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Neuroscience Center and Kresge Hearing Laboratories at Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA
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Abstract |
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45% of the way through the membrane electric field was calculated. Channel length was estimated as 31 Å and the width of the pore mouth at < 17 Å. Each effective antagonist had a minimal diameter, measured about the penetrating amine, of less than the pore diameter, with a direct correlation between IC50 and minimal diameter. The IC50 was also directly related to the length of the amine side chains, further validating the proposed pore blocking mechanism. Data provided by these two approaches support a hypothesis regarding channel permeation and block that incorporates molecular dimensions and ion interactions within the pore.
(Received 15 January 2004;
accepted after revision 2 June 2004;
first published online 4 June 2004)
Corresponding author A. J. Ricci: Neuroscience Center and Kresge Hearing Laboratories, 2020 Gravier St Suite D, LSU Health Sciences Center, New Orleans LA 70112, USA. Email: aricci{at}lsuhsc.edu
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Introduction |
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The MET channel exhibits a non-specific cation conductance with a high calcium permeability (Ohmori, 1985; Crawford et al. 1991; Lumpkin & Hudspeth, 1995; Ricci & Fettiplace, 1998). MET channels pass large molecules such as tetraethyl ammonium and the membrane dye FM1-43 (Corey & Hudspeth, 1979; Gale et al. 2001). Single channel conductance estimates vary from 10 to 300 pS (Ohmori, 1985; Holton & Hudspeth, 1986; Crawford et al. 1991; Geleoc et al. 1997; Ricci et al. 2003). No voltage dependence of the channel (Ohmori, 1987; Crawford et al. 1991) has been reported. Biophysical characteristics suggest the channel is related to the broad class of non-specific cation channels that include the transient receptor potential channels (TRP), a subgroup of which are the vanniloid receptors (TRPV), cyclic nucleotide gated channels (CNG), mechanically gated channels, the nicotinic family of channels and even gap junctional hemi-channels. The presence of both CNG type and TRP type channels in hair cells (Kolesnikov et al. 1991; Liedtke et al. 2000; Drescher et al. 2002) make these channel classes prime candidates for the MET channel. With the identification of TRP-like channels regulating mechanosensation in Drosophila (Walker et al. 2000) and zebrafish (Sidi et al. 2003), a great deal of attention is being paid to the TRP family (Corey, 2003; Strassmaier & Gillespie, 2003). However, existing data cannot clearly classify the channel type.
Pharmacological data regarding MET channels are limited in that the blockers were not specific to channel classes, nor do they provide insight into the properties of the channel pore. For example, aminoglycoside antibiotics that can antagonize calcium channels, are open MET channel blockers (Ohmori, 1985; Kroese et al. 1989; Kimitsuki & Ohmori, 1993; Ricci, 2002). Amiloride and its derivatives, which block some CNG channels as well as channels of the epithelial sodium channel (ENAC) family (Frings et al. 1992; Kellenberger & Schild, 2002) also antagonize MET channels of hair cells as well as oocytes (Jorgensen & Ohmori, 1988; Lane et al. 1993; Rusch et al. 1994; Ricci, 2002). Tubocurarine, a nicotinic antagonist, also blocks the MET channel (Glowatzki et al. 1997). Cisplatin is yet another antagonist of the MET channel (Kimitsuki et al. 1993).
Present work creates a pharmacological profile of the MET channel pore to serve as a basis for comparisons with other known channel types and a template for the molecular identification and characterization of this channel. Eight new MET channel blockers were identified demonstrating the channel has broad similarities to other non-specific cation channels including the TRP and CNG channels. Furthermore, the molecular dimensions of the channel were estimated and support a novel open channel blocking hypothesis for charged amines.
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Methods |
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Auditory papillae were prepared as previously described (Ricci & Fettiplace, 1997). Red-eared sliders (Trachemys scripta elegans), carapace length 8 to 15 cm were decapitated and the inner ear organs removed using procedures approved by the ACUC committee at LSU Health Sciences Center and conforming to standards established by NIH guidelines. The inner ear organs were placed into external solution containing (mM): 125 NaCl, 0.5 KCl, 2.8 CaCl2, 2.2 MgCl2, 2 each of pyruvate, creatine, lactate, ascorbate, 6 glucose and 10 Hepes. The solution was buffered to pH 7.6 and had a final osmolality of 275 mosmol kg–1. The tissue was pinned to the bottom of a Sylgard-coated dish with minutien pins with the auditory papilla facing upward. The tectorial membrane was exposed and treated with protease type XXIV (Sigma) 0.02–0.04 mg ml–1 for 5–20 min depending on enzyme activity. The tectorial membrane was removed with an etched tungsten wire and the enzyme removed with multiple rinses of external solution. The papilla was trimmed and placed into a coverslip-bottomed recording chamber and held in place with three single strands of dental floss. The recording chamber was perfused at a rate of 0.5–1 ml min–1 with external solution supplemented with 100 nM apamin (Calbiochem) to block the caesium-permeable SK calcium-activated potassium current (Tucker & Fettiplace, 1996). A peristaltic pump (Gilson, Middleton, WI, USA) was used for the bath perfusion.
Drug application
Drugs were applied to the apical surface through a 2 mm diameter pipette whose tip was pulled to an external diameter of 75 µm. The pipette was placed about 100 µm away from the cell being recorded. Flow was perpendicular to the sensitive axis of the hair bundle (Ricci & Fettiplace, 1997). Flow rate was controlled using a Gilson peristaltic pump coupled through miniature solenoid valves (Lee Valves), the rate was maintained at 1–3 ml h–1. Complete exchange of the apical fluid took 1.5 min. Measurements were made between 5 and 10 min after drug application. Toxins were pressure applied through a pipette of tip diameter 3–5 µm using a picospritzer (General Valve). Osmolality was varied by first lowering NaCl by 25 mM for the low osmolar solution and replacing with sucrose to raise osmolality to 275 (control) or 325 mosmol kg–1 (high osmolar solution). This method allowed ionic strength to remain constant during changes in osmolality. Drugs were purchased from Fisher, Calbiochem and Sigma when necessary. Pseudecatoxin was a generous gift from Dr Moretti at Meiji Pharmaceutical University, Japan.
Recording procedures
A large blunt pipette, filled with extracellular solution, was advanced into the papilla from the abneural edge while applying pressure to the back end of the pipette, making a hole from which one to three cells could be removed to ensure good access. The location of the hole (d) is the relative position measured from the papilla apex. Unless otherwise stated, measurements were made from a high frequency location (n= 136, d= 0.61 ± 0.01), while the average low frequency position was 0.39 ± 0.05 (n= 50). Whole cell recordings were obtained as has been previously described (Ricci & Fettiplace, 1997). An Axopatch 200 (Axon Instruments) was used for all recordings. The internal solution contained (mM): 110 CsCl, 3 MgATP, 5 creatine phosphate, 1 BAPTA, 10 Hepes, 2 ascorbate; pH was 7.2. Series resistances averaged 3.0 ± 0.2 M
(n= 186) after up to 70% compensation. Cell capacitance was 12.5 ± 0.2 pF (n= 186) giving voltage-clamp speeds of 38 µs. A junction potential of –4 mV was measured and corrected offline, as was any residual series resistance. Cells with leak currents greater than 50 pA, measured as non-mechanically gated inward current at –80 mV were excluded. Minimal leak subtraction was performed with the amplifier circuit. Cells were excluded if series resistance varied by more than 25% during the recording. All experiments were performed between 19 and 22°C.
Upon reaching whole cell configuration, calcium currents increased in amplitude for the first 10–15 min (Schnee & Ricci, 2003). Mechano-electric transducer (MET) currents also showed some limited run-up that was accompanied by a speeding up of adaptation and a slight leftward shift in the current–displacement plots. To account for these changes, data were not collected until the peak current had stabilized, about 10 min after break through.
Mechanical stimulation
Hair bundles (see example in Fig. 1A) were stimulated with a stiff glass probe attached to a piezo-electric element (Ricci & Fettiplace, 1997). The voltage step to the piezo was filtered, with an 8-pole Bessel filter, at either 2 or 5 kHz to prevent exciting the intrinsic resonance of the ceramic. Probe motion was calibrated with a photodiode motion detector (Crawford & Fettiplace, 1985; Ricci et al. 2000). The glass probe, 1–1.5 µm in diameter, was placed near the bottom third of the bundle on the short stereocilia side so that the bundle was effectively pushed to open channels and pulled to close channels. Probe adherence to the bundle was enhanced by acid washing prior to use. On occasion, motion of the bundle was measured as a control to ensure probe adherence to the bundle (using the photodiode detector, Ricci et al. 2000). Activation protocols were driven by the Cambridge Electronic Device (CED) signal software. In some instances a piezo-electric stack was used (Jena), particularly for experiments where current–voltage relationships were investigated. The stack motion is axial compared to the ceramic, which is perpendicular to axial. The stimulus direction of the stack allowed for a larger diameter probe with better bundle contact that resulted in more reliable negative deflections which were essential for accurate measurements of MET currents at positive potentials.
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All data are presented as means ±S.E.M. The number of cells (n) is given with each set of data, usually for dose–response curves near the data point in the figure. Unless otherwise stated, current traces illustrated were averages of 16 sweeps for activation protocols and 4 sweeps for depolarization protocols. Data were collected with Signal software (CED) and exported to Origin (Microcal) or IGOR (Wavemetrics) for analysis. Origin uses the Levenberg-Marquardt algorithm for fitting. Where appropriate, correlation coefficients are given as r2 values. Unless otherwise stated, Student's two-tailed t tests were used to assess statistical significance (P < 0.01).
Maximal currents were obtained by adding the current amplitudes measured from saturating positive and negative hair bundle displacements (Fig. 1B). Responses were determined to be saturating by fitting the peak current plotted against stimulus amplitude with the equation for a double Boltzmann function (see Fig. 1B; Ricci & Fettiplace, 1997):
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| (1) |
Current ratios, Iion/INa, rather than reversal potential shifts were used for both comparison of monovalent permeabilities and amine permeabilities. This method is necessary because removing calcium irreversibly abolishes transduction (Assad et al. 1991; Crawford et al. 1991) and lowering calcium can alter the single channel conductance (Ricci et al. 2003). It was necessary to maintain calcium at a constant level while altering the species of monovalent ion. It is assumed that part of the current carried under each condition was calcium, a hypothesis supported by the maintenance of some adaptation in the presence of amines or antagonists. Current ratios can be used as estimates of permeability as long as the independence principal is satisfied (Hille, 1973). Monovalent ions have not been shown to interact in the pore, thereby meeting the criteria, though divalents have. The interaction between divalents and monovalents is assumed a constant for these experiments. Further experimental justification of this methodology is presented in the results.
Dose–response curves were fitted with a Hill equation:
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| (2) |
Molecular dimensions
The dimensions of the amine compounds and drugs were determined by building Corey-Pauling-Koltun (CPK) space filling models (Adams et al. 1980). The orientation of the models was first determined using energy minimalization in Chem3D software (Cambridge, Cambridge, MA, USA). For the amine derivatives, the diameter was measured as the diameter of the smallest circle that the molecule could pass through. Three measurements were made from the antagonists, the minimal diameter of the region of the molecule proposed to penetrate the channel pore, the maximal width proposed to prevent permeation of the compound and the distance between these two measurements. The first two measurements were made in an identical manner to that of the simple amine compounds, the latter was measured with a calliper calibrated for the size of the model molecules.
Pore diameter
In Fig. 10C two theoretical fits are given to the plot of ionic radius against relative permeability. The first assumes a circular pore and spherical amines. The relationship between relative permeability and ionic radius is then equivalent to:
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| (3) |
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| (4) |
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A single site blocking model was fitted to current–voltage data obtained in the presence of different antagonists and n-alkyl-amine compounds of the form:
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| (5) |
is the fractional distance through the membrane electric field traversed by the amine, Vr is the reversal potential and Vs reflects the steepness of rectification (Woodhull, 1973; Kros et al. 1992; Rusch et al. 1994; Gale et al. 2001). Although the MET channel is a multi-ion pore where interactions between ions have been suggested (Lumpkin et al. 1997; Ricci & Fettiplace, 1998), this simple single energy barrier model adequately fits the data presented. Pore length
Knowing the single channel conductance and the diameter of the pore allows for the estimate of the length of the channel. This estimate assumes a cylindrical shape to the pore. Rearrangement of the original equations (Hille, 1968) gives:
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| (6) |
is the resistivity of the solution (100 cm), g is the single channel conductance, D is the diameter of the pore, L is the length of the channel. |
Results |
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Ion substitution experiments were performed by replacing Na+ ions with the test monovalent ion in the apical perfusion solution (Fig. 1D). Replacement with K+, Cs+ and Li+ demonstrated that the channel followed a permeation order directly correlated with the ion's hydration energy (Fig. 1E) (Edsall & McKenzie, 1978). The order Cs+ > K+ > Na+ > Li+ followed an Eisenmann series XI, in good agreement with previous investigations using reversal potential measurements (Ohmori, 1985) and indicative of a channel with a negatively charged external mouth (Adams et al. 1980). The agreement with reversal potential estimates supports the accuracy of using current ratios in these investigations. The minimally rectifying, non-specific conductance of the channel was also verified by measuring peak currents at various membrane potentials (Fig. 1F and G). Depolarizations reduce the driving force for calcium entry, resulting in a leftward shift of the MET activation curve (Eatock et al. 1987; Crawford et al. 1989). The example in Fig. 1F illustrates this phenomenon at +90 mV. Here, outward MET current turns on during depolarization as the activation plot shifts leftward due to the decrease in intraciliary Ca2+. Mechanical deflection of the hair bundle away from the kinocilium turns the MET current off, seen as a reduction in outward current. Comparison of the relative current turned off and on mechanically at ± 90 mV illustrates the large leftward shift in activation. Current amplitudes for the current–voltage plots (Fig. 1G) were measured as the net current evoked from positive and negative hair bundle deflections (Fig. 1F). The reversal potential was 7 ± 1 mV (n= 7; eqn 5) suggesting a non-specific cation channel. The comparable currents obtained at positive and negative potentials demonstrate the minimally rectifying nature of the current. These results are in good agreement with previous investigations (Ohmori, 1985; Crawford et al. 1989). In the presence of a drug, eqn (5) estimates the distance through the electric field the drug travels until bound (). In control, however, Ca2+ is the blocking agent and eqn (5) gives an estimate of the distance Ca2+ travels to its blocking site (Kros et al. 1992; Gale et al. 2001). An estimate of 0.45 ± 0.01 was obtained, suggesting the binding site is near the middle of the electric field and in reasonable agreement with previous work in chick (Kimitsuki & Ohmori, 1993), bullfrog (Kroese et al. 1989) and mouse (Kros et al. 1992; Gale et al. 2001). This estimate supports previous arguments that the blocking site for Ca2+ and the binding site for fast adaptation may be different (Crawford et al. 1991; Ricci et al. 1998).
Cyclic nucleotide-gated (CNG) channel antagonists
CNG channel antagonists were investigated first using the benzothiazepine derivative diltiazem and the acetonitrile derivative D600, normally considered L-type calcium channel blockers, but also potent antagonists of CNG channels (Frings et al. 1992; Kleene, 2000). Diltiazem's effect on MET currents is presented in Fig. 2A, where responses to different amplitude stimuli are shown in the absence and presence of two doses of the drug. Both fast and slow adaptation were slowed by the antagonist. The effect on fast adaptation is more clearly illustrated in Fig. 2B, an expanded view of the response to the smallest bundle stimulus (Fig. 2A). As the diltiazem concentration was increased, adaptation was slowed. Effects on adaptation were best observed from responses to small mechanical hair bundle deflections. With larger deflections, used to obtain maximal currents, a fast decay in current was observed in the presence of diltiazem that was not observed in the absence of the drug (Fig. 2A and C). This type of response is often, though not always, observed with open channel blockers (Armstrong, 1971; French & Shoukimas, 1981). It was previously argued that the ability to detect the drug-induced decay in current was in part determined by the kinetics of the channel (Ricci, 2002). Larger deflections of the hair bundle result in the channel open time increasing, thus promoting the observation of the open channel block (Ricci et al. 2003) and supporting the hypothesis regarding kinetics.
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, obtained from the exponential fit) was plotted against dose for each compound in Fig. 3G and best fitted with a linear regression indicative of a first order reaction where the slope of the line represents the rate constant. Slope values of 1.8 ± 0.2 and 2.4 ± 0.2 ms–1 mM–1 were obtained for D600 and diltiazem, respectively. The fastest values for each compound were not included in the fit as they were limited by the voltage-clamp speed of the recording. A first order reaction is consistent with an open channel block. The block by D600 and diltiazem was voltage-dependent, with little of the MET current being blocked at +80 mV (data not shown). The voltage dependence of block was also investigated for both diltiazem and D600 (Fig. 3C and F, n= 5). In both cases block was removed as the cell was depolarized, supporting the argument of an open channel blocker. Slight removal of block was also observed at hyperpolarized potentials. Although this might be interpreted as representing a permeable block by these compounds, it is unlikely (see Discussion). For D600, complete block of the current was not obtained at 1 mM and so the increased current may simply reflect the increased driving force for this unblocked portion. Higher doses were attempted but solubility became an issue and so those data are not included. For diltiazem, the release is postulated to reflect calcium or monovalent ions being forced passed the compound in the pore of the channel (see Discussion for explanation).
Both D600 and diltiazem are L-type calcium channel blockers that bind to overlapping regions of the channel's 
-subunit (Kraus et al. 1996), a similar location to the dihydropyridine binding site. Because dihydropyridines block L-type calcium channels but not CNG-gated channels (though see Zufall & Firestein, 1993), nimodipine was applied to the apical surface at 5 µM. No significant effect was observed on the MET current (n= 3), suggesting the ability of diltiazem and D600 to antagonize the MET current was not a function of a similarity between the MET channel and L-type calcium channels (Fig. 3H).
Local anaesthetics such as tetracaine also block CNG channels, a block postulated to occur by binding to the channel's closed state (Fodor et al. 1997). Tetracaine potently antagonized the MET current (Fig. 4A). Figure 4B shows the dose–response relationship and Hill equation fit with values for IC50 of 579 ± 39 µM and Hill coefficient of 2.7 ± 0.4, values consistent with the block of a CNG channel. No decay in current during the stimulus was observed.
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Transient receptor potential (TRP) channels
TRP channels represent a broad and growing class of non-specific cation channels (Clapham et al. 2001; Benham et al. 2002; Montell et al. 2002). Due to their diversity and novelty, the pharmacology of these channels has not been well characterized. However, there are several known antagonists. Trivalent ions such as lanthanum antagonize TRP channels (Halaszovich et al. 2000). La3+ antagonized the MET current quite potently, with an IC50 value of 3.8 ± 0.7 µM and a Hill coefficient of 0.7 ± 0.1 (Fig. 5A and B). Gadolinium, another trivalent compound, also blocked the MET current (Kimitsuki et al. 1996). Both of these trivalent molecules are not TRP selective and block other mechanically gated channels, including those of baroreceptors (Kraske et al. 1998), TRAAK channels (Maingret et al. 1999) and oocyte mechano-gated channels (Wilkinson et al. 1998). The overlap in blocking ability may represent similarities in the pores of mechanically gated channels.
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The vanilloid receptor channels constitute a subclass of the TRP family (Gunthorpe et al. 2002). Recent evidence demonstrates the presence of an osmotically activated vanilloid receptor in hair cells (Liedtke et al. 2000). The effects of osmolality, pH and capsaicin on MET currents were investigated (n= 3 for each). Changes of ± 50 mosmol kg–1 using sucrose substitution did not alter the MET response. pH variations of ± 1 unit also did not alter the MET current, and capsaicin application (200 nM) had no effect on the MET current (see Table 1). These data suggest the MET channel is not one of the known vanilloid channels.
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Several properties of the MET channel, including single channel conductance, calcium block and permeability, as well as non-rectification, are similar to gap junctional and hemi-gap junctional channels (DeVries & Schwartz, 1992). Although these channels are not typically thought of as mechanically gated, the pharmacology was investigated because the biophysical properties of the pore suggested there might be similarities. Quinine, a compound with a variety of actions, both opens and antagonizes hemi-gap junctional channels (Srinivas et al. 2001). Quinine also has effects in the auditory system, causing tinnitus and hearing loss (Tange, 1998; Kaltenbach, 2000; Zheng et al. 2001). Applied to the apical hair bundle, quinine antagonized the MET current with an IC50 of 10 ± 2 µM and a Hill coefficient of 1.2 ± 0.1 (Fig. 6A and B). However flufenamic acid, another antagonist of hemi-gap junctional channels (Harks et al. 2001; Srinivas & Spray, 2003), had no effect on the MET current (Fig. 6C, n= 4). So here too results were somewhat ambiguous, suggesting similarities between the hemi-gap pore and the MET pore, but not conclusively identifying the channel type.
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At this point the pharmacological data do not clearly classify the channel, as antagonists from each class of compound tested were effective. In an attempt to identify a common mode of action of these compounds, space-filling models were constructed (Fig. 7A). Previous findings of permeable block by FM1-43 (a charged amine derivative; Gale et al. 2001) and block by curare (a quarternary amine; Glowatzki et al. 1997) indicated that a partially charged amine might be capable of entering the pore electrochemically. The arrows in Fig. 7 point to the partially charged amine for each compound. Note that in each case the amine is in a region of the molecule that is structurally small in size. In addition, it was reasoned that a bulky side chain would be needed to impede flow of the compound through the channel. The side chains are outlined by a rectangle (Fig. 7). Compounds that did not work had sterically hindered amine groups postulated not to be able to enter the channel pore (like nimodipine) or else did not have the partially charged amine group to drive the compound into the channel (Fig. 7B). The similarity in structure led to the hypothesis that the compounds were acting as open channel blockers, with the amine penetrating the pore and the side chain preventing permeation. The open channel nature of the block shown in Figs 2 and 3 supports this idea. To test this hypothesis two additional compounds were investigated, curare and methylene blue (Fig. 7C). Whereas both compounds have a charged amine to drive the compound into the pore, curare has a side chain to prevent permeation and methylene blue does not, so it is predicted that methylene blue would act as a permeable blocker of the channel.
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) versus dose. The linear plot is indicative of a first order reaction and supportive of the open channel nature of the block (Fig. 8C) (Armstrong, 1971). The cork-in-bottle model proposed here is first order in that one drug molecule is postulated to interact with one channel to give a blocked channel. A further test of the open channel nature of the block was the voltage dependence of the block (Fig. 8D–F) where only inward current was antagonized. This result supports the hypothesis that curare acts as an open channel blocker, driven into and out of the pore electrochemically. The lack of release from block at hyperpolarized potentials indicates that curare is not a permeable blocker of the pore (Zarei & Dani, 1994; Gale et al. 2001). The control traces (Fig. 8D) illustrate the non-specific, minimally rectified nature of the channel while in the presence of curare the response becomes rectified. Inward current was completely blocked by curare while outward current at positive potentials was only slightly impeded.
The effect of curare on MET currents from low frequency (d= 0.3) cells was also investigated to test whether MET channels varied pharmacologically across frequency positions. Previous work found differences in sensitivity to streptomycin but not to amiloride (Ricci, 2002). No difference in curare efficacy was observed for low frequency cells (Fig. 8B). The quarternary ammonium compounds atropine and hexamethonium were also used to further validate the open channel block hypothesis. Both blocked the MET current with an IC50 of around 1 mM (see Table 1).
Another test of the open channel hypothesis was performed using methylene blue (Fig. 9), a tertiary amine and known guanylyl cyclase antagonist with no known ion channel effects. Methylene blue was chosen because it has two tertiary amines predicted to penetrate the pore but no large R-group (see Fig. 7, rectangles) to prevent permeation; therefore it is predicted to be a permeant blocker much like FM1-43. Methylene blue antagonized the MET current almost completely at 1 mM, showing a hint of an open channel block (Fig. 9A and B). Simultaneously with current block the cell turned blue, suggesting that methylene blue was a permeant blocker of the channel. As methylene blue eventually stains all cells, its ability to access the cell cytosol is not limited to passage through the MET channel. However, when MET channels were active the cells stained in 1–3 min whereas when MET channels were not open (due to mechanical disruption during dissection) at least 10 min was required.
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A third test of methylene blue acting as a permeant blocker was the voltage dependence of the block. As mentioned earlier, relief of block at both extremes of potential is indicative of a permeant channel blocker. Protocols like those used in Figs 1, 3 and 8 were used again to examine the voltage dependence of block of methylene blue. In contrast to the effects with curare, methylene blue block was relieved at both positive and negative potentials, supporting the conclusion that methylene blue was a permeant blocker. As the cells turned blue during these experiments it is possible to conclude that the current was carried by methylene blue, in contrast to the previous experiments with diltiazem and D600. The reversal potential shifted –25 mV in the presence of methylene blue, suggesting divalent permeation was blocked before monovalent. The relative distance through the electric field was not different from control (0.51 ± 0.06 and 0.48 ± 0.04 for control and in the presence of methylene blue, respectively, n= 3). These results are similar to those observed with FM1-43, another permeant blocker of the MET channel (Gale et al. 2001).
Pore width
Together the data suggest that large amines can act as open channel blockers of the MET channels and that drug sensitivities may be in part a function of the presence of an amine group serving to drive the compound into the pore, while a large R-group prevents further permeation. The ability of the charged amine to reach the narrow portion of the channel and the relative size of the channel pore compared to the size of the amine group are predicted to determine drug efficacy. To better quantify the relationship between the molecular dimensions of the drugs and that of the channel pore, experiments were performed to more directly assess channel pore dimensions. The ability of simple amine compounds to permeate channels allows for estimates of channel diameter (Hille, 1971). Simple amines such as methylammonium, trimethylammonium, triethylammonium, tetramethylammonium, tetraethylammonium, tetrapropylammonium and tetrabutylammonium were substituted for sodium and current ratios obtained in order to estimate the size of the MET channel pore. Examples of the currents obtained are displayed in Fig. 10A. Relatively large amines showed partial permeability except for the tetrabutyl ammonium. A plot of molecular weight against current ratio (Fig. 10B) yields an exponential relationship, demonstrating that the size of the molecule and not binding within the pore dictates current flow and that from the point of view of amine permeability, the pore can be regarded as a molecular sieve (Dwyer et al. 1980; McCleskey & Almers, 1985; Sun et al. 1997). CPK models of the amines allowed for the minimum cross-section of the molecule to be determined and its diagonal used as an estimate of the molecular diameter. Plotting the radius of the molecule against current ratio allowed for an estimate of pore diameter. The continuous line fitted to these data (eqn (3)) points to the current ratio being a function of the volume of the amine relative to the radius of the pore. This fit corresponds to a pore diameter of 11.6 ± 0.6 Å. The parabolic nature of the fit has the slope of the curve becoming positive for larger amines, a result not observed in the data. A slightly better fit was obtained by including a viscous drag component (eqn (4)) proportional to the size of the amine compound (dashed lines). From this model a pore diameter of 12.5 ± 0.8 Å was obtained. The viscous drag component flattens the parabolic fit, better representing the data. A problem with this analysis is that Ca2+ was required in the external solution during measurements in order to maintain the integrity of the transduction process. As Ca2+ most likely binds in the channel pore and affects permeation, these estimates may have some inherent error. If Ca2+ permeation was a constant percentage of current flow, regardless of the monovalent ion, then the error will be negligible (based on eqn (4)). If the monovalent permeation is a non-linear function of calcium then we cannot assess the degree of error. However, lowering external Ca2+ to 50 µM did not alter the apparent permeability of the amine (see below), supporting the conclusion that the methodology was reasonable.
Penetration into the membrane electric field
To further explore the pore region, current–voltage plots were obtained while mechanically stimulating the bundle to maximal ‘current on’ and ‘current off’ positions (Fig. 10D–E) in the presence of different tetra-n-alkyl amines. The current traces of Fig. 10D demonstrate that the inward currents (at negative potentials) were reduced much more than the outward currents (positive potentials). Plots of the total MET current normalized to the control current in Na+ at –120 mV were plotted for each of the tetra-n-alkyl amines in Fig. 10E to further illustrate the rectification. These plots were fitted by eqn (5) in order to estimate the relative distance through the membrane electric field travelled by the cation before blocking or binding. The distance travelled into the membrane electric field in the presence of Na+ was 0.48 ± 0.03 (n= 6) (presumably Ca2+ is the blocking compound). Similar distances were obtained for the amine compounds: 0.41 ± 0.02 (n= 6) for tetramethylammonium, 0.39 ± 0.08 (n= 6) for tetraethylammonium and 0.32 ± 0.07 for tetrapropylammonium (n= 6), suggesting the molecules approached a similar region of the pore. A funnel-shaped pore region is indicated by the slight trend for smaller molecules to penetrate further than larger amines. That each compound could reach a similar position indicates that the mouth of the channel must open rapidly to a vestibule that can accommodate compounds of these larger sizes, 14 Å. The reversal potential obtained from these measurements also shifted to hyperpolarized values: 9 ± 4 mV for control (n= 6), –12 ± 2 mV for tetramethylammonium (n= 6), –19 ± 9 mV for tetraethylammonium (n= 6) and –15 ± 7 mV for tetrapropylammonium (n= 6). Tetrabutylammonium antagonized the current completely at all potentials. Careful examination of the plots of Fig. 10E illustrates the difficulty in using reversal potential measurements with this channel. The plot flattens with the amine compounds in a manner similar to that observed with methylene blue, making the estimate of reversal potential difficult; this is also illustrated by the large errors associated with the measurement. An unexpected finding was the reduction of outward current at positive potentials, a phenomenon also observed in other non-specific cation channels where it was argued that the increased hydrophobicity of the larger amines favoured hydrophobic interactions within the channel (French & Shoukimas, 1985; Sanchez et al. 1986). Thus entering the channel is more energetically favourable than exiting because the hydrophobic vestibule stabilizes the amine. This type of interaction within the pore might also explain the tetrabutylammonium results, implicating a hydrophobic vestibule in the pore region.
Pore length
Assuming a cylindrical pore, an estimate of channel diameter and single channel conductance allows an estimate of channel length to be made (eqn (6)). Recent single channel conductance estimates for this papilla location were 150 pS in 2.8 mM Ca2+, while lowering external Ca2+ to 50 µM resulted in a much larger single channel conductance of 300 pS (Ricci et al. 2003). The mechanism responsible for this variation in conductance is unknown. Because experiments were done at 2.8 mM Ca2+, the initial estimate of pore length used 150 pS as the single channel conductance in eqn (6), yielding a length of 72 Å, about three times the width of the plasma membrane. Using 300 pS, however, gave an estimate of 31 Å, similar to that for other channels including mechanically gated channels (Cruickshank et al. 1997). Assuming a channel length of 72 Å is incorrect suggests that differences in measured values of single channel conductance were not due to a change in the diameter of the pore. That is, perhaps the single channel conductance is a constant but the apparent lower value in high Ca2+ was due to rapid flickering behaviour that the recording speed could not resolve. Caution must be exercised with this interpretation because although the channel appears non-rectified in 2.8 mM Ca2+ it is clearly rectified in lower Ca2+ concentrations (Crawford et al. 1991; Ricci et al. 2003). This observation suggests that the pore structure is more complex than a simple cylinder. To further test whether pore diameter changed with external Ca2+ the relative permeability of tetramethylammonium was measured in normal 2.8 mM and low 50 µM Ca2+, the prediction being that the relative current would increase as extracellular Ca2+ was lowered due to a change in pore width from 9 Å (estimated from rearranging eqn (6) to solve for diameter) to 12.5 Å. No difference in relative current flow was observed, however. Lowering extracellular Ca2+ relieved the Ca2+ block of the channel so the total current amplitude increased (Ricci & Fettiplace, 1998). The ratio of current in 0.05 mM to that in 2.8 mM Ca2+ with either Na+ or tetramethylammonium as the monovalent ion did not differ (1.4 ± 0.3 versus 1.5 ± 0.4: P > 0.05, n= 6), indicating that the permeability of the amine was unaffected by external Ca2+.
Single channel measurements show an increase in conductance with papilla location, suggesting a difference in pore diameter (Ricci et al. 2003). A test for a difference in diameter comparing current ratios obtained with tetramethylammonium at high (0.18 ± 0.01, n= 7) and low (0.20 ± 0.02, n= 6) frequency positions did not reveal any statistical difference (P > 0.05). What then might be regulating the single channel conductance? It is possible that the methodology is not sensitive enough to resolve a difference. More experiments are required to better resolve this issue.
Regardless of absolute value, the data suggest the MET channel has a long pore region, consistent with previous measurements indicating the channel may show anomalous mole fraction behaviour (Lumpkin et al. 1997).
Open channel block
The pharmacological data can now be re-evaluated in terms of molecular dimensions as opposed to channel classification. The hypothesis tested earlier was that amine-containing compounds like diltiazem, D600 and tetracaine, acted as open channel blockers where the partially charged amines penetrated the channel pore and the large R-groups attached to the amines prevented further penetration into the channel, like a cork in a bottle. Data obtained with curare, methylene blue, atropine and hexamethonium all support this hypothesis. Cross-sectional areas of each compound were determined from CPK models (Fig. 11A). Minimal diameters were measured about the region of the drug thought to penetrate the channel. As with the amine experiments, the diameter of a circle that the portion of the compound thought to penetrate the channel could pass through was taken as the minimal diameter of the amine. All of the antagonists had a minimal diameter smaller than that of the channel pore, suggesting the compounds could enter the channel and approach its narrowest region (see Table 1 and Fig. 11B). A plot of the minimal diameter against IC50 shows a good correlation (r2= 0.91) (Fig. 11B). This relationship suggests that the size of molecule binding in the pore dictates occlusion of the pore. Note that atropine, which has its amine in a ring structure, was an outlier in this plot. Perhaps the rigidity of the ring structure sterically limited atropine's ability to penetrate the pore.
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Further support for the open channel block theory comes from the correlation between the IC50 of the block and the length of the blocking agent (Fig. 11C). This relationship suggests the longer the compound, the more potent the block. Here, the idea is that the distance between the charged amine and the bulky R-group determines how close the charged amine can get to the minimal diameter of the pore. However, this correlation only held for the charged amine derivatives and not for other compounds (indicated by open symbols). In fact, the estimated channel length (31 Å) and the estimated distance through the electric field (0.45) give an approximate distance 14 Å into the pore that the drugs need to travel in order to reach the binding site of. Despite these being only crude estimates, the most effective amine compounds had lengths longer than this value, suggesting the amine could reach the binding site while the bulkier portion of the compound could remain near the external face of the channel.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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MET channel: diltiazem, D600, tetracaine, lanthanum, ruthenium red, quinine, curare, atropine, hexamethonium and methylene blue. Five compounds were ineffective as antagonists and neither pH nor osmolality had significant effects on the MET current. This pharmacological profile indicates that the MET channel is unique relative to other non-specific cation channels in that antagonists of CNG, TRP and hemi-gap channels, as well as nicotinic channel blockers, were effective antagonists at hyperpolarized potentials. Although the profile does suggest that the MET channel is most similar to CNG and TRP channels, a caveat to this conclusion is that many of the newly identified TRP and CNG channels have not had complete pharmacological evaluations. A hypothesis is presented regarding drug actions that incorporates the molecular dimensions of the pore. The model also postulates a mechanism for Ca2+ permeation and block of the channel. The quantitative estimates of channel dimensions serve to underscore the proposed mechanism of drug action and so are discussed first.
Molecular dimensions
n-Alkyl amines were used to estimate both the minimal pore diameter and its position in the membrane electric field. A diameter of 12.5 ± 0.8 Å is considerably larger than that reported for other cation channels including nicotinic channels (9.2 Å; Dwyer et al. 1980; Dani, 1989), calcium channels (6 Å; McCleskey & Almers, 1985), NMDA channels (7.2 Å; Zarei & Dani, 1995), voltage-gated potassium channels (4.7 Å; Bezanilla & Armstrong, 1972), voltage-gated sodium channels (6.1 Å; Hille, 1971) and CNG channels (9.2 Å; Balabramamian et al. 1995). Moreover, the diameter of the MET channel is considerably smaller than that reported for other mechanically gated channels (40 Å) (Cruickshank et al. 1997). Although making predictions regarding molecular dimensions from macroscopic recordings is not the most precise method, work on gramicidin channels and alamethacin channels has given estimates remarkably close to crystallized structures (Eisenberg et al. 1977; Rosenberg & Finkelstein, 1978). The model assumes a symmetrical cylindrical pore that is most likely an oversimplification but allows for comparisons between known channels. The model does not take into account charges within the pore that might serve as a binding site for the amines, though the relationship between the current ratio and molecular weight suggests binding is not an issue (see Sun et al. 1997). That the channel acts as a molecular sieve is also supported by the rather small differences in monovalent ion permeabilities (Fig. 1D and E), despite following an Eisenmann series XI suggestive of a highly electronegative pore (Adams et al. 1980).
Another possible source of error is the requirement for extracellular [Ca2+] to maintain functional channels, resulting in the use of current ratios as opposed to reversal potential changes to
, in the absence of blocker; •, in the presence of blocker). The fits to eqn (5) for control data are shown as continuous lines where K= 0.29 ± 0.02 and 0.29 ± 0.02, 
) had sensitivities similar to that of high frequency cells. Low frequency position was d= 0.34 ± 0.04 (n= 6). Dose–response curves were fitted to the peak (continuous curve) and steady-state (dashed curve) resulting in IC50 values of 16 ± 1 µM and 6.3 ± 0.8 µM and Hill coefficients of 2.1 ± 0.3 for both peak and steady state currents, respectively (r2= 0.99). C, the rate constant was estimated from the fit to the current decay (seen in A) at each dose and plotted against concentration. A linear fit with an intercept of 0.11 ± 0.004 ms–1 and a slope of 0.02 ± 0.002 ms–1µM–1 was applied to the data, indicative of a first order reaction (r2= 0.99). D–F, the open channel nature of the block was also confirmed by the reduced block at depolarized potentials. D and E, examples of the maximal transducer currents obtained at hyperpolarized and depolarized membrane potential in the absence (D) and presence (E) of 100 µM curare; mechanical stimulus shown above current traces. Depolarization indicated by dashed line. F, a plot of transducer current amplitude against membrane potential in the absence (
) of MB. MB block was reduced at both extremes of polarization. Fits (continuous curve) to the single site binding model (eqn (5)) were k= 0.8 ± 0.6 and 0.007 ± 0.003, 