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(Circulation Research. 2003;93:726.)
© 2003 American Heart Association, Inc.

Cellular Biology

Proton Permeation Through the Myocardial Gap Junction

Massimiliano Zaniboni, Alessandra Rossini, Pawel Swietach, Nurindura Banger, Kenneth W. Spitzer, Richard D. Vaughan-Jones

From the Burdon Sanderson Cardiac Science Centre (M.Z., A.R., P.S., N.B., R.D.V.-J.), University Laboratory of Physiology, Oxford, UK; Dipartimento di Biologia Evolutiva e Funzionale (M.Z., A.R.), University of Parma, Parma, Italy; Nora Eccles Harrison Cardiovascular Research and Training Institute (K.W.S.), University of Utah, Salt Lake City, Utah.

Correspondence to Prof Richard D. Vaughan-Jones, Burdon Sanderson Cardiac Science Centre, University Laboratory of Physiology, Parks Road, Oxford OX1 3PT, UK. E-mail richard.vaughan-jones{at}physiol.ox.ac.uk

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 
Although protons can directly or indirectly gate solute permeability of the myocardial gap junction, there is little information regarding their own permeation, despite their importance in the regulation of myocardial contractility and rhythm. By pipette-loading of acid into guinea pig isolated, ventricular myocyte pairs while imaging pH_i confocally using SNARF fluorescence, we have observed that protons permeate the junctional region. Permeation is inhibited by glycyrrhetinic acid, an agent that also increases intercellular electrical resistance, suggesting H⁺ permeation via gap junctions. The rate of spread of acid between cells appears to be limited by junctional permeation rather than by cytoplasmic diffusion. Mathematical analyses, combined with experiments using SNARF as a proton carrier, suggest that gap junctional H⁺ transmission may be accomplished physiologically by the permeation of intrinsic "proton-porter" molecules. We propose that proton flux through gap junctions will contribute to the dissipation of regional acid loads within the myocardium. This represents a mechanism for the local control of myocardial pH_i.

Key Words: H⁺ permeation • gap junctions • cardiac myocytes

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 
Changes of intracellular pH (pH_i) affect both the contractile and electrical behavior of the heart.^1–4 While stimulation of sarcolemmal acid extrusion normally compensates for an intracellular acidosis,⁵ this need not be the only regulatory mechanism. Indeed, under circumstances of ischemia, the activity of sarcolemmal acid transporters appears to be compromised⁶ so that other means for removing intracellular acid become more important. When acid generation is localized, as occurs during regional ischemia,⁷ its effects on pH_i may conceivably be dissipated by simple H⁺_i diffusion into adjacent, nonaffected cells. Recent work, however, has shown that diffusion of intracellular H⁺ ions is slow,^8,9 being retarded about 200-fold by cytoplasmic buffers, which may slow pH_i compensation via this route.

A further barrier to spatial H⁺_i dissipation is likely to be the intercalated disk. Gap junctions are multimers of connexin (Cx) proteins that assemble between adjacent myocytes, the dominant connexin in ventricular tissue being Cx43.¹⁰ The assembly forms an intercellular channel, allowing passage of many intracellular solutes, including ions, thus establishing a chemical and electrical syncytium in the heart.¹¹ While protons may permeate the channel, the question of junctional H⁺ transmission has not hitherto been investigated. Gap junctional permeability to solutes is reduced by a fall of pH_i,^12–14 potentially limiting H⁺ translocation. Full junctional inhibition, however, requires very low pH_i (<6.5 U) and has been attributed partly to a simultaneous elevation of Ca²⁺_i.^15,16 Less extreme physiological conditions may therefore favor significant H⁺ ion permeation. Given that H⁺_i diffusion occurs via a shuttling among buffers such as amino acids, peptides, and proteins,^8,9 and given that various amino acids and small peptides translocate the gap junction,^17,18 it is possible that physiological H⁺ transmission between myocytes is set by the junctional permeability of intracellular buffer molecules rather than by pH_i-dependent gating.

We have used confocal imaging of pH_i to investigate intercellular transmission of acid in pairs of ventricular myocytes, digested enzymically from guinea pig hearts. Acid was introduced directly into one cell from a glass micropipette, and its passage into the second cell was monitored using an intracellular pH fluorophore, carboxy SNARF-1. We explored the contribution of SNARF, itself, to any acid transmission, given that the dye acts as a mobile carrier of protons.⁸ All results were analyzed using a two-dimensional model of H⁺ diffusion that incorporates a term representing the apparent intercellular H⁺ permeability constant, P_H^app. The above combination of experimental and computational methodology allowed an assessment of the role that gap junctions may play in regulating the spread of acid within the myocardium.

A preliminary report of this work has been published.¹⁹

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 
Cell Isolation
Ventricular myocytes were isolated, as described previously,¹⁶ from albino guinea pigs weighing 350 to 450 g, supplied by D. Hall Ltd, Bicester, UK (killed humanely, according to UK Home Office recommendations, by concussion and cervical dislocation), using a combination of enzymic and mechanical dispersion (0.7 mg/mL collagenase, Roche and 0.04 mg/mL protease, Sigma). The cells were finally suspended in HEPES-buffered Dulbecco’s modified Eagle’s medium at room temperature (20°C) until use. The yield of cell pairs, variously connected (end-to-end, side-by-side, and intermediate), was usually low (5%). Experiments were performed within 24 hours of isolation.

Solutions
Normal Tyrode’s contained (in mmol/L) NaCl 135, KCl 4.5, MgCl₂ 1, CaCl₂ 2, glucose 11, and HEPES 20, adjusted to pH 7.4 with 1 mol/L NaOH at 37°C. The pipette filling solution used in electrical-coupling measurements contained (in mmol/L) KCl 113, NaCl 10, glucose 5.5, K₂ATP 5, MgCl₂ 0.5, KOH 11, and HEPES 10, adjusted to pH 7.1. In order to pipette-load acid into myocytes, suction pipettes were filled with an isotonic solution containing (in mmol/L) KCl 140, MgCl₂ 0.5, glucose 5.5, and HCl 1, ie, pH 3.0. When pipette-loading myocytes with unesterified carboxy-SNARF-1, 400 µmol/L of its free-acid form (Molecular Probes) was incorporated in a filling solution containing (in mmol/L) KCl 140, MgCl₂ 1, and HEPES 10, adjusted to pH 7.1 with 1 mol/L NaOH. 18- or ß-glycyrrhetinic acid obtained from Sigma-Aldrich. Cariporide was kindly supplied by H.W. Kleemann of Aventis (Germany).

Confocal Imaging of pH_i
pH_i was imaged^9,20,21 at 37°C in superfused myocyte pairs, preloaded for 10 minutes (room temperature) with 10 µmol/L carboxy-SNARF-1-AM (acetoxymethyl ester; Molecular Probes). A Leica-DM IRBE confocal microscope (with Leica TCS-NT software and a x40-NA 1.25 oil-immersion planoapochromat objective lens) was used for imaging. SNARF was excited at 514 nm from an argon laser. Emission was measured at 580±20 nm and >610 nm, respectively, by two photomultiplier tubes, and expressed as a ratio (580/>610; NIH Image software). A transmitted light detector provided a nonfluorescent image of the cells. Images (256x256 pixels) were acquired online at a frame rate of 0.5 Hz. The pH dependence of the ratio was calibrated using the nigericin technique, as described previously.²²

Intracellular Solute Loading and Electrophysiology
Micropipettes, when filled, had a resistance of 1 to 4 M. Transmembrane potential (V_m) was monitored and current injection performed, using two bridge circuits of an Axoclamp 2B amplifier (Axon Instruments).

Intercellular Electrical Coupling
R_j was estimated in cell pairs held in double whole-cell configuration. Hyperpolarizing current (0.15 nA, 70 ms) was injected into one cell and then the other, while measuring electrotonic voltage displacements in both. By using these data, the equivalent delta circuit was solved for R_j (a lumped parameter that includes a small contribution from cytoplasmic resistance).²³

Estimating H⁺_i Mobility and Junctional Permeability
When considering mobility, we refer to H⁺ rather than counter OH^- ion diffusion although, at physiological pH values, these movements cannot be distinguished.^8,9,24 The same convention is adopted for intercellular H⁺ (as opposed to OH^-) permeation. The spatiotemporal characteristics of [H⁺]_i during pipette loading were analyzed, using the finite element method (FEM), by solving the diffusion equation in two dimensions, as described previously for single myocytes^9,24:

where u is the rise in H⁺_i concentration above control levels, D_H^app is apparent H⁺ diffusion coefficient, and Q is apparent rate of H⁺ loading by pipette, assumed to be constant (pipette pH<<cytoplasmic pH). An array of coordinates (x, y) is generated by applying Delaunay triangulation (MATLAB; Mathworks Corporation) within a domain, , defined by the outline of the cell pair (Figure 1C). Boundary points are ascribed to the junctional region (_junct) and the nonjunctional region (_out).

View larger version (51K): [in this window] [in a new window]   Figure 1. A, Transmission image of end-to-end cell pair; note pipette tip at lower end. B, Ratiometric, pseudocolored confocal image of intracellular SNARF fluorescence from cell pair shown in A (decreasing acidity: orange, yellow, white); position of pipette tip is circled. C, Delaunay-triangulated outline of same cell pair with junctional region indicated in green.

Boundary conditions across _junct are conditioned as

where P_H^app is the junctional H⁺ permeability constant.

Nonjunctional boundary conditions feature H⁺_i reflection (ie, zero transporter-mediated H⁺ extrusion):

Since the diffusion equations are linear, the FEM solutions for [H⁺]_i in time or space may be scaled to fit the amplitude of the data (equivalent to scaling the H⁺ injection rate, Q). This is repeated for different pairs of values of D_H^app and P_H^app until the least-squares best fit is obtained, where ² giving P>0.9 was deemed acceptable.

Statistical Analysis
Results are presented as mean±SEM. Statistical analysis was performed using Student’s t test for unpaired data.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 
Imaging Junctional H⁺ Permeation
Figure 1A shows the transmission image of a cell pair superfused with Tyrode’s (pH 7.40) containing 30 µmol/L cariporide, a potent NHE-1 inhibitor (to prevent sarcolemmal acid extrusion). A glass micropipette (filled with isotonic KCl at pH 3.0) was sealed, in whole-cell configuration, toward the end of the first cell. Figure 1B illustrates a ratiometric image of SNARF_i fluorescence obtained from the same cell pair. Figure 1C shows the outline with Delaunay triangulation applied. This is required (see Materials and Methods) for running the FEM model of H⁺_i diffusion/permeation in order to derive values for D_H^app and P_H^app.

Figure 2A plots the time course of fall of pH_i averaged in four downstream regions of interest (ROIs) after break-in by the pipette. The horizontal dashed line represents a fall in pH_i of 0.1 U, equivalent to a rise in [H⁺]_i of about 20 nmol/L. This level of acidification was observed at the distal end of cell 1, 46 seconds after break-in, and about 35 seconds later in cell 2. Figure 2B shows data converted into increases of [H⁺]_i. Also plotted here are the best-fit time courses obtained from the FEM algorithm (see Materials and Methods). Figure 2C shows data averaged for 6 cell pairs where acid loading was followed for nearly 5 minutes after break-in (similar results were obtained for two other cell pairs monitored for 80 seconds each after break-in). In a total of 8 cell pairs (4 end-to-end and 4 side-by-side/intermediate pairs), D_H^app estimated using the FEM algorithm, was 12.43±3.02x10^-7 cm²/s, not significantly different from that determined recently⁹ in guinea pig single ventricular myocytes (12.1x10^-7 cm²/s). Mean P_H^app was 2.03±0.66x10^-4 cm/s (n=8). Results therefore demonstrate that acid permeates the junctional region.

View larger version (22K): [in this window] [in a new window]   Figure 2. A, Time course of change in pHi after break-in by acid-filled pipette; data averaged in four circular ROIs, diameter 10 µm, positioned (see inset) down central, longitudinal axis of cell pair image (ROI centers 11, 69, 100, 164 µm from pipette, pipette at 12 µm from cell edge). Superfusate contained 30 µmol/L cariporide. B, Data for first 110 seconds of acid loading shown in A, converted to [H+]i increase (nmol/L); continuous lines are best fits by FEM model, for DHapp=12.2x10-7 cm2/s and PHapp=4.1x10-4 cm/s. C, pHi averaged for cell 1 and cell 2 (ie, pre- and postjunctional ROIs were set to equal whole-cell area) plotted versus time for 6 cell pairs perfused with cariporide (30 µmol/L).

Figure 3A shows results of an experiment where the profile of [H⁺]_i was measured down the longitudinal axis of a cell pair. After break-in, there was an elevation of [H⁺]_i that was largest close to the pipette. After 60 to 100 seconds, a spatially localized fall of [H⁺]_i (20 nmol/L) was evident across the junctional region, connecting with a lower level in the second cell. This indicates that, with respect to H⁺ movement within the cell pair, its permeation across the junction was rate-limiting. For ease of comparison, the spatiotemporal predictions for [H⁺]_i obtained from the FEM model have been plotted on identical coordinates in Figure 3B, whereas Figure 3C superimposes original data with results of the model. Similar results were seen in 13 cell pairs analyzed in this way (n=8, in the presence, and n=5 in the absence of 30 µmol/L cariporide). The good agreement between experiment and model strongly suggests that proton movement within a cell pair is diffusive, coupled to passive intercellular permeation.

View larger version (48K): [in this window] [in a new window]   Figure 3. A, Spatiotemporal behavior of [H+]i during local acid loading by pipette, measured in a longitudinal ROI (10 µmx190 µm) incorporating the gap junction (see gray bar in top diagram); pipette positioned 10 µm from end of cell. Green arrow indicates moment of break-in (time zero, seconds); red arrow indicates position of gap junction (130 µm along ROI). B, Result of best fit to data by FEM model, giving DHapp=12.0x10-7 cm2/s and PHapp=1.0x10-4 cm/s. C, Data in A and B have been superimposed. Note abrupt fall of [H+]i across junctional region.

Contribution of Intracellular SNARF to Junctional H⁺ Permeation
Given that the pK_a for SNARF is 7.6, it will act as an intracellular mobile buffer. Nevertheless, because of its low intracellular concentration in cardiac cells (400 µmol/L) and relatively low mobility coefficient (1 to 3x10^-7 cm²/s), it accounts for <1% of H⁺_i mobility.^8,9 The extent of its contribution to acid flux across the junctional region has not, however, been assessed.

Figure 4A shows results of an experiment where diffusion of SNARF from a micropipette into a cell pair was imaged confocally. The longitudinal profile of [SNARF]_i was recorded at various times after break-in. The spatiotemporal behavior of SNARF_i was well described by the same FEM model as used for analyzing H⁺_i diffusion (Figure 4B). Figure 4C superimposes results of experiment and model. In 4 cell pairs, apparent intracellular SNARF mobility (D_SNARF^app) was 3.7±0.6x10^-7 cm²/s and apparent junctional SNARF permeability (P_SNARF^app) was 0.4±0.2x10^-4 cm/s (n=4). The value for D_SNARF^app is similar to that reported previously⁹ for the guinea pig single ventricular myocyte (3x10^-7 cm²/s). The value for P_SNARF^app is of the same order of magnitude as that reported previously for other fluorescent dyes of similar molecular weight, such as lissamine rhodamine and Lucifer yellow (0.74 and 2.8x10^-4 cm/s, respectively²⁵). Interestingly, the value is also comparable to that derived above for intracellular H⁺ ions (2.0x10^-4 cm/s).

View larger version (43K): [in this window] [in a new window]   Figure 4. A, Spatiotemporal behavior of [SNARF]i during local loading by pipette, measured in a longitudinal ROI (5 µmx82 µm; see cartoon at top; pipette positioned 65 µm from end of cell). Ordinate shows [SNARF]i (µmol/L), calibrated by assuming fluorescence intensity (emission at 640 nm), is proportional to dye concentration, and that steady-state [SNARF]i=400 µmol/L (concentration in pipette). B, Results of best fit to data by FEM model, giving DSNARFapp=1.5x10-7 cm2/s and PSNARFapp=0.4x10-4 cm/s. C, Experimental and model data superimposed. Note abrupt fall of [SNARF]i across junctional region.

As outlined in the Appendix, the contribution of intracellular SNARF to the value derived for P_H^app may be estimated using the "proton permeability" equation:

assuming that ß_SNARF<<ß_int, where P_H^app is the increase caused by junctional H⁺_i shuttling on SNARF, ß_int is the cell’s intrinsic buffer capacity (about 27 mmol/L at pH_i 7.1), and ß_SNARF is the intracellular buffer capacity of the fluorophore (160 µmol/L at pH_i 7.1). The predicted increase of P_H^app is 1.1x10^-6 cm/s, an increase of <1.0%. In the present work, junctional permeability to SNARF will therefore have exerted a negligible influence on the total junctional flux of acid.

Pathway for Junctional H⁺ Permeation
An important question is whether H⁺ transmission occurs through connexin channels or whether other routes are involved. For example, NHE proteins are highly expressed at the intercalated disk,²⁶ but their possible contribution can be excluded as H⁺ transmission persists in the presence of 30 µmol/L cariporide.

Effect of Glycyrrhetinic Acid on H⁺ Transmission
Figure 5A shows an experiment where acid was pipette-loaded in the presence of the gap junctional inhibitor, ß-glycyrrhetinic acid (ßGA; 60 µmol/L). No H⁺ transmission was observed. A similar result was found when applying 60 µmol/L GA (not illustrated).

View larger version (16K): [in this window] [in a new window]   Figure 5. A, Block of acid transmission by 60 µmol/L ßGA. Rise of [H+]i averaged in four 5-µm-diameter ROIs 6, 57, 85, and 143 µm downstream from an acid-filled pipette (see cartoon at top right). Note lack of H+i rise in distal cell. Continuous lines best fit by FEM model (DHapp=2.7x10-7 cm2/s, PHapp=0.0x10-4 cm/s). B, Mean values derived for PHapp with and without GA in the superfusate. *Significantly different from control, P<0.05. C, Mean values for DHapp. All superfusates contained 30 µmol/L cariporide.

Time-course data for intracellular acid loading in the presence of or ßGA were fitted using the FEM model, in order to derive values for P_H^app (Figure 5B) and D_H^app (Figure 5C). GA reduced mean P_H^app about 20-fold while ßGA reduced it about 10-fold. In contrast, neither agent significantly affected the value derived for intracellular D_H^app. These results suggest that and ßGA block H⁺ movement through gap junctions while having no effect on its movement in cytoplasm.

Effect of GA on Junctional Resistance
Figure 6 illustrates a double-patch experiment on a cell pair (Figure 6A) in order to estimate junctional resistance, R_j (see Materials and Methods). Figure 6B illustrates the cycle of current pulses applied through the pipettes. Toward the middle of each cycle, a brief depolarizing current pulse was used to trigger an action potential (AP) in cell 1, to see if it propagated to cell 2.

View larger version (35K): [in this window] [in a new window]   Figure 6. Measurement of intercellular electrical resistance (Rj). A, Cell pair with two suction pipettes attached; arrow indicates junctional region. B, Protocol (repeated at 1 Hz) for current injection through pipettes. Pulses a and b (0.15 nA, 70 ms) elicit electrotonic voltage displacements (V) in both cells (labeled a and b in panel C). Pulse c (3 ms) elicits an action potential (AP) in cell 1 that propagates to cell 2. C, Time course for changes of V amplitude in both cells; for clarity, results of only every 40th current-injection cycle have been displayed (triggering of AP also not shown); Rj estimated at arrows is listed below; insets at bottom show (black trace) AP triggered in cell 1 and (red trace) electrical response in cell 2.

Figure 6C shows the time course for changes in electrotonic potential amplitude (V) elicited by current injection after superfusing 40 µmol/L ßGA. At 450 seconds, V had increased in the injected cell but had decreased in the follower cell, indicating a rise in junctional resistance. This was associated with delay in junctional propagation of the evoked AP. At 810 seconds, junctional propagation of the AP was inhibited. The control estimate for R_j (before ßGA) was 6 M, and rose to 570 M after 810 seconds of exposure to the drug. Despite intrinsic limitations in current-clamp estimates of R_j,²³ the experiment clearly shows electrical uncoupling, reflected in changes of subthreshold responses and action potential propagation. Such behavior is far greater and faster than the rate of spontaneous uncoupling reported for guinea pig ventricular cell pairs²⁷ and must be due to effects of ßGA. Interestingly, there was little effect on action potential amplitude or resting membrane potential in either cell (resting potential hyperpolarized from -80 to -84 mV; not shown). Similar results were observed in 2 further experiments that lasted for a shorter time (450 seconds), enough for ßGA to raise R_j to 80 to 110 M, about 16 times the resting R_j. Overall, the results are consistent with ßGA inhibiting H⁺ transmission by reducing gap junctional conductance.

Effect of GA on Sarcolemmal Acid Extrusion
To check further the selectivity of GA, we tested its effect on the time course of whole-cell pH_i recovery from an intracellular acid load (induced by prepulsing an isolated single ventricular myocyte with 30 mmol/L extracellular ammonium chloride⁵; not shown). In cells superfused with HEPES-buffered Tyrode’s, pH_i recovery was unaffected by the presence or absence of 60 µmol/L GA (n=38), demonstrating no effect on sarcolemmal NHE activity.

Effect of pH_i on Junctional H⁺ Permeability
Given that intercellular resistance is increased by acidosis,^12,13 we investigated if P_H^app was affected by the level of acid loading encountered in our experiments. In the present work, P_H^app was quantified for the first 60 seconds after break-in by the pipette (see Figure 2). Figure 7 shows P_H^app recorded in 8 cell pairs, plotted as a function of prejunctional pH_i, measured in ROI-2 (see inset at top of Figure 2) 60 seconds after break-in. Over the pH_i range, 7.20 to 6.79, there was no correlation between P_H^app and pH_i (correlation coefficient=0.095). This suggests that a modest acidosis (up to 0.40 pH units) may not significantly affect P_H^app.

View larger version (8K): [in this window] [in a new window]   Figure 7. Relationship between pHi and PHapp. Superfusates contained 30 µmol/L cariporide.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 
The present work establishes that intracellular H⁺ ions permeate the cardiac gap junction. While cytoplasmic H⁺_i mobility is low,^8,9,24 analysis of the longitudinal [H⁺]_i profile down a cell pair reveals that the rate-limiting step for H⁺ translocation is the junctional transfer itself. This implies that when a localized acidosis is both large and rapid, intercellular H⁺ transmission will occur, but H⁺ ions will also back up in the affected cell, as demonstrated in Figure 3. The resulting local fall of pH_i will activate H⁺ extrusion on sarcolemmal transporters such as NHE, thus attenuating further transfer of acid into adjacent cells. Intercellular dissipation of acid, and its regulation by the gap junction, therefore imposes a local control on pH_i that may be of functional importance to the myocardium.

Possible Mechanism of H⁺ Transmission: A Permeant Proton Porter
Within the cytoplasmic compartment, intrinsic H⁺_i movement occurs via a shuttling on intracellular mobile buffers, with little parallel diffusion of H⁺ ions.^24,28,29 Junctional H⁺ permeation may therefore operate via a similar mechanism. Figure 8 illustrates a cycle of protonation and deprotonation occurring within cytoplasm (note competition for H⁺ binding from fixed intrinsic buffers). Protonated mobile buffer then passively permeates the gap junctional pore, moving down a concentration gradient, the H⁺ ion dissociating on encountering a more alkaline cytoplasmic region in the distal cell. The shuttle is completed by permeation back of the deprotonated buffer. On such a model, H⁺ flux through the gap junction is via permeant "proton-porter" molecules.

View larger version (53K): [in this window] [in a new window]   Figure 8. Proposed model for intercellular H+ transmission. Source of local acid is a cell-attached pipette. Protons locally titrate intracellular mobile buffer (M) or fixed buffer (F). Diffusion of protonated mobile buffer (HM) followed by distal deprotonation mediates cellular mobility of H+ ions. Permeation of HM through gap junctions (proton-porter step) followed by deprotonation in the distal cell mediates junctional H+ transmission.

Gap junctional channels, unlike classical ion channels, have pores of large diameter (at least 12Ź¹) compared with 4Å within the selectivity filter of the KcsA K⁺ channel³⁰ and so may be permeable to intracellular mobile buffers, although permeation may also be influenced by the molecule’s electrical charge.^18,31 The fluorophore, SNARF (molecular weight, 453; pK_a 7.6), may be regarded as a surrogate (albeit nonphysiological) mobile buffer, and it permeates the gap junction as shown in the present work. While we have no direct evidence for the operation of a junctional proton porter, it is striking that our estimate of the apparent junctional permeability for H⁺ ions is of the same order of magnitude as that for SNARF (5-fold difference). In contrast, the K⁺ ion, an unbuffered lower molecular weight solute, has a junctional permeability about 40-fold higher than for Lucifer yellow,³¹ a dye whose molecular weight (457) and junctional permeability are comparable to SNARF (25, with the present work).

The similarity of P_H^app and P_SNARF^app is suggestive of H⁺ permeation via a proton porter of molecular weight approaching SNARF. We have eliminated SNARF, itself, as the proton porter in our experiments, as its intracellular buffering capacity (0.16 mmol/L) and mobility (3.0x10^-7 cm²/s) are too low to support the observed junctional flux of acid. The proton porter could, however, be one or more of the cytoplasmic mobile buffers (eg, inorganic phosphate, taurine, and the dipeptides homocarnosine and acetylanserine^8,9). Their molecular weights are 100 to 300, and their intracellular concentrations are in the millimolar range. Moreover, various amino acids and small peptides have previously been shown to be junctionally permeant.^17,18 The summed capacity of intrinsic mobile buffer is 40% of total intrinsic buffering, and its capacity probably remains relatively constant (about 11 mmol/L/pH unit) over the physiological pH range.⁹ Mobile cytoplasmic buffer would therefore be an ideal candidate for the junctional proton porter.

Mathematical analysis of the proton porter model is presented in the Appendix. On such a model, junctional H⁺ permeability, like intracellular H⁺ mobility in cytoplasm,^24,28,29 is governed principally by the mobile buffer fraction (ß_mob/ß_int), in this case by the fraction of total intrinsic buffer capacity that is permeant, defined by the "proton-permeability" equation:

where P_mob is the junctional permeability of the proton porter pooled for all mobile buffers. At a resting pH_i of 7.10, when (ß_mob/ß_int) is about 0.40 (see above), this equation predicts a mean P_mob of about 6x10^-4 cm/s.

H⁺_i Gating of H⁺ Transmission?
A pH_i-dependent gating of gap junctions,^13,14 by increasing the probability of connexin channel closure during acidosis, would slow the intercellular permeation of H⁺ ions, thus potentiating prejunctional acid extrusion. In the present work, no correlation was detected between P_H^app and pH_i but, given the limited pH_i range (7.20 to 6.79), our experimental technique may have lacked sufficient resolution to detect small effects on P_H^app. Averaged data shown in Figure 2C, however, indicate that [H⁺]_i eventually rose at a similar, near-constant rate in both cells, suggesting little or no reduction of P_H^app during the course of an experiment. Nevertheless, we do not exclude inhibition of proton flux at lower pH_i values, particularly when these are associated with elevations of Ca²⁺_i. This would be consistent with previous work on cardiac gap junctions in situ^12,15,32,33 and on homotypic Cx43 channels expressed in Xenopus oocyte pairs^13,14 where decreases of pH_i to <6.50 can induce electrical uncoupling.

Possible Role of Carbonic Buffer
We have yet to investigate if H⁺ transmission is affected by the presence of CO₂/HCO₃^- (carbonic) buffer. This facilitates cytoplasmic H⁺ diffusion within the cardiomyocyte,^9,34 and so may conceivably accelerate proton traffic through the gap junction. Both CO₂ and HCO₃^- are likely to permeate connexin channels, but any effect on pH_i will be complicated by the fact that CO₂ released from the protonation of bicarbonate will also pass between cells via a parajunctional route, owing to its ability to permeate the lipid bilayer.³⁵ For a given cell pair, permeation of the distal cell by CO₂, followed by its hydration and dissociation to H⁺ and HCO₃^-, would be expected to acidify the postjunctional region. In contrast, permeation of connexin channels by HCO₃^- anions that subsequently combine with H⁺ would be expected to alkalinize the postjunctional region. The summed effect of CO₂ and HCO₃^- on intercellular acid transmission is therefore difficult to predict. Whatever its effect, however, the junctional shuttling of H⁺_i on intrinsic buffers will operate independently, representing a significant means for trafficking acid between cells.

Modulation of Junctional Coupling by Glycyrrhetinic Acid
The finding of electrical uncoupling by GA confirms a brief report of 75 µmol/L GA or ßGA decreasing intercellular conductance in mouse cardiac myocyte pairs.³⁶ GA also reduces junctional permeation of Lucifer yellow in heart,³⁷ while GA or ßGA decreases junctional coupling in various noncardiac tissues (see Reference ³⁶ for review). Of the two GA compounds used in the present work, we noticed that myocytes exposed to 60 µmol/L GA survived longer (up to 60 minutes) than those exposed to the same dose of ßGA (up to 30 minutes), suggesting that GA may be less cytotoxic. Although both agents exhibit nonselective actions, particularly at high concentration,³⁶ in the present work ßGA had little effect on resting membrane potential or action potential amplitude.

Junctional Control of Local pH_i
After a localized intracellular acidosis, junctional H⁺ permeability will determine the balance between sarcolemmal H⁺ extrusion and intercellular H⁺ transmission. Although H⁺ transmission may appear to be slow, total junctional flux of acid is considerable, given that most acid is buffered intracellularly. Junctional flux during a pipette-loading experiment is given by the term (dpH_i[d]/dtxß_int) where dpH_i[d]/dt is the rate of change of pH_i averaged for the whole of the distal cell. From data pooled in Figure 2C, this flux is estimated to be 1.7 mmol/L per min (ß_int is 30 mmol/L at pH_i 7.10 to 6.80).⁵ Over the same pH_i range, sarcolemmal acid extrusion via Na⁺-H⁺ exchange when not pharmacologically inhibited, increases from 0.15 to 2.5 mmol/L per min (rising to 3.5 mmol/L per min if Na⁺-HCO₃ cotransport activity is also factored in).⁵ Junctional flux is therefore comparable to sarcolemmal flux, indicating that dissipation of acid through gap junctions will play an important role in the local regulation of pH_i.

Regional ischemia is associated with localized intracellular acidosis,⁷ caused by the generation of lactic acid. It will therefore be of interest to determine if gap junctional routes for acid flux exist from ischemic to nonischemic myocardium. These would complement any spatial dissipation of acid mediated via CO₂ diffusion.³⁵ Although junctional permeability may be moderated by the large pH_i reduction and [Ca²⁺]_i elevation that occur during ischemia (eg, Dekker et al³⁸), it is far from certain whether these are sufficient to produce junctional closure. Indeed, during simulated ischemia, fluorescent dyes such as Lucifer yellow still appear to pass readily between cardiomyocytes.³⁷

	Appendix

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 
P_H^app Derived for a "Proton-Porter" Model
The flux of H⁺ over space variable x, with diffusion coefficient D_H, junctional-permeation constant P_H, and junctional H⁺ gradient H⁺, is given by

For intracellular protons, the equation is expanded to include unbuffered and buffered proton movement (the latter divided into permeant, HP, and impermeant buffers, HI, with diffusion and permeation constants of D_P, D_I, and P_P, P_I, respectively).

Assuming protonated permeant and impermeant buffer concentration is a function of total buffer concentration B_P and B_I, where a_P and a_I are functions of proportionality:

We take (where denotes the change above initial conditions H⁺₀ and a)

We assume that a is proportional to the change in proton concentration (H⁺), where is a constant:

Assuming that impermeant intracellular buffer is fixed and that mobile buffer is permeant, D_I, and P_I are zero:

From the definition of buffering capacity ß,

substituting and dividing by total intrinsic buffering capacity, ß_int,

Since the LHS expression in the bracket is by definition D_H^app (eg, Reference ²⁹), the RHS expression can be called P_H^app:

At physiological pH_i, this may be simplified to a "proton-permeability" equation:

where junctional H⁺_i permeability is proportional to the fraction of intracellular buffer that is permeant.

	Acknowledgments

 
This work was supported by grants from the British Heart Foundation and Wellcome Trust to R.D.V.-J., a Wellcome Trust studentship and Overseas Research Scheme award (UK) to P.S., grants from the National Heart, Lung, and Blood Institute (HL-42873) and Nora Eccles Treadwell Foundation to K.W.S., and CIRC Compagnia di San Paolo to M.Z. and A.R. and MIUR to M.Z.

	Footnotes

 
Original received May 23, 2003; resubmission received July 9, 2003; revised resubmission received August 22, 2003; accepted August 22, 2003.

	References

Top Abstract Introduction Materials and Methods Results Discussion Appendix References

 

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