The fourth alpha helix, S4, contains positive charges represented by three arginine residues (R205, R208, and R211) in the characteristic VSD repeats. This channel is thought to be a functional dimer formed by two subunits comprising an intracellular N-terminus, a bundle of four transmembrane helices (TMHs, S1–S4) in the VSD fold, and a long intracellular alpha helix that mediates a coiled-coil interaction, mainly responsible for dimerization and cooperative activation ( Koch et al., 2008 Lee et al., 2008 Tombola et al., 2008). Also, H V1 is involved in different pathologies such as B cell malignancy ( Hondares et al., 2014), breast cancer ( Wang et al., 2012), and post-ischemic brain injury ( Wu, 2014 Yu et al., 2020) consequently, in recent years, H V1 has emerged as a possible pharmacological target ( Zhao et al., 2018 Zhang et al., 2022).Īmong all voltage-gated proton channels sequenced, the human ortholog, hH V1, is the most widely studied ( Musset et al., 2008). The functions of H V1 channels are diverse, including intracellular pH regulation ( Ma et al., 2022), charge compensation during immune response ( Ramsey et al., 2009), modulation of flagellar beating in spermatozoa ( Lishko and Kirichok, 2010), bioluminescence ( Eckert and Sibaoka, 1968), and possible roles in calcification processes in marine organisms ( Taylor et al., 2011 Rangel-Yescas et al., 2021). H V1 is thought to form ion channels activated by voltage and employing a mechanism of activation similar to the VSDs of canonical voltage-gated potassium, sodium, and calcium channels ( Gonzalez et al., 2012). These VSDs are also encountered in voltage-sensitive phosphatases (VSPs) and voltage-gated ion channels (VGICs), and their principal function is to detect the membrane potential difference and translate it into a conformational change that activates VSP and opens VGIC ( Catacuzzeno and Franciolini, 2022). Voltage-gated, proton-permeable ion currents in a large variety of cell types and organisms are produced by the H V1 gene (HVCN1 in humans), which encodes a membrane protein that is a member of the superfamily of voltage-sensing domains (VSDs Sasaki et al., 2006 Ramsey et al., 2006). These results shed light on the complexities of voltage-dependent opening of human H V1 channels. Our experiments also suggest that the voltage sensor can move after channel opening and that the absolute value of the pH can influence the channel opening step. The kinetics of the fluorescence signal reveal the existence of a very slow transition in the deactivation pathway, which seems to be singularly regulated by ΔpH. We present data that indicate that Anap incorporated in the S4 helix is quenched by an aromatic residue located in the S2 helix and that motion of the S4 relative to this quencher is responsible for fluorescence increases upon depolarization.
Simultaneous recording of currents and fluorescence signals allows for direct correlation of these parameters and investigation of their dependence on voltage and the pH gradient (ΔpH). To circumvent this problem, we have implemented patch-clamp fluorometry in combination with the incorporation of the fluorescent non-canonical amino acid Anap to monitor channel opening and movement of the S4 segment. Voltage-dependent gating of the voltage-gated proton channels (H V1) remains poorly understood, partly because of the difficulty of obtaining direct measurements of voltage sensor movement in the form of gating currents.
0 kommentar(er)
