Peña-Pichicoi A et al. (2023), N-terminal region is responsible for mHv1 chann...

XB-ART-60377

Front Pharmacol 2023 Jan 01;14:1265130. doi: 10.3389/fphar.2023.1265130.

Show Gene links Show Anatomy links

N-terminal region is responsible for mHv1 channel activity in MDSCs.

Peña-Pichicoi A , Fernández M , Navarro-Quezada N , Alvear-Arias JJ , Carrillo CA , Carmona EM , Garate J , Lopez-Rodriguez AM , Neely A , Hernández-Ochoa EO , González C .

???displayArticle.abstract???
Voltage-gated proton channels (Hv1) are important regulators of the immunosuppressive function of myeloid-derived suppressor cells (MDSCs) in mice and have been proposed as a potential therapeutic target to alleviate dysregulated immunosuppression in tumors. However, till date, there is a lack of evidence regarding the functioning of the Hvcn1 and reports on mHv1 isoform diversity in mice and MDSCs. A computational prediction has suggested that the Hvcn1 gene may express up to six transcript variants, three of which are translated into distinct N-terminal isoforms of mHv1: mHv1.1 (269 aa), mHv1.2 (269 + 42 aa), and mHv1.3 (269 + 4 aa). To validate this prediction, we used RT-PCR on total RNA extracted from MDSCs, and the presence of all six predicted mRNA variances was confirmed. Subsequently, the open-reading frames (ORFs) encoding for mHv1 isoforms were cloned and expressed in Xenopus laevis oocytes for proton current recording using a macro-patch voltage clamp. Our findings reveal that all three isoforms are mammalian mHv1 channels, with distinct differences in their activation properties. Specifically, the longest isoform, mHv1.2, displays a right-shifted conductance-voltage (GV) curve and slower opening kinetics, compared to the mid-length isoform, mHv1.3, and the shortest canonical isoform, mHv1.1. While mHv1.3 exhibits a V0.5 similar to that of mHv1.1, mHv1.3 demonstrates significantly slower activation kinetics than mHv1.1. These results suggest that isoform gating efficiency is inversely related to the length of the N-terminal end. To further explore this, we created the truncated mHv1.2 ΔN20 construct by removing the first 20 amino acids from the N-terminus of mHv1.2. This construct displayed intermediate activation properties, with a V0.5 value lying intermediate of mHv1.1 and mHv1.2, and activation kinetics that were faster than that of mHv1.2 but slower than that of mHv1.1. Overall, these findings indicate that alternative splicing of the N-terminal exon in mRNA transcripts encoding mHv1 isoforms is a regulatory mechanism for mHv1 function within MDSCs. While MDSCs have the capability to translate multiple Hv1 isoforms with varying gating properties, the Hvcn1 gene promotes the dominant expression of mHv1.1, which exhibits the most efficient gating among all mHv1 isoforms.

???displayArticle.pubmedLink??? 37915407
???displayArticle.pmcLink??? PMC10616795
???displayArticle.link??? Front Pharmacol
???displayArticle.grants??? [+]

Genes referenced: hvcn1

???attribute.lit??? ???displayArticles.show???

	FIGURE 1. Predicted alternative splicing for the Hvcn1 gene could lead to the expression of transcripts exhibiting voltage-dependent function. (A) Images of rectangular boxes represent the length and exonic composition of the six transcript variants predicted to be expressed by the voltage-gated proton channel gene in mice, Hvcn1. Their ORF length is highlighted with a light blue bar. All the exons from 9 to 13 are predicted to be conserved among all the Hvcn1 transcripts. Transcript variants 1, 2, and 3 were validated and reported in vitro, while transcript variants x, x1, and x2 are still predicted. From these transcripts, it is predicted to express mHv1.1 (black), mHv1.2 (blue), and mHv1.3 (red) isoforms at a membrane, pointing out the additional amino acids each isoform could have in comparison with the canonical mHv1.1. They show their main domains, such as the transmembrane segments (S) 0 to 4, the intracellular arranged coiled-coil region (CCR), the C-terminal domain (C’T), and the N-terminal domain (N’T), that focus the distinctive amino acidic differences among these isoforms. (B) MDSC Hvcn1 transcripts were amplified by RT-PCR. All predicted variants were expressed besides the reported variants 1–3. The marker for Gr1 was used as the MDSC control. GAPDH was used as the positive control, while RT (−) (amplification without the retrotranscriptase enzyme) and NTC (non-template control) were used as negative controls. Arrow colors are related to the color symbology used for mHv1 isoforms in (A). (C) Voltage-dependent outward currents from the different ORFs cloned from transcripts expressed from Hvcn1. Representative family of currents from oocytes expressing mHv1.1 (left, black), mHv1.2 (middle, blue), and mHv1.3 (right, red). Upper left inset: image representation of a macro-patch in inside-out configuration at ΔpH = 2. Bottom inset: voltage protocol applied during the experiment. Upper right inset: scale bar of intensity versus time.
	FIGURE 2. Predicted transcript variants exhibit biophysical hallmarks of mammalian mHv1 isoforms. (A) ∆pH dependence of IV curves for mHv1.2 (blue) and mHv1.3 (red) currents, respectively. The parameters of the fit of the data to the normalized Boltzmann function in Eq. 2, V0.5, and zδ are tabulated in Table 3. (B) Representative results from nonstationary noise analysis at ΔpH = 1 (pHin:6; pHex:7) of mHv1.2 (blue, left) and mHv1.3 (red, right) at 100 mV and 80 mV, respectively. (top) Time course of the mean current and the variance of mHv1.2 and mHv1.3, whose noise exhibits a biphasic behavior, characteristic of the probabilistic opening process of ion channels, with the first part pointing to a variance maximum, which corresponds to the point where half of the channels in the ensemble are open, and the second part pointing to the effective maximum open probability of the isoforms (bottom). Variance versus mean current plots for mHv1.2 and mHv1.3, with both data showing a parabolic behavior. These data were fitted to the parabolic Eq. 3, obtaining microscopical parameters of mHv1.2 and mHv1.3, such as the number of channels, with 9,477 mHv1.2 channels and 106,900 mHv1.3 channels; the unitary current, with 28 fA for mHv1.2 and 12.7 fA for mHv1.3; the maximum open probability, with 0.82 for mHv1.2 and 0.73 for mHv1.3; and the unitary conductance, with 177 fS for mHv1.2 and 91.9 fS for mHv1.3. (C) (upper) Representative currents for mHv1.2 (left, blue) and mHv1.3 (right, red) at ∆pH1 and with a depolarizing shortened protocol that avoids depletion (middle inset). The red rectangle is delimiting the fast ramp that is used to estimate the reversal potential (bottom). Magnification of the delimited red rectangle, showing the crossing of the proton currents and the minimum variance (bold curve) achieved where the currents are reversed (vertical black straight line) for mHv1.2 (left, blue) and mHv1.3 (right, red). (D) Reversal potentials plotted against pH gradients (∆pH) for mHv1.1, mHv1.2, and mHv1.3. Circles represent the experimental data; the black, blue, and red straight lines symbolize the fit to the data; and the dotted gray straight line represents the prediction of the Nernst equation of the equilibrium of protons.
	FIGURE 3. The longer isoforms of mHv1 possess a differential gating of their function. (A) Multiple alignment between the predicted primary sequences of the identified mHv1 isoforms, showing amino acid differences at the beginning of the N terminus. (B) Normalized conductance values at ΔpH = 2 (pHin:6; pHex:8) for isoforms of mHv1 (mHv1.1 in black, mHv1.2 in blue, and mHv1.3 in red) computed as G (V) = I/(V-Vrev). The data are expressed as mean ± SEM (n = 3) and fitted to a Boltzmann function (continuous line), G (V) = Gmax/1+exp [-zδF (V-V0.5)/RT] (Student’s t-test; *p < 0.005). (C) Time constants plotted against voltage for isoforms of mHv1 obtained from macroscopic Hv1 currents. The values are shown as mean ± SEM (n = 3) (Student’s t-test; p < 0.01 for mHv1.1 vs. mHv1.2 and mHv1.1 vs. mHv1.3; mHv1.2 vs. mHv1.3 exhibited p < 0.6).
	FIGURE 4. The N-terminal beginning directly modulates gating of mHv1 channels. (A) Image representation of the truncation of 20 amino acids from the N-terminal beginning in mHv1.2 to produce mHv1.2 Δ20N. (B) Multiple alignment between mHv1.1 and mHv1.2 and its 20-amino acid truncated form, mHv1.2 Δ20N. (C) Representative mHv1.2 Δ20N currents, by an inside-out macro-patch clamp at ΔpH = 2. (D) Fits shown in Figure 3B at ΔpH = 2 (pHin:6; pHex:8) for mHv1.1 (in black), mHv1.2 (in blue), and normalized conductance for mHv1.2 Δ20N (in green), computed as G (V) = I/(V-Vrev). The data are expressed as mean ± SEM (n = 3) and fitted to a Boltzmann function (continuous line), G (V) = Gmax/1+exp [-zδF (V-V0.5)/RT] (Student’s t-test; p < 0.05 for mHv1.1 vs. mHv1.2 Δ20N and mHv1.2 vs. mHv1.2 Δ20N). (E) Time constants plotted against voltage for isoforms of mHv1 and mHv1.2 Δ20N obtained from macroscopic Hv1 currents. [Student’s t-test; p < 0.05 for mHv1.1 vs. mHv1.2 Δ20N and mHv1.2 vs. mHv1.2 Δ20N; results were expressed as mean ± SEM (n = 3)].
	FIGURE 5. The Hvcn1 gene of MDSCs expresses up to three functional isoforms alternatively spliced at their N terminal. (A) The data obtained in the characterization of the macroscopic currents of the different isoforms of mHv1 allow us to conclude that a shorter N terminal promotes the efficiency of the channel gating. Thus, depending on the functional requirements, the MDSCs could modulate the activity related to proton extrusion by preferring isoforms with N terminals of different lengths. (B) Image representation of an MDSC expressing the Hvcn1 gene and alternatively splicing different transcripts that express as mHv1 isoforms. The alternative splicing regulates the extension of the N-terminal portion of mHv1 isoforms, which regulates the conductance of protons and opening kinetics in these channels.

References [+] :

Alvarez, Counting channels: a tutorial guide on ion channel fluctuation analysis. 2002, Pubmed