Published online before print March 15, 2007, doi: 10.1161/01.RES.0000264071.11619.47
© 2007 American Heart Association, Inc.
Cellular Biology |
H+ Ion Activation and Inactivation of the Ventricular Gap Junction
A Basis for Spatial Regulation of Intracellular pH
From The Burdon Sanderson Cardiac Science Centre (P.S., A.R., R.D.V.-J.), Department of Physiology, Anatomy and Genetics, University of Oxford, UK; and The Nora Eccles Harrison Cardiovascular Research and Training Institute (K.W.S.), University of Utah, Salt Lake City.
Correspondence to Richard D. Vaughan-Jones, PhD, Burdon Sanderson Cardiac Science Center, University Laboratory of Physiology, Parks Rd, Oxford OX1 3PT, UK. E-mail richard.vaughan-jones{at}dpag.ox.ac.uk
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Abstract |
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H+ ions are powerful modulators of cardiac function, liberated during metabolic activity. Among their physiological effects is a chemical gating of cell-to-cell communication, caused by H+-mediated closure of connexin (Cx) channels at gap junctions. This protects surrounding tissue from the damaging effects of local intracellular acidosis. Cx proteins (largely Cx-43 in ventricle) form multimeric pores between cells, permitting translocation of ions and other solutes up to
1 kDa. The channels are essential for electrical and metabolic coordination of a tissue. Here we demonstrate that, contrary to expectation, H+ ions can induce an increase of gap-junctional permeability. This occurs during modest intracellular acid loads in myocyte pairs isolated from mammalian ventricle. We show that the increase in permeability allows a local rise of [H+]i to dissipate into neighboring myocytes, thereby providing a mechanism for spatially regulating intracellular pH (pHi). During larger acid loads, the increased permeability is overridden by a more familiar H+-dependent inhibition (H+ inactivation). This restricts cell-to-cell H+ movement, while allowing sarcolemmal H+ transporters such as Na+/H+ exchange, to extrude the acid from the cell. The H+ sensitivity of Cx channels therefore defines whether junctional or sarcolemmal mechanisms are selected locally for the removal of an acid load. The bell-shaped pH dependence of permeability suggests that, in addition to H+ inactivation, an H+ activation process regulates the ensemble of Cx channels open at the junction. As well as promoting spatial pHi regulation, H+ activation of junctional permeability may link increased metabolic activity to improved myocardial coupling, the better to meet mechanical demand.
Key Words: connexin 43 • gap junction channel • intracellular pH • ventricular myocytes
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Introduction |
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Protons generated within respiring tissue exert a profound influence on cellular function. In the heart, this includes effects on intracellular Ca2+ signaling,1 contraction,2 and electrical excitation.3 Indeed, much of the contractile failure and electrical arrhythmia associated with myocardial ischemia has been attributed to the ensuing decrease of pHi.4,5 In cardiac ventricular myocytes, an acute displacement of pHi from its normal value of
7.2 is typically corrected by the sarcolemmal movement of H+ ions, or their equivalent, on ion-transport proteins, such as Na+/H+ exchange (NHE) and Cl–/HCO3– exchange.6 These operate with high transport capacity during large changes of pHi, although their overall activity is low within a more physiological range of pHi values (6.95 to 7.25).6 Recent work on coupled mammalian ventricular myocytes shows that a localized intracellular acidosis also induces passive cell-to-cell H+ flow through junctional channels,7 suggesting an additional mechanism for regulating pHi. Gap junctions may dissipate localized acid/base disturbances, helping to maintain a more uniform pHi within the myocardium.
Gap junctions consist of 2 hemichannels (connexons) coupled in series between adjacent myocytes. In ventricular tissue, the hemichannels are typically made of connexin 43 (Cx-43) subunits,8 arranged as a hexamer around a central pore,9 permitting passage of solutes <1 kDa.10,11 H+ permeation through ventricular gap junctions occurs readily but not via the movement of free H+ ions. Rather, it occurs via the permeation of mobile buffer molecules that carry the H+ ion through the channel.7 Typical mobile buffers are histidyl dipeptides, such as homocarnosine and acetylcarnosine (of molecular mass, 100 to 200 Da12). Because intracellular mobile buffer concentration is relatively high (15 mmol/L12–14), significant quantities of H+ ions can be shuttled passively through the channel in response to a modest transjunctional pHi gradient.7
One problem with the suggested role of myocardial gap junctions in mediating the spatial dissipation of H+ ions is that intracellular acidosis closes connexin channels.15–23 Such chemical gating is believed to be mediated directly by H+ titration of specific sites on the cytoplasmic C terminus of each Cx-43 subunit21–23 and indirectly by an H+-induced rise of Ca2+i that also gates the channel.19,20 The extent to which this influences junctional H+ permeation is not known. By using techniques of cellular imaging, flash photolysis of an intracellular caged H+ compound, local cellular microperfusion, and cell-to-cell current injection, we have measured the ability of H+ ions and other solutes to flux through the gap junctions of isolated ventricular myocyte pairs, when pHi was manipulated to various levels. Implications of the results for the spatial control of myocardial pHi have then been explored using computational modeling. An intriguing finding is that an increase of [H+]i can enhance as well as inhibit gap-junctional permeability to various solutes, including the H+ ion itself. The results prompt a reevaluation of the chemical control of Cx channels by H+ ions and a reassessment of the functional effects of acidosis on metabolic and electrical coupling within the myocardium.
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Materials and Methods |
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Cell Preparation and pHi Imaging
Cell pairs were enzymically isolated from rat or guinea pig ventricles.6,7 Cells were loaded with 10 µmol/L acetoxymethyl (AM) carboxy-SNARF-1 or -4 (pK
7.5 and pK6.5, respectively) for 10 minutes.24 In some experiments, cells were coloaded with 100 µmol/L BAPTA-AM to provide additional intracellular Ca2+ buffering. Myocytes were then transferred to a poly-L-lysine–coated coverslip in a superfusion chamber, mounted on an inverted Leica TCS confocal microscope. Intracellular dye was excited at 514 nm (argon laser line), and fluorescence was measured at 580 nm and 640 nm (256x256 pixels every 2.1 seconds), ratioed, and converted to pHi.6,7 In experiments performed in the presence of CO2/HCO3– buffer, cell pairs were superfused with Tyrode containing 120 mmol/L NaCl, 4.5 mmol/L KCl, 22 mmol/L NaHCO3, 11 mmol/L glucose, 2 mmol/L CaCl2, 1 mmol/L MgCl2. Solutions were bubbled with 5% CO2 at 37°C (pH=7.4). In experiments performed in the absence of CO2/HCO3– buffer, NaCl was raised to 135 mmol/L and NaHCO3 replaced by 20 mmol/L Hepes (pH 7.4).
Photolytic Uncaging of H+ Ions
Rat ventricular myocytes were superfused with solutions containing 1 mmol/L membrane permeant, 2-nitrobenzaldehyde, a caged H+ donor.24 A small region of interest (ROI) (5x5 µm) in one cell was exposed to a double-flash of UV light (separation time, 1 second; 351 nm; 10 µs/pixel) once every 9 seconds. Between flash events, pHi was imaged and the time course of H+ loading averaged in four 15x15 µm regions in the cell pair (Figure 1A). Time courses were fitted with diffusion–permeation equations7 to estimate the apparent H+ junctional permeability constant (PHapp). PHapp is determined by junctional permeability to mobile buffer (Pmob) and by the fraction of total buffering that is mobile (fmob), PHapp=Pmob · fmob. Previous measurements of fmob13 were used to estimate Pmob from PHapp. The total rise of [H+]i in the cell pair was used to calculate H+ injection rate (Jinj=–dpH/dt · buffering capacity), whereas the [H+]i rise in the distal cell was used to compute junctional H+ flux (Jjunc), also expressed as a fraction of Jinj (for equal sized myocytes, maximum Jjunc=0.5 · Jinj).
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Inducing Steady-State Junctional H+ Flux
One pole of an end-to-end guinea pig myocyte pair was exposed (Figure 2A) to 10 to 30 mmol/L NH4Cl-containing Hepes-buffered Tyrode solution (with appropriate reduction of [NaCl] to maintain isotonicity). Such partial perfusion was performed using a square-bore, double-barreled micropipette (interstream boundary, <10 µm). The regional intracellular alkalosis evoked by local NH3 influx25,26 induces H+ flux across the junction, which stabilizes within 30 seconds.26 This produces a standing longitudinal H+i gradient for the duration of dual microperfusion. The rate-limiting H+ flux results in a step-junctional H+ discontinuity (Figure 2Aii) that can be analyzed26 to give a measure of Pmob, using the steady-state boundary condition: Dmob · 
[H+]=Pmob · 
[H+]. Dmob (mean diffusion constant for intracellular mobile buffer) has been measured previously in guinea pig myocytes13 (6.48 · 10–6 cm2/sec), and the size of the H+i discontinuity ([H+]) and the average of the [H+]i slopes ([H+]) leading into and out of the junction (linear regression over a 30-µm length) were calculated from the individual H+i profiles.26 Junctional pH (pHj) was calculated at mid-[H+]. In some experiments performed from a low-preset pHi, NH4Cl was replaced with 40 to 120 mmol/L Na/acetate (Figure 2B) to generate a local acidosis that drives an opposite junctional H+ flux.
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Junctional Permeability to SNARF
SNARF-1 (453 Da) was loaded into guinea pig cell pairs via a cell-attached patch pipette7 (ruptured patch; filling solution, 113 mmol/L KCl 10 mmol/L NaCl, 5.5 mmol/L glucose, 5 mmol/L K2ATP, 1 mmol/L MgCl2, 10 mmol/L Hepes, 400 µmol/L carboxy-SNARF-1 [free-acid], pH adjusted to 7.15 with 12 mmol/L KOH). Dye flux across the gap junction was imaged confocally from the rise of intracellular fluorescence in both cells (Figure 3A) and quantified in terms of a permeability constant, PSNARF.7
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Junctional Electrical Resistance
Junctional permeability was probed in guinea pig myocyte pairs by measuring junctional conductance to simple inorganic ions (principally K+ and Cl–, the main charge carriers) using junctional current injection27 (Figure 4A). This involved injecting small hyperpolarizing current pulses (200 to 500 pA, 300 ms) alternately into each cell (every 2 seconds) via suction pipettes (ruptured patch; Corning-8250 glass, resistance 1 to 2 M
; filling solution: 113 mmol/L KCl, 10 mmol/L NaCl, 5.5 mmol/L glucose, 5 mmol/L K2ATP, 1 mmol/L MgCl2, 10 mmol/L Hepes, pH adjusted to 7.15 with 12 mmol/L KOH) connected to a AxoClamp 2A amplifier (Axon Instruments) in bridge mode. Rj was calculated from current (I) and the electrotonic changes in membrane potential (Em).27 To elucidate the pHi dependence of Rj, the superfusion medium was changed, at constant pHo, to one containing 15 mmol/L NH4Cl or 20 mmol/L trimethylamine to raise pHi uniformly, or to one containing 80 mmol/L Na/acetate (plus 30 µmol/L cariporide) to reduce pHi uniformly.
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Presetting pHi
It was often necessary to preset pHi to different levels before an experiment. This was achieved by prepulsing the cell pair with 10 to 30 mmol/L NH4Cl-containing or 40 to 80 mmol/L Na/acetate-containing solutions for 3 to 8 minutes. On removal of these compounds, a uniform intracellular acidosis and alkalosis, respectively, was generated within 1 minute. The new pHi level was then clamped by inhibiting membrane H+-equivalent transport. In some experiments, Cl–/HCO3– exchange and Na+/HCO3– cotransport (NBC) were blocked by using CO2/HCO3–-free superfusates, and Na+/H+ exchange inhibited by adding 30 µmol/L cariporide (as Cl–/OH– exchange activity is low, <2 mmol/L per minute, no inhibitory measures were taken). When using CO2/HCO3– buffered superfusates, NBC was inhibited pharmacologically by 10 µmol/L S0859,28 and base transport at high pHi was blocked by replacing superfusate Cl– with gluconate (and raising [Ca2+] to 8.5 mmol/L to compensate for Ca2+ binding to gluconate).
Modeling Spatial pHi Regulation
Spatial pHi regulation was simulated in a model myocyte expressing sarcolemmal transporters and gap junctions. See Appendix for details.
Statistics and Curve Fitting
Quantitative data presented as mean±SEM; asterisks denote significance (t test; 5% significance level). Data presented in Figures 1B, 2C, and 3
B were fitted with biphasic curves that define junctional permeability (P) with H+-binding constants K and Q: 
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Results |
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Local Intracellular H+ Uncaging to Measure Junctional H+ Permeability
Repetitive UV flash photolysis of 2-nitrobenzaldehyde, a membrane-permeant caged H+ compound, was used to introduce H+ ions24 (
15 mmol/L/min) into one cell of an isolated pair of rat ventricular myocytes (Figure 1). Either side-by-side or end-to-end cell pairs were used. Progressive acidification of the distal cell of the pair indicated that cell-to-cell H+ transmission was occurring (Figure 1A), the time course of which was fitted with a diffusion–permeation algorithm to estimate PHapp, the apparent junctional H+ permeability.7 By presetting the resting pHi of cell pairs to different values (see Materials and Methods) and then locally uncaging H+ ions, the pHi dependence of PHapp was revealed. In the presence of CO2/HCO3– buffer, a reduction of pHi from 7.5 to 7.1 increased PHapp 5-fold (Figure 1B). A further reduction of pHi from 7.1 to 6.3 reversed this effect. Note that, although permeability was 12-fold higher in end-to-end compared with side-by-side cell pairs, the fractional changes of permeability with pHi were the same. Comparable results were obtained when experiments were performed in the absence of CO2/HCO3– (Figure 1B), indicating that this buffer is not essential for the changes of PHapp. Reduced solute permeability during acidosis is well documented for gap junctions,15–23 but an increase has not commonly been reported.
As described in the introduction, almost all H+ ions that permeate the cardiac gap junction do so while bound reversibly to mobile buffers.7 Figure 1E illustrates this for H+ permeation on an intrinsic buffer such as acetylanserine, acetylcarnosine, or homocarnosine, whose collective intracellular concentration is 15 mmol/L.12,13 The size of these buffers (100 to 200 Da) is well within the permeability limit of Cx-43 channels (1 kDa).10,11 The slightly higher peak permeability seen with CO2/HCO3– (Figure 1B) indicates that carbonic buffer may also assist H+ permeation. Interpreting changes of H+ ion permeability can be problematic, as buffer capacity is pHi dependent,6 which will affect the pHi sensitivity of PHapp. Using the mean pK for mobile and fixed intrinsic buffers,12,13 and pHi data gathered in the absence of CO2/HCO3–, we computed the average junctional permeability to the buffer molecules themselves7 (Pmob; see Materials and Methods). Any pHi sensitivity detected here is likely to be independent of the degree of buffer protonation. Results plotted in Figure 1C show that Pmob displays a biphasic dependence on pHi. Consistent with these permeability changes, the cell-to-cell H+ flux measured during the H+-uncaging procedure also varied biphasically with pHi (Figure 1D). Thus a modest fall of pHi from rest (gray arrow) enhanced H+ transmission, whereas a larger decrease impaired it.
Steady-State pHi Sensitivity of Junctional H+ Permeability
H+-uncaging experiments provide an estimate of PHapp and Pmob during dynamic changes of pHi. We also measured Pmob under steady-state pHi conditions, using a different technique. One end of a ventricular cell pair (this time, from a guinea pig rather than rat heart) was exposed to Hepes-buffered solution containing 30 mmol/L NH4Cl to elevate pHi locally25,26 (Figure 2Ai). Within 30 seconds, this establishes an intracellular H+ gradient along the cell pair, which drives transjunctional H+ flux on the mobile buffer shuttle. The [H+]i gradient is remarkably stable over time (many minutes) and displays a sharp discontinuity at the gap junction, where H+ flux is rate limiting (Figure 2Aii). In other experiments, NH4Cl was replaced by Na/acetate (Figure 2Bi) to generate local acidosis25,26 and drive H+ flux in the opposite direction. This establishes a longitudinal pHi gradient, distributed inversely to that seen with NH4Cl (Figure 2Bii). In both cases, the amplitude of the H+i change across the junction was used to calculate Pmob, at a constant junctional pH.26
Presetting pHi in the cell pair to various levels, and then measuring Pmob (during local NH4Cl or Na/acetate application), revealed that it varied biphasically with junctional pH (Figure 2C), matching that seen during dynamic H+ uncaging (Figure 1C). The permeability changes did not occur in the presence of 60 µmol/L 
-glycyrrhetinic acid, a selective gap-junctional inhibitor7 (Figure 2C), confirming that permeation was via Cx channels. The changes were established within 1 to 3 minutes (the minimum time for presetting pHi and then applying a local [H+]i gradient). They were also reversible, as they were independent of the direction in which pHi was preset in a cell pair (ie, from 7.5 to 6.2 or 6.2 to 7.5; see Figure I in the online data supplement, available at http://circres.ahajournals.org). Furthermore, the changes persisted after Ca2+i was clamped by intracellular BAPTA (loaded as the acetoxymethyl-ester; Figure 2C), although the decrease of Pmob in the pHi range from 6.95 to 6.20 was attenuated at pHi values <6.5. Thus, as reported previously, inactivation of junctional permeability at very low pHi probably depends on a rise of [Ca2+]i19, 20 as well as a fall of pHi.15,16 In contrast, however, the activation of Pmob as pHi declines from 7.5 to 6.95 was unaffected by BAPTA, suggesting that activation is Ca2+ independent.
pHi-Sensitive Junctional Permeability Measured by Fluorescent-Dye Transfer
We investigated the pHi sensitivity of junctional permeability applied to solutes other than H+ ions or intrinsic mobile buffer. In one set of experiments (Figure 3A), we monitored cell-to-cell permeation of a fluorescent marker dye by introducing SNARF-1 (453 Da) into one cell of a guinea pig ventricular pair from a cell-attached pipette.7 The rise of fluorescence in both cells was fitted with a diffusion–permeation algorithm to quantify junctional permeability to SNARF (PSNARF).7 By presetting pHi uniformly to different levels, PSNARF was found to follow a biphasic relationship with pHi (Figure 3B), similar in shape to the pHi–Pmob relationship (Figure 2C).
pHi-Sensitive Junctional Permeability Measured by Current Injection
In other experiments (Figure 4Ai), we monitored electrical junctional resistance (Rj) by passing current (carried largely by intracellular K+ and Cl– ions) between coupled cells27 while uniformly superfusing them with NH4Cl (Figure 4Aii), trimethylamine-chloride, or Na/acetate to displace pHi. Data pooled in Figure 4B show that Rj also varied biphasically with pHi, rising at both high and low pHi. By plotting Rj (inverted axis) and SNARF permeability on a common pH axis (Figure 4C), it is apparent that both parameters display a similar pH sensitivity. The biphasic pHi sensitivity of permeability is thus likely to be a general feature of the ventricular gap junction.
Computational Modeling of Cell-to-Cell H+ Movement
The H+-dependent activation and inactivation of junctional permeability has important implications for the spatial regulation of myocardial pHi. Figure 5A superimposes the pH dependence of junctional permeability (Pmob) on the known pHi dependence of sarcolemmal H+-equivalent transport that classically mediates pHi regulation in cardiac cells.6,28 It is notable that the pHi range for peak junctional permeability coincides with minimum transporter activity and vice versa. We have incorporated these parameters into a computational model of a myocyte surrounded by, and coupled to, other myocytes (see Appendix). Reducing pHi of the central cell in the model from 7.25 (resting pHi) to 6.0 induces an H+ efflux from that cell (Figure 5B). This is predicted to occur through gap junctions into surrounding cells (green trace; Jjunc) and on sarcolemmal transporters into the extracellular space (red trace; Jsarc). In the ventricular myocardium, gap junctions couple 1 myocyte to as many as 11 neighbors.29 In the model, junctional efflux (Jjunc) predicted for maximal cell-to-cell coupling (continuous green trace) rises steeply to a peak at approximately pHi=6.85, facilitated by activation of gap-junctional permeability. It declines at lower pHi values because of inactivation of junctional permeability, whereas sarcolemmal H+ efflux (Jsarc) activates steeply. At pHi values 7.25 to 6.20, Jjunc exceeds Jsarc, indicating that most H+ efflux from the locally acidified zone is via junctional pathways. The dashed green line shows results predicted when only one end of the central cell is coupled in the model to the surroundings (analogous to the cell pair experiments shown in Figure 1). Even under these restricted circumstances, Jjunc is significantly larger than Jsarc for much of the pHi range (7.25 to 6.50). Junctional H+ coupling in the intact myocardium is likely to lie between the two extremes illustrated in Figure 5B (continuous and dashed green lines). The model therefore predicts that, following a local fall of pHi, junctional H+ permeability in the myocardium will provide a major mechanism for dispersing the acid load.
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Spatial Regulation of pHi: Junctional Versus Sarcolemmal Control
We have shown that H+ ions within ventricular tissue can activate as well as inactivate gap-junctional permeability. An important molecule that normally permeates the junction is the intracellular mobile H+ buffer, which serves to carry H+ ions between adjacent myocytes.7 Thus intracellular H+ ions, by attaching to mobile buffers and also by modulating gap junctional permeability, can regulate their own cell-to-cell movement. The pHi sensitivity of junctional permeability provides a sophisticated mechanism for spatially regulating myocardial pHi. Our computational model indicates that a modest local H+i load (eg, pHi 6.90) in 1 or more cells (induced, for example, by local reduction of capillary perfusion) will enhance gap-junctional permeability, permitting the load to dissipate passively into neighboring cells: a "permissive" mode of local pHi regulation (Figure 5Ci). In contrast, a local H+i "overload" (eg, pHi 6.3; caused, for example, by extreme metabolic stress) will inhibit gap-junctional permeability, restricting the spatial spread of H+ ions: a "nonpermissive" mode of pHi regulation, as much junctional H+ transmission is barred (Figure 5Cii). Spatial dissipation of a modest H+i load will dilute it into an extended and highly buffered intracellular volume and will thus be an energy-efficient form of pHi regulation. The dispersed H+ ions will eventually be exported at a low rate across the sarcolemma. In contrast, following the isolation of a local H+i overload by gap-junctional inactivation, the H+ ions will be pumped rapidly into the extracellular space, as sarcolemmal extrusion is greatly stimulated at low pHi (Figure 5A). Given that H+i overload is potentially dangerous to the heart,4,5 restricting its spread while simultaneously removing it at source would seem a sensible strategy. But for more modest H+i loads, the pH sensitivity of gap junctions will enhance rather than inhibit the exchange of H+ ions between cells, tending to promote a myocardial pHi syncytium.
H+ Activation and Inactivation of the Ventricular Gap Junction
The element orchestrating junctional versus sarcolemmal modes of pHi regulation is the pH sensitivity of the ventricular gap junction. We have yet to investigate if similar pH sensitivity is observed in other regions of the heart, such as the atria and conduction system. Although chemical H+ inactivation of Cx-43 and Cx-45 channels, the dominant isoforms in ventricular myocardium,8,29 has long been known,21–23,30 the possibility of chemical H+ activation of junctional permeability has received little attention. It is notable, however, that early work on amphibian blastomeres31 and on Cx-43 channels expressed in oocyte pairs21,22 documented a paradoxical and unexplained rise of junctional conductance during progressive acidosis that preceded the anticipated fall. H+ activation could account for such anomalous behavior.
How may H+ ions both activate and inactivate junctional permeability? The bell-shaped pH sensitivity of junctional permeability (Figures 2C and 3B) is consistent with distinct, overlapping H+ activation and H+ inactivation processes (Figure 6A) of similar pK (7.0), producing a steady-state window of ensemble Cx channel permeability (Figure 6B). In such a model, H+ titration of activation sites would increase junctional permeability, whereas simultaneous titration of inactivation sites would decrease it. Although acute H+ inactivation has been linked structurally to the cytoplasmic C terminus of Cx-43 proteins,21–23 the molecular correlates of H+ activation have yet to be identified. Preliminary work suggests that junctional permeability in HeLa cell pairs heterologously expressing Cx-43 channels shows a biphasic dependence on pHi, qualitatively similar to that seen here in myocyte pairs.32 Furthermore, H+ activation and inactivation of junctional permeability appears to be greatly attenuated when mutant Cx-43 channels with truncated C termini are expressed.32 The H+-induced increase of junctional permeability in myocytes, like the H+-induced decrease, may therefore be related to a structural motif in the channel protein itself.
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Acute H+ inactivation largely reflects the closure (ie, gating) of Cx channels.21–23 In contrast, there are at least three possible mechanisms for H+ activation that cannot yet be distinguished: increased Cx channel conductance, increased channel opening (or decreased closure), and insertion of additional Cx channel proteins from a mobilizable source,33,34 ie, a form of junctional remodeling. Indeed, the primary H+ activation site need not necessarily be located on the Cx channel itself. It could, for example, be located remotely on an accessory protein (as represented schematically in Figure 6A) or be part of a biochemical cascade, which targets the channel. H+ activation, however, must ultimately interact with the dynamic behavior of Cx channels to generate increased junctional permeability. The relatively rapid kinetics and magnitude of H+ activation (up to 4-fold permeability increase within 1 to 3 minutes; Figure 2) suggest a regulation of channel gating and conductance, although a fast increase in channel density cannot be excluded. In contrast, H+-induced and ischemia-induced remodeling of glial34 and cardiac cell junctions33 is a slow process (>30 minutes) and has so far involved internalization rather than plasmalemmal insertion of Cx channels. The mechanism for H+ activation of Cx channels therefore remains unresolved.
Wider Implications for H+ Control of Junctional Coupling
A variety of connexin gap junctions, and functionally similar channels composed of innexin or pannexin proteins, is widely expressed in tissues throughout the animal kingdom.35 Whether H+ activation of junctional permeability is a common feature for these channels remains to be established. The possibility arises that, in addition to heart, H+ control of junctional coupling may direct the spatial distribution of pHi in neural and glial networks, in smooth muscle, and in epithelial tissue. H+ activation may also help to explain recent reports that hypoxia and simulated ischemia can paradoxically open gap-junctional hemichannels in neurones36 and cardiac myocytes,37 as these conditions promote a fall of pHi.
The acute increase of gap-junctional permeability with modest acidosis implies that, in ventricular myocardium, metabolic stimulation may commonly enhance electrical and biochemical communication between cells. This may be one means of increasing cardiac efficiency to match metabolic activity and hence mechanical demand. Only with more profound intracellular acidosis, such as occurs during severe ischemia, would this then be replaced by electrical and metabolic uncoupling.
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We used a mathematical model to assess the relative role of junctional and sarcolemmal H+-equivalent fluxes in controlling pHi of a myocyte, coupled to neighboring cells in 3 dimensions, during a localized injection of acid (Figure 5). The simulations were performed assuming CO2/HCO3– buffering. The model was based on a 3D mesh of nodes (spacing=2.5 µm) with cylindrical symmetry, over which a diffusion equation was solved (MATLAB, Mathworks Co):
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where r and x are the radial and longitudinal axes, and DHapp is the pH-dependent apparent H+ diffusion coefficient measured previously.24 The model cell assembly consisted of a central cell (Figure 5B inset; dark cylinder, length=100 µm, diameter=25 µm) surrounded by a compartment that simulates a layer of neighboring cells (length=300 µm, diameter=75 µm). Buffering capacity was defined as the sum of intrinsic and carbonic buffering.6,14 Junctional permeation H+ fluxes (Jjunc) were defined as follows (note [mmol/L · sec–1] for all equations hereafter):
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where PHapp is the pH-dependent apparent H+-permeability constant (Figure 2C). The coefficient accounts for permeation anisotropy (Figure 1B; =12.1). The constant 
is the surface-area-to-volume ratio6 (=2017 cm–1). Sarcolemmal H+-equivalent transport fluxes (NHE,28 NBC,28 Cl–/OH– exchange [CHE],6 and Cl–/HCO3– exchange [AE]6) were defined as follows:
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In the simulation, H+ ions are injected into the whole volume of the central cell at a rate (Jinj) up to 125 mmol/L per minute. During injection, some H+ ions are buffered, some extruded by sarcolemmal carrier mechanisms (Jsarc), and some transmitted to neighboring cells through gap junctions (Jjunc). In the analysis shown in Figure 7A, the central cell was H+ loaded at 100 mmol/L per minute. In the absence of sarcolemmal or junctional recovery mechanisms, this would predict a fall of central pHi by >1.7 U within 1 minute. The activation of Jsarc and Jjunc reduces this acidosis to 0.8 U. The regulatory routes taken by the injected H+ ions were followed by plotting the cumulative rise in the amount of H+ ions in three domains (Figure 7B): (1) central-cell buffers, (2) neighboring cells (junctional transmission), and (3) the extracellular space (sarcolemmal extrusion). Over the first 60 seconds, Jjunc is far larger than Jsarc. By repeating the simulations for a range of Jinj values, it was possible to quantify, over a range of central pHi values, the relative importance of Jjunc and Jsarc for limiting the acid load (Figure 5B). These simulations reveal that Jjunc is the dominant regulatory mechanism during a physiological acid load (eg, pHi 7.25 to 6.60), whereas Jsarc becomes increasingly important at lower, more pathological pHi values (eg, pHi 6.60 to 6.00). Thus both Jjunc and Jsarc will be key local controllers of pHi.
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Acknowledgments |
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We thank Nurindura Banger and Philip Cobden for technical assistance.
Sources of Funding
This work was supported by a British Heart Foundation Programme grant (to R.D.V.-J.) and NIH Method to Extend Research in Time award 5R37HL042873 (to K.W.S.).
Disclosures
None.
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Footnotes |
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Original received January 12, 2007; revision received February 15, 2007; accepted March 6, 2007.
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References |
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