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

Frontiers in Pharmacology

Pharmacology

Juan J. Alvear-Arias¹  Antonio Peña-Pichicoi¹  Nieves Navarro-Quezada¹  Alan Neely¹  Christian A. Carrillo¹  Miguel Fernández¹  Carlos González²  Erick O. Hernández-Ochoa³  Angelica M. Lopez-Rodriguez⁴  Jose Garate⁵  Emerson M. Carmona⁶ 

[1] Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile;Millennium Nucleus in NanoBioPhysics, Universidad de Valparaíso, Valparaíso, Chile;Centro Interdisciplinario de Neurociencia de Valparaíso, Universidad de Valparaíso, Valparaíso, Chile;Millennium Nucleus in NanoBioPhysics, Universidad de Valparaíso, Valparaíso, Chile;Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, FL, United States;Department of Biochemistry and Molecular Biology, School of Medicine, University of Maryland, Baltimore, MD, United States;Facultad de Ciencias Químicas, Universidad Juárez del Estado de Durango, Durango, Mexico;Millennium Nucleus in NanoBioPhysics, Universidad de Valparaíso, Valparaíso, Chile;Facultad de Ingeniería y Tecnología, Universidad San Sebastián, Santiago, Chile;Texas Tech University Health Sciences Center, Lubbock, TX, United States;

关键词: voltage-gated proton channel;    cloning;    mHv1.1;    mHv1.2;    mHv1.3;    myeloid-derived suppressor cells;    immunosuppression;   

DOI  :  10.3389/fphar.2023.1265130

 received in 2023-07-22, accepted in 2023-09-25,  发布年份 2023

来源: Frontiers

【 摘 要 】

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.

【 授权许可】

Unknown   
Copyright © 2023 Peña-Pichicoi, Fernández, Navarro-Quezada, Alvear-Arias, Carrillo, Carmona, Garate, Lopez-Rodriguez, Neely, Hernández-Ochoa and González.