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Comparative Study
. 2014 Dec 16;111(50):18078-83.
doi: 10.1073/pnas.1411390111. Epub 2014 Nov 25.

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

Elayne Hondares  1 , Mark Adrian Brown  1 , Boris Musset  2 , Deri Morgan  3 , Vladimir V Cherny  3 , Christina Taubert  1 , Mandeep K Bhamrah  4 , David Coe  5 , Federica Marelli-Berg  5 , John G Gribben  6 , Martin J S Dyer  7 , Thomas E DeCoursey  8 , Melania Capasso  9
Affiliations
Comparative Study

Enhanced activation of an amino-terminally truncated isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells

Elayne Hondares et al. Proc Natl Acad Sci U S A. .

Abstract

HVCN1 (Hydrogen voltage-gated channel 1) is the only mammalian voltage-gated proton channel. In human B lymphocytes, HVCN1 associates with the B-cell receptor (BCR) and is required for optimal BCR signaling and redox control. HVCN1 is expressed in malignant B cells that rely on BCR signaling, such as chronic lymphocytic leukemia (CLL) cells. However, little is known about its regulation in these cells. We found that HVCN1 was expressed in B cells as two protein isoforms. The shorter isoform (HVCN1S) was enriched in B cells from a cohort of 76 CLL patients. When overexpressed in a B-cell lymphoma line, HVCN1S responded more profoundly to protein kinase C-dependent phosphorylation. This more potent enhanced gating response was mediated by increased phosphorylation of the same residue responsible for enhanced gating in HVCN1L, Thr(29). Furthermore, the association of HVCN1S with the BCR was weaker, which resulted in its diminished internalization upon BCR stimulation. Finally, HVCN1S conferred a proliferative and migratory advantage as well as enhanced BCR-dependent signaling. Overall, our data show for the first time, to our knowledge, the existence of a shorter isoform of HVCN1 with enhanced gating that is specifically enriched in malignant B cells. The properties of HVCN1S suggest that it may contribute to the pathogenesis of BCR-dependent B-cell malignancies.

Keywords: Hv1; chronic lymphocytic leukemia; gating kinetics; phosphorylation; proton currents.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
HVCN1S is an alternative isoform of the voltage-gated proton channel HVCN1 enriched in malignant B cells. (A, Left and Center) Immunoblots showing expression of two isoforms of HVCN1, HVCN1L (“L”), and HVCN1S (“S”) in B-cell lines, primary B cells, and CLL samples. R (RI1), U (U2932), and H (HBL1) are diffuse large B-cell lymphoma cell lines; E (EJM) is a multiple myeloma cell line, and Ra (RAJI) is a Burkitt lymphoma cell line. (Right) Densitometry analysis of protein expression of HVCN1S in B cells from healthy donors (n = 7) and CLL patients (n = 76). Protein expression levels as determined by Western blot, relative to loading control (β-actin or α-tubulin) and normalized to a positive control used across different blots (cell line HBL1). Statistical analysis carried out with Mann–Whitney U test. (B, Left and Center) In vitro transcription-translation assays with recombinant HVCN1L and mutated ttgHVCN1L. (Right) Expression of ttgHVCN1L transduced in LK35.2 cells. (C) PCR on HBL1 mRNA to identify the three mRNA sequences reported for human HVCN1 (SI Appendix, Fig. S1). (Left) Duplex PCR with primers designed for mRNA variant 2, which amplifies a band of 432 bp, and primers recognizing all variants (228 bp). (Right) PCR performed with different annealing temperatures with primers designed to recognize HVCN1 mRNA variants 1 and 3. The expected bands for isoforms 1 and 3 are 433 and 393 bp, indicated by arrows. (D) Immunoblots of a CLL sample and LK35.2 cells overexpressing HVCN1L and HVCN1S with an anti-HVCN1 that recognizes residues 26–46 (Left) and residues 1–20 (HVCN1L-specific, Right).
Fig. 2.
Fig. 2.
HVCN1S responds more strongly to PMA stimulation than HVCN1L. Perforated-patch voltage clamp was used to evaluate the electrophysiological properties of the two HVCN1 isoforms. Families of currents in 10-mV increments up to 70 mV from Vhold = −40 mV are shown in representative LK35.2 cells expressing HVCN1L (A) or HVCN1S (B) before stimulation, after application of the PKC activator PMA, and after inhibition of PKC by GF 109203X (GFX). Between the families are superimposed currents obtained during test pulses to 60 mV (for HVCN1L) or 40 mV (for HVCN1S) applied at 30-s intervals before and after addition of PMA or GFX to the bath solution. (C and D) Proton conductance–voltage relationships, gH–V. The current amplitude was determined by extrapolating a single exponential fitted to the rising current, and gH was calculated from the current using Vrev measured in each solution. Measurements were made in symmetrical pH 7.0 solutions containing 50 mM NH4+ to clamp pHi (9).
Fig. 3.
Fig. 3.
HVCN1S is phosphorylated more by PKC-δ than HVCN1L. PKC-δ in vitro kinase assay showing phosphorylation of HVCN1L and HVCN1S wild type (“WT”), single mutants T29A (“29”) or T9A for HVCN1S (“9”), S97A (“97”) or S77A for HVCN1S (“77”), and double mutants T29A/S97A and T9A/S77A for HVCN1S (“DM”). The assay was carried out with recombinant HVCN1L and HVCN1S expressed in HEK293T cells and immunoprecipitated with an anti-myc antibody. Cells transfected with an empty vector (“EV”) were used as negative control. The myc immunoblot indicates loading. Bars represent the densitometry analysis of the 32P-HVCN1 versus myc-HVCN1 of three independent experiments (mean ± SEM). NS, not significant.
Fig. 4.
Fig. 4.
HVCN1S is associated and internalized with the BCR to a lesser extent compared with HVCN1L. (A, Left) Confocal images of A20 D1.3 cells overexpressing HVCN1L or HVCN1S at steady state (“resting” panels) or 30 min after activation with an anti-IgM crosslinking F(ab')2 fragment (“activated” panels). Merge panels represent IgM and HVCN1 staining. White arrows indicate cointernalized HVCN1 and IgM. (Right) Quantification of percentage of internalized HVCN1 (Upper graph) and colocalization coefficient for HVCN1 and IgM (Lower graph). Each symbol represents a single cell [n = 78 (Upper); n = 67 (Lower)], and horizontal lines indicate the mean. Pearson’s colocalization coefficient (0, no colocalization; 1, total colocalization). (B) Coimmunoprecipitation of overexpressed HVCN1L or HVCN1S and endogenous CD79B (Ig-associated-β or Ig-β). Proteins were coimmunoprecipitated from A20 D1.3 cells and analyzed by immunoblot in nonreducing conditions. EV, cells transduced with empty vector; IN, input cell lysate (2% of the cell lysate used for immunoprecipitation); Ig, negative control beads conjugated to mouse or rat IgG; IP, immunoprecipitation; IB, immunoblot. Graphs represent densitometry analysis of both co-IP experiments (mean ± SEM, three independent experiments).
Fig. 5.
Fig. 5.
HVCN1S expression modulates BCR signaling, cell proliferation, and migration. (A) BCR stimulation with 20 μg/mL F(ab')2 anti-IgM in A20 D1.3 cells overexpressing empty vector (EV), HVCN1L, and HVCN1S. Immunoblot showing phosphorylated Erk (p-Erk) and total Erk. Bars indicate the ratio of the densitometry analysis of p-Erk versus total Erk from three independent experiments (mean ± SEM). (B) Proliferation of A20 D1.3 cells assessed by EdU incorporation after 3 h of incubation. Results are shown as percentage of Edu+ cells versus total number of GFP+ cells of two independent experiments (mean ± SEM). (C) Transwell chamber assay of migration toward the chemokine CXCL12. Migrated cells were counted after 4 h. Results are shown as fold increase of migrated cells in the presence of CXCL12 versus media alone. Data represent the average of two experiments (mean ± SEM).
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