.

To develop spheroplasts from M. magnetotacticum the protocol used for sphero-plast preparation in E. coli bacteria was first tried (24). Ten milliliters of pure culture wasadded to 35 mL of MSGM medium in a 45-mL sterile, screw-capped, Beckman centrifugetube. Cephalexin (160 µL @ 10 mg/mL) was added to achieve a final concentration of0.035 g/L. The culture was then placed in a water bath at 30°C. Filament developmentwas monitored by taking 15-µl samples and observing filament length under a phase-contrast microscope. This procedure was continued until the filament length was con-sidered to be great enough (,80 µm) to achieve reasonable spheroplast preparations.

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Table 1. Results from Different Spheroplast Preparation Protocols for M.Magnetotacticuma

No. of EDTA Lysozyme TRISMedium hours (mL) (mL) (mL) Observations

*Anaerobic 27.5 150 120 150 Few and smallAnaerobic 27.5 300 120 150 Mod. freq. and smallAnaerobic 27.5 300 240 150 Mod. freq. and smallAnaerobic 27.5 450 120 150 Few and smallAnaerobic 27.5 450 240 150 Very few and smallAerobic 23 300 120 150 Mod. freq. and smallAerobic 24 300 240 150 Few and smallAerobic 25 150 240 150 Mod. freq. and smallAerobic 25 300 240 150 Few and small-mediumAerobic 25 450 240 150 Few and small-mediumAerobic 26 75 120 200 Few and small-mediumAerobic 26 150 120 200 Few and medium*Aerobic 26 300 120 200 Mod. freq and medium

a ‘‘Medium’’ specifies oxygen conditions under which inoculation culture was developedand ‘‘no. of hours’’ is the number of hours that filaments were left to develop in thepresence of cephalexin. The two groups marked with an asterisk were the sources of bacte-ria that were successfully patched.

The average time for this was 27 h; however, different incubation times were tested(Table 1).

The filaments were harvested by centrifugation at 3,500 rpm for 10 min using aBeckman Instruments J-68 centrifuge (19), the supernatant was discarded by aspiration,and the pellet was resuspended in 2.5 mL of 0.8 M sucrose. The following reagents wereadded in order: 1 M Tris Cl (pH 7.8); lysozyme (5 mg/mL); DNAase (5 mg/mL); and0.125 M EDTA (pH 8.0). Varying amounts of Tris, lysozyme, and EDTA were added inan attempt to optimize spheroplast development for the M. magnetotacticum (DNAasewas 30 µL in all cases). Table 1 shows different incubation times for the snake-like fila-ments and different amounts of the reagents used in the development of spheroplasts. Itshould be noted that M. magnetotacticum cells were prepared under both aerobic andanaerobic conditions (see ref. 23); however, all bacteria were non-magnetite-producing.

Electrophysiologic Recordings

All recordings were made using the patch clamp technique for single ion channelrecording (26). Pipettes used for patch clamping were made from borosilicate glass(Sigma-Aldrich) and were pulled using a Flaming/Brown Micropipette puller (P-87, Sut-ter). The pipettes were made as required and gave bubble numbers of approximately 3.4in 100% ethanol, corresponding to an average resistance of 4 MΩ.

Pipette tips were filled by suction and then backfilled with pipette solution (200 mMKCl, 40 mM MgCl2, 10 mM CaCl2, 0.1 mM EDTA, and 5 mM HEPES—pH adjustedto 7.2 with KOH). Brief tapping of the pipette ensured removal of any bubbles trappedbetween the tip and the barrel of the pipette. Pipettes were mounted on a suction pipetteholder, which created an electrical connection to the pipette solution and the Ag/AgCl

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MS ION CHANNELS IN MAGNETOTACTIC BACTERIA 85

electrode and allowed for suction or pressure to be applied to the interior of the pipette. Thepipette holder was attached to a Leitz micromanipulator and Sensin ZX-118DZ pressuretransducer. Spheroplasts (1.5–3.0 µL) were placed in a 0.5-mL bath containing 250 mMKCl, 90 mM MgCl2, 10 mM CaCl2, 0.1 mM EDTA, and 5 mM HEPES—pH adjustedto 7.2 with KOH. Spheroplasts could be viewed on the phase-contrast microscope.

Using the micromanipulator, the pipette tip was positioned near a spheroplast insolution, and upon slight application of negative pressure (by mouth) to the interior ofthe pipette a low resistance seal formed—approximately 50 MΩ. Upon further suctionthe seal was improved to the order of gigaohms, usually 2–3 GΩ. With this high resistanceseal a constant voltage (between 50 and 250 mV) was applied across the membrane,and channels were then activated by applying suction to the interior of the pipette via a10-mL syringe. Single-channel currents were filtered at 2 kHz and digitized at 5 kHz usingthe Axon Instruments pCLAMP data acquisition program and analyzed using the AxonInstruments Axoscope for Windows program.

A total of five M. magnetotacticum spheroplasts were patched; however, only twowere stable at more than one voltage. These two recordings were used to construct voltage-current graphs for comparison with E. coli. All spheroplasts that were patched came fromthe two preparation protocols marked with an asterisk in Table 1.

RESULTS

MscS in E. coli

In E. coli, the MS channel of small conductance (MscS) is more sensitive to pressurechanges than the MS channel of large conductance (22). To characterize MscS in E. colifor comparison with M. magnetotacticum, the conductance of this channel was determined.In plotting the current steps of each channel at a range of different voltages, the slope ofthe graph in Figure 1a gives the conductance of the channel (V 5 IR 5 I/C, where V isvoltage, I is current, R is resistance, and C is conductance). For the data shown in Fig-ure 1 the conductance was found to be 995 pS at positive voltages and 560 pS at negativevoltages.

MscS in Magnetospirillum magnetotacticum

The most successful spheroplast preparations from M. magnetotacticum resultedin spheroplasts that were generally smaller than those formed by E. coli—approximately2–3 µm in diameter. In addition, there were indications that M. magnetotacticum filamentshad been broken down by the lysozyme and EDTA and had not developed into sphero-plasts. When attempting to seal the spheroplasts onto the pipette tip, achieving a seal ofsufficiently high resistance took some time and required the slow application of negativepressure to the inside of the pipette before a gigaohm seal would form. All the M. magne-totacticum ion channel recordings were taken as on-cell patches. However, when negativepipette pressure was applied, after the formation of the seal, the spheroplast collapsed.The cell collapse was indicated in the ion channel trace as a large increase in current flowthrough the pipette tip with the seal immediately reforming. It was after the cell collapsethat ion channels were evident (Fig. 2).

To characterize the MS ion channels present in M. magnetotacticum, the conduc-

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FIGURE 1. Current-voltage plots for (a) MscS in E. coli showing a conductance of 995 pS atpositive voltages and 560 pS at negative voltages (n 5 4). (b) An MscS-like channel in M. magne-totacticum showing a conductance of 988 pS at positive voltages and 597 pS at negative voltages(n 5 2).

tance of these channels was determined and compared with E. coli (Fig. 1b). The conduc-tance was calculated as 988 pS at positive voltages and 597 pS at negative voltages.

DISCUSSION AND CONCLUSIONS

Using an adapted protocol for the production of E. coli spheroplasts, we have demon-strated that it is possible to produce spheroplasts from a pure culture of M. magnetotac-ticum. Generally, spheroplasts suitable for patch clamping should be greater than 3 µm.Spheroplasts produced from M. magnetotacticum were only just this size, approximately2–3 µm in diameter. In spite of the small size of the spheroplasts, however, we wereable to patch and record ion channel activity successfully in these bacteria. Although this

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MS ION CHANNELS IN MAGNETOTACTIC BACTERIA 87

FIGURE 2. Ion channel traces of (a) MscS in E. coli and (b) an MscS-like channel in M. magne-totacticum. Both traces are at 630 mV.

technique requires further refinement, it should form the basis for patch clamp investiga-tions of M. magnetotacticum in the future.

MscS examined in E. coli spheroplasts displayed pressure-sensitive behavior withrectifying behavior at positive voltages, as previously described by Martinac and others(24) and Berrier and others (27). Patch clamp data gathered for M. magnetotacticum revealevidence of the presence of MscS-like channels. The channel conductivity appears uniformacross ion channel traces from two spheroplasts under the same pressure and voltageconditions. Comparison of current-voltage plots for MscS channels in E. coli with thoseseen in M. magnetotacticum shows that both channels exhibit very similar conductancesat both positive and negative voltages—988 pS for M. magnetotacticum compared with995 pS for E. coli at positive voltages and 597 pS compared with 560 pS at negativevoltages. Both types of bacteria also displayed rectifying behavior at positive pipette volt-ages. This appears to confirm the presence of an MscS-type channel, as this rectifyingbehavior is only seen in MscS and not the other two types of MS channels commonlyobserved in E. coli (27).

Although M. magnetotacticum has been studied in great detail since its discovery,

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prior to this work no studies of MS ion channels in these bacteria had been undertaken.The ability of M. magnetotacticum to synthesize intracellular particles of magnetite (23)and the presence of MscS ion channels mean that M. magnetotacticum provides a naturallyoccurring model system for in vitro study of the effects of magnetic fields on magne-tosomes and MS channels, such as those proposed by Kirschvink (12) and Dobson andSt. Pierre (13), as well as for studies of remote magnetic activation of MS ion channels.

ACKNOWLEDGMENTS

We would like to thank Dr. Tim St. Pierre and Professor Mike Fuller for helpfuldiscussions as well as Anna Kloda, Dr. Alex Le Dain, Dr. Nathalie Saint, and Jay Steerfor their assistance in the laboratory.

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