Function of the HVCN1 proton channel in airway epithelia and a naturally occurring mutation, M91T

Abstract

Airways secrete considerable amounts of acid. In this study, we investigated the identity and the pH-dependent function of the apical H(+) channel in the airway epithelium. In pH stat recordings of confluent JME airway epithelia in Ussing chambers, Zn-sensitive acid secretion was activated at a mucosal threshold pH of approximately 7, above which it increased pH-dependently at a rate of 339 +/- 34 nmol x h(-1) x cm(-2) per pH unit. Similarly, H(+) currents measured in JME cells in patch clamp recordings were readily blocked by Zn and activated by an alkaline outside pH. Small interfering RNA-mediated knockdown of HVCN1 mRNA expression in JME cells resulted in a loss of H(+) currents in patch clamp recordings. Cloning of the open reading frame of HVCN1 from primary human airway epithelia resulted in a wild-type clone and a clone characterized by two sequential base exchanges (452T>C and 453G>A) resulting in a novel missense mutation, M91T HVCN1. Out of 95 human genomic DNA samples that were tested, we found one HVCN1 allele that was heterozygous for the M91T mutation. The activation of acid secretion in epithelia that natively expressed M91T HVCN1 required approximately 0.5 pH units more alkaline mucosal pH values compared with wild-type epithelia. Similarly, activation of H(+) currents across recombinantly expressed M91T HVCN1 required significantly larger pH gradients compared with wild-type HVCN1. This study provides both functional and molecular indications that the HVCN1 H(+) channel mediates pH-regulated acid secretion by the airway epithelium. These data indicate that apical HVCN1 represents a mechanism to acidify an alkaline airway surface liquid.

Figure 1.

Orientation of epithelium, pH electrode, and titrator burette in Ussing chamber with serosal HEPES-buffered and mucosal unbuffered solutions during pH stat experiments. Solutions were continuously gassed with O₂ (serosally) and N₂ (mucosally) to mix the solutions and prevent air CO₂ from entering.

Figure 2.

Acid secretion by JME epithelial cultures. (A) Rates of acid secretion in the absence (filled symbols) and presence (open symbols) of 10 µM ZnCl₂. Serosal pH 7.4 and mucosal pH was adjusted to 7, 7.4, and 8; n = 5. *, significantly different from control; t test. (B) Zn-sensitive rates of acid secretion (calculated as the difference from data in A) resulted in an average slope of 339 ± 33.6 nmol × h⁻¹ × cm⁻² × pH⁻¹ and a threshold pH of 6.90 (by linear regression).

Figure 3.

Effect of outside pH (pH_o) on H⁺ currents in JME cells. (A–D) Whole cell currents elicited by voltage pulses (as shown in top inset in A) at pH_o 6, 7, and 8 (A–C), and in the presence of 10 µM ZnCl₂ at pH_o 8 (D). All recordings are from one cell; pH_i 6. Y axis and 10-s time bar in A are the same for B–D. Selected voltage steps are labeled. The bottom insets in A–C are respective expanded tail currents on a scale as shown in A. (E) Average steady-state current–voltage relations at pH_o 6, 7, and 8 as indicated. Currents were normalized to membrane capacitance. The number of experiments is given in F in parentheses and is the same for E–H. (F) Reversal potential (E_rev) was determined from tail currents resulting in a slope of −53 ± 5.1 mV/pH. (G) Threshold potential (V_th) as determined from 8-s voltage steps resulted in a slope of −31.5 ± 1.9 mV/pH. (H) Relationship between V_th and V_rev (using data from F and G) resulted in a slope of 0.62 ± 0.12 mV/mV and an offset of 35.5 ± 7.4 mV.

Figure 4.

Effect of HVCN1 siRNA on H⁺ currents in JME cells. (A) HVCN1 mRNA levels relative to GAPDH mRNA. Treatment with siRNA significantly reduced HVCN1 mRNA levels (P = 0.002; Kruskal-Wallis ANOVA on ranks). Bars, average; symbols, individual measurements. (B–D) H⁺ currents measured in single JME cells evoked by depolarizing pulses (as shown in inset); pH_i 6 and pH_o 7. (E) H⁺ current density after activation by an 8-s, 80-mV pulse. Treatment with siRNA significantly reduced HVCN1 mRNA levels (P = 0.003; Kruskal-Wallis ANOVA on ranks). Currents measured in siRNA-treated cells were very small (corresponding to a conductance of 79 ± 15 pS/cell) and not voltage activated in the used voltage range.

Figure 5.

pH dependence of acid secretion of primary human tracheal cultures. Serosal pH 7.4 and mucosal target pH were set to step-wise increasing values over a range of pH 6.0 to 8. (A) Cultures expressing wild-type HVCN1. Filled symbols, untreated; open symbols, 10 µM ZnCl₂ mucosally. (B) Primary human tracheal cultures expressing M91T HVCN1. (C) Activation of Zn-sensitive acid secretion by mucosal alkalinization in wild-type (filled symbols) and M91T HVCN1 (open symbols). Data were calculated as the difference of values before-minus-after Zn addition from data in A and B. *, significantly different from control; t test.

Figure 6.

Patch clamp recordings of H⁺ currents after recombinant expression of wild-type HVCN1, M91T HVCN1, and empty vector in COS-7 cells. (A) Typical overview recording for this set of experiments. Top trace shows current measured at holding potentials as shown in the bottom trace. Baseline voltage was −80 mV, pulsed every 40 s to +20 mV, and a voltage step protocol from −40 to +80 mV, step 20 mV, was applied before and after 10 µM ZnCl₂ was added to the bath; pH_i 6 and pH_o 7. Note the recovery of depolarization-activated currents after the first current–voltage steps. (B–D) Recording from cells expressing recombinant wild-type HVCN1. Current pulse to 0 mV is indicated in B, and voltage step protocol is depicted in the inset; recordings in B and C are from the same cell, and the y-axis scaling is the same. (E–G) Recording from a cell expressing recombinant M91T HVCN1. Current pulse at 20 mV is indicated in E; recordings in E and F are from the same cell, and the y-axis scaling is the same. (H–J) Mock-transfected cells expressed no significant currents. The y axis in I is the same as in H. The time bar in B is the same for all current recordings (excluding A). Recordings in H and I are from the same cell, and the y-axis scaling is the same. D and G are reported in percent of maximal current, and the x axis is the same as in J. The number of experiments is given in parentheses.

Figure 7.

Threshold potentials of H⁺ current activation across recombinantly expressed wild-type and M91T HVCN1 in COS-7 cells. (A and C) Wild-type HVCN1. Example recordings of depolarizing steps from −80 mV to potentials near V_th at pH_o 7.1 (A) and 7.5 (B) from one cell. Clamped voltages are given next to traces. Tail currents are shown on an expanded time scale (note the break in time axes); pH_i 6. (B and D) Corresponding recording from an M91T HVCN1 at pH_o 7.1 (B) and 7.65 (D) recorded from same cell; pH_i 6. pH_o was determined from aliquots taken from bath solution for every condition during experimental runs. (E) Relationship between V_th and pH_o. Linear regression analysis for wild-type and M91T resulted in similar slopes of wild type (−28.1 ± 3.6 mV/pH_o) and M91T (−30.3 ± 3.1 mV/pH_o). Correspondingly, M91T required significantly higher pH_o (7.08 ± 0.05) than wild type (6.43 ± 0.08; P < 0.001). The number of experiments is given in parentheses. (F) Relationship between V_th and E_rev for wild-type and M91T HVCN1. Regression analysis yielded the following: wild-type, V_th = 0.78 ± 0.06 E_rev + 2.1 ± 1.9 mV; M91T, V_th = 0.81 ± 0.09 E_rev + 34 ± 4.7 mV. Offset, but not slope, was significantly different (P < 0.001; t tests).

Figure 8.

Alignment of the HVCN1 region around M91 for several species. Position 91 is boxed. Identical residues are marked dark gray, and similar residues are marked light gray. Alignment with the human sequence was done using BlastP (http://blast.ncbi.nlm.nih.gov).