Trexler EB et al. (1999), Rapid and direct effects of pH on connexins rev...

XB-ART-13083

J Gen Physiol 1999 May 01;1135:721-42.

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Rapid and direct effects of pH on connexins revealed by the connexin46 hemichannel preparation.

Trexler EB, Bukauskas FF, Bennett MV, Bargiello TA, Verselis VK.

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Species referenced: Xenopus laevis
Genes referenced: gja1 gja3 isyna1

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	Figure 2. Acidification-induced uncoupling in pairs of Cx46-transfected Neuro-2a cells. A and B show whole-cell patch recordings of junctional current (Ij) in cell 1 in response to a series of brief Vj steps applied to cell 2. (A, I) A recording from a cell pair with Ij in response to alternating Vj steps of +30 and −30 mV. Application of 100% CO2-equilibrated medium for 45 s reduced Ij to undetectable levels. Recovery was delayed for ∼3 min, and only reached 10–15% of the original value in the subsequent 2 min. (A, II) Two recordings from a second cell pair with Ij in response to Vj steps of +25 mV. Application of 100% CO2-equilibrated medium for 25 s reduced Ij to undetectable levels. Wash solution was applied when Ij decreased to undetectable levels. Recovery was delayed for ∼60 s on washout, and Ij increased slowly to ∼50% of the original value. A shorter exposure to 100% CO2 (15 s) significantly but not completely reduced Ij. Washout began soon after the initial decline in Ij. Recovery was delayed less with this shorter exposure and was essentially complete (>90%) within 2 min. (B) Three recordings from a third cell pair with Ij in response to alternating Vj steps of +20 and −20 mV. Ij decreased steadily during application of medium acidified with HCl to pH 6.5 (∼3 min), pH 6.0 (∼3 min), and pH 5.5 (∼4 min). Recovery from low pH applications began soon after washout with pH 7.5 solution. Ij reached >90% of the original values for pH 6.5 and 6.0, but with the longer exposure to pH 5.5, Ij recovered only to ∼70% of the original value.
	Figure 3. Measurement of intracellular pH in Cx46-transfected Neuro-2a cells. pHi was measured using the acidic form of BCECF. Single cells were whole-cell patch clamped with BCECF added to the patch pipette solution. (A) Application of CO2-equilibrated medium for 90 s decreased pHi to ∼5.8 within 50 s. However, recovery was much slower and lasted for almost 300 s. (B) pHi steadily decreased from 7.25 to ∼6.0 with a 300-s application of a medium acidified to pH 5.5 with HCl. Recovery after washout was complete within ∼100 s.
	Figure 4. Cx46 hemichannels in Xenopus oocytes are sensitive to both internal and external pH. Shown are consecutive depolarizing voltage steps to +30 mV applied to a Xenopus oocyte expressing Cx46. The cell was held at −70 mV between voltage steps. The duration of each voltage step was 40 s and the interval between voltage steps was 50 s. The periods the oocyte was exposed to either 100% CO2-equilibrated medium or HCl-acidified pH 5.5 medium are bounded by rectangles. Hemichannel currents responded rapidly to application of 100% CO2 solution in the fourth depolarization and were abolished within ∼80 s of the application. Washing with pH 7.5 bathing media led to full recovery within four depolarizations. Hemichannel currents were also decreased with application of pH 5.5 HCl acidified medium during the 13th depolarization. By the next depolarization, Cx46 currents were reduced more. Hemichannel currents recovered by the third depolarization after solution exchange with original pH 7.5 bathing medium. The solid horizontal line marks the level of the activation of Cx46 hemichannels before any treatment. The dashed line approximates the increase in peak current with time, which is probably due to continued expression of new Cx46 hemichannels.
	Figure 5. Measurement of pHi in Cx46 expressing Xenopus oocytes during application of medium (A) equilibrated with 100% CO2 and (B) adjusted to pH 6.3 with HCl. pHi was monitored with BCECF and recorded at five distances from the edge of the oocyte, ranging from 50 to 450 μm. Experimentally, these values were obtained by placing 100-μm circular “regions of interest” (ROI) centered at the specified distances on the acquired image of the oocyte and replaying the saved acquisition. During image acquisition, the objective was focused on the oocyte's perimeter. With a large, round oocyte, this focal plane would pass through the center of the oocyte, although with considerable light scatter. The ratiometric determination of pH in this plane will include some signal from the periphery above and below the center of the oocyte, but the image from the perimeter of the oocyte will include entirely peripheral cytoplasm. Thus, there is a significant difference in the amount of signal contributed from deep within the oocyte cytoplasm as the ROI is moved away from the perimeter. The slower change in pH towards the center of the oocyte was observed consistently, but eventually the pH reached the same value in the center as at the periphery. (A) A 4-min application of 100% CO2-equilibrated medium lowered pHi from ∼7.6 to ∼5.8. An enlarged view (A1) shows how the kinetics of intracellular acidification slows with increasing distance from the oocyte periphery. The same steady state value of pHi was reached in all cases before recovery was initiated by washing. Similar kinetics was seen with recovery (A2). (B) An ∼3-min application of HCl-acidified pH 6.3 medium also lowered pHi. Again, the effects were faster at the edge of the oocyte. With the duration of the application shown, pHi decreased less at the center of the oocyte.
	Figure 6. Cx46 hemichannels in excised patches retain sensitivity to pH. (A) An inside-out patch that was placed in a stream composed of pH 7.5 IPS-A. With the holding potential of −50 mV, hemichannel openings are downward deflections in current. Rapid switching to pH 6.0 IPS-A led to a sharp decrease in the open probability of the four active hemichannels in the patch. This example shows no transitions to the fully open state while the patch was in the pH 6.0 stream. The exploded view of one application reveals the rapid closure of two open channels and no subsequent openings for the duration of the application. Upon switching back to pH 7.5, the hemichannels returned to normal gating behavior. I = 0 pA denotes the leak conductance of the patch. (B) A fit of the Hill equation to averaged data from several patches. The mean and SD of I/I7.5 are plotted against pH. The number of low pH exposures used to obtain mean current ratios is displayed above each point. The Hill equation fit to the data was of the form: The parameters from the fit were k = 4.04 × 10−7 M (pKa of 6.4) and n = 2.3.
	Figure 7. Cx46 hemichannels are equally sensitive to pH with [Ca2+] at 100 nM and 10 μM. IPS-B (2 mM BAPTA) was used for all solutions, with added CaCl2 adjusted to give 100 nM and 10 μM [Ca2+]. IPS-B with 100 nM [Ca2+] was used as the pipette filling solution in both cases. (A) Rapidly switching from pH 7.5 to 6.0 with 100 nM [Ca2+] significantly lowered the open probability of the three active channels in the patch. Few full reopenings are seen during the low pH application. I/I7.5 = 0.1. (B) With 10 μM [Ca2+] solutions, Cx46 hemichannel open probability was similarly reduced with pH 6.0 applications. I/I7.5 = 0.08. I = 0 pA denotes the leak conductance of the patch; channel openings are downward deflections in current.
	Figure 8. Ensemble currents show the time course of pH- induced closure. (A) An inside-out patch held at −30 mV and containing a single Cx46 hemichannel was subjected to repeated 2-s applications of pH 6.0 IPS-A (region within the rectangle). Hemichannel openings are downward deflections in current. The beginning of each 12-s trace immediately followed the end of the previous trace. (B) Summation of the currents from the patch shown in A, together with 112 more traces from four other patches at −30 mV containing multiple channels. (C) A sum of two exponentials fitted to the pH 6.0 induced decay of ensemble currents. The time constants of the decay were 110 and 935 ms, and the constant parameter of the fit was −232 pA. (D) An enlarged view showing the time of solution switching (denoted by the dashed line) in comparison with the onset of ensemble current decay. There was no delay between pH 6.0 application and current decrease.
	Figure 9. Ensemble currents show the effect of the duration of acidification on the extent of recovery. (A) An inside out patch held at −30 mV and containing at least 20 Cx46 hemichannels was subjected to two applications of pH 6.0 for 5 s each. After each application, the number of active channels was markedly reduced. (B) Ensemble currents from multiple patches were normalized to the average of the currents over the 5 s before acidification. The 1-s ensemble is the sum of 96 pH 6.0 applications from three patches, the 2-s ensemble is the sum of 132 applications from five patches (the same as in Fig. 8), and the 5-s ensemble is the sum of 31 applications from a total of five patches. Recovery from 1- and 2-s applications is nearly 100%, whereas recovery from 5-s applications is only ∼80%.
	Figure 10. (A) Application of low pH to the cytoplasmic face of Cx46 hemichannels in inside-out patches decreases channel open probability whether the application occurs when the Cx46 hemichannel is open or closed. In this example, taken from the same patch depicted in Fig. 6, rapid solution switching to pH 6.0 followed a spontaneous closure. During the remainder of the application, channel open probability was reduced, and no full reopenings were evident. Opening is downward and I = 0 pA is the leak conductance of the patch. (B) The open probability of Cx46 hemichannels during pH 6.0 applications exhibits moderate voltage dependence. An inside-out patch held at +40 mV was exposed to pH 6.0 for a brief time. Closure occurred soon after application, but there were several brief reopenings to a reduced conductance during the low pH exposure. At −40 mV, there were no complete openings while the patch was exposed to pH 6.0. I = 0 pA is the leak conductance of the patch.
	Figure 11. Ensemble currents from an outside-out patch reveal a much lower sensitivity to extracellular pH 6.0 applications. (A) An outside-out patch containing a single Cx46 hemichannel was repeatedly exposed to pH 6.0 IPS-A. Hemichannel open probability is not as reduced as with pH 6.0 applications to the cytoplasmic face of the channel. (B) Summation of the 20 traces displayed in A with 30 additional traces from the same patch (not shown). (C) An exponential fitted to the pH 6.0 induced ensemble current decrease. The time constant of decay was 241 ms, and the constant parameter of the fit was −336 pA.
	Figure 12. Comparison of Cx46 hemichannel closed times during application of pH 7.5 and 6.0 to an outside-out patch. (A) Example of two current idealizations obtained using the Sublevel Hinkley detector set to low sensitivity. Channel openings are downward deflections in current. Traces are from the same patch as in Fig. 11. Closed times were categorized as pH 7.5 closed times or pH 6.0 dwell times according to the condition in which the event began. (B) Dwell-time histogram of the closed times at pH 6.0 compiled for all 50 traces of the same patch. The histogram was fitted to a single exponential to give a mean closed time of 600 ms. (C) The recovery of the ensemble currents shown in Fig. 8 B after rapidly switching to pH 7.5 from a 2-s application of pH 6.0 was fitted by a single exponential with a time constant of 636 ms, which represents the mean latency to first opening. (D) Dwell-time histogram of the closed times at pH 7.5. A single exponential fit to the distribution gave a mean closed time of 170 ms. Due to the smaller number of events at pH 6.0, binning was increased to 200-ms intervals, compared with 100-ms intervals for pH 7.5 events. (E) Comparison of pH 6.0 applications to an outside-out patch held at +40 and −40 mV. There was an ∼10–15% single channel conductance decrease at +40 mV, but very little change in the open probability of the two channels in the patch (I/I7.5 ≅ 0.91). The pH 6.0 application at −40 mV occurred after one of the channels had already closed. The open probability of the two channels was markedly reduced (I/I7.5 ≅ 0.4). The opening events during the pH 6.0 application may be attributable to both channels. I = 0 pA denotes the leak conductance of the patch.
	Figure 13. Transitions from fully open to fully closed states are slow for both pH-induced and hyperpolarization-induced closures. (A) A single Cx46 hemichannel in an inside-out patch held at −50 mV transited from fully open to fully closed over the course of ∼50 ms. Numerous transitions among discrete substates are evident. Dash-dot lines indicate putative substate levels. (B) Rapidly switching from pH 7.5 to 6.0 IPS-A caused full closure of a single Cx46 hemichannel in an outside-out patch. Closure did not initiate for >100-ms after the solution exchange, and required >100 ms to complete. The channel reopened fully during this application (not shown). The full closing transition is very noisy with no clearly resolvable substates.
	Figure 14. Mutations shown to affect the pH sensitivity of Cx43 have little effect on Cx46. Hemichannel currents were induced by a series of 20-s depolarizing voltage steps separated by 70 s; the displayed interval between steps is truncated to 10 s for clarity. Voltage was held at −60 mV between depolarizing steps. In all cases, medium equilibrated with 100% CO2 was applied between the second and third depolarizations, and was washed out after the fourth depolarization. Wild-type Cx46 currents were nearly abolished with medium equilibrated with 100% CO2. Cx46ΔCT259, a mutant lacking 82% of the COOH terminus, and the mutant Cx46H95D hemichannel behaved similarly to wild-type Cx46. Full recovery for these three hemichannel types occurred, on average, by the fourth episode after washout. The small variation in the time to full recovery seen among these three different hemichannel types was similar to that seen for oocytes expressing the same hemichannel type (n = 4, data not shown). In contrast, the mutant Cx46H95C hemichannel, while similarly sensitive to CO2 application, recovered considerably more slowly upon washout, requiring twice as long as the three previous hemichannel types. Hemichannel currents were elicited by voltage steps to +5 mV for wild-type Cx46 and Cx46H95D, +10 mV for Cx46ΔCT259, and +15 mV for Cx46H95C.

References [+] :

Arellano, Calmodulin acts as an intermediary for the effects of calcium on gap junctions from crayfish lateral axons. 1988, Pubmed