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Recently, three proton channels (HV) have been identified and characterized in Aplysia californica (AcHV1‐3). Focusing on AcHV1 and AcHV2, analysis of Transcriptome Shotgun Assembly and genomic databases of 91 molluscs identified HV homologous channels in other molluscs: channels homologous to AcHV1 and to AcHV2 were found in 90 species (56 full‐length sequences) and in 33 species (18 full‐length sequences), respectively. Here, we report the discovery of a fourth distinct proton channel family, HV4. This new family has high homology to AcHV1 and AcHV2 and was identified only in bivalvian molluscs (13 species, 12 full‐length sequences). Typically, these channels possess an extracellular S1–S2 loop of intermediate size (~ 20 amino acids) compared to the shorter loops of molluscan HV1 channels (~ 13 amino acids) and the much larger loops of molluscan HV2 channels (> 65 amino acids). The characteristic voltage‐sensor motif in S4 possesses only two arginine residues with the common third arginine being replaced by a lysine. Moreover, HV4 channels are much smaller with only around 200 amino acids in total length. The smallest functional channel found so far in nature (189 amino acids) is expressed in the pacific oyster Crassostrea gigas (CgHV4) and might be considered an archetypical minimal proton channel. Functional expression and electrophysiological characterization demonstrated that CgHV4 shares distinctive hallmarks of other investigated proton channels as high proton selectivity, slow activation, and pH‐ and voltage‐regulated gating. This work is the first description of a HV4 type channel, adding a new member to the recently expanded family of proton channels. [ABSTRACT FROM AUTHOR]

Voltage‐gated ion channels, whose first identified function was to generate action potentials, are divided into subfamilies with numerous members. The family of voltage‐gated proton channels (HV) is tiny. To date, all species found to express HV have exclusively one gene that codes for this unique ion channel. Here we report the discovery and characterization of three proton channel genes in the classical model system of neural plasticity, Aplysia californica. The three channels (AcHV1, AcHV2, and AcHV3) are distributed throughout the whole animal. Patch‐clamp analysis confirmed proton selectivity of these channels but they all differed markedly in gating. AcHV1 gating resembled HV in mammalian cells where it is responsible for proton extrusion and charge compensation. AcHV2 activates more negatively and conducts extensive inward proton current, properties likely to acidify the cytosol. AcHV3, which differs from AcHV1 and AcHV2 in lacking the first arginine in the S4 helix, exhibits proton selective leak currents and weak voltage dependence. We report the expansion of the proton channel family, demonstrating for the first time the expression of three functionally distinct proton channels in a single species. [ABSTRACT FROM AUTHOR]

Aspartate key to proton selectivity Voltage-gated proton channels are considered completely selective for protons -- no evidence exists for the permeation of ions other than H.sup.+. A study of the underlying mechanism of the high selectivity of the human voltage-gated proton channel reveals an Asp112 residue as the selectivity filter. When this residue was changed to a neutral amino acid, the mutant channel lost proton selectivity and either became anion-selective or did not conduct any ions. The ion selectivity of pumps and channels is central to their ability to perform a multitude of functions. Here we investigate the mechanism of the extraordinary selectivity of the human voltage-gated proton channel.sup.1, H.sub.V1 (also known as HVCN1). This selectivity is essential to its ability to regulate reactive oxygen species production by leukocytes.sup.2,3,4, histamine secretion by basophils.sup.5, sperm capacitation.sup.6, and airway pH.sup.7. The most selective ion channel known, H.sub.V1 shows no detectable permeability to other ions.sup.1. Opposing classes of selectivity mechanisms postulate that (1) a titratable amino acid residue in the permeation pathway imparts proton selectivity.sup.1,8,9,10,11, or (2) water molecules 'frozen' in a narrow pore conduct protons while excluding other ions.sup.12. Here we identify aspartate 112 as a crucial component of the selectivity filter of H.sub.V1. When a neutral amino acid replaced Asp 112, the mutant channel lost proton specificity and became anion-selective or did not conduct. Only the glutamate mutant remained proton-specific. Mutation of the nearby Asp 185 did not impair proton selectivity, indicating that Asp 112 has a unique role. Although histidine shuttles protons in other proteins, when histidine or lysine replaced Asp 112, the mutant channel was still anion-permeable. Evidently, the proton specificity of H.sub.V1 requires an acidic group at the selectivity filter., Author(s): Boris Musset [sup.1] , Susan M. E. Smith [sup.2] , Sindhu Rajan [sup.3] , Deri Morgan [sup.1] , Vladimir V. Cherny [sup.1] , Thomas E. DeCoursey [sup.1] Author Affiliations: [...]

The effects of low concentrations of extracellular ATP on cytosolic [Ca.sup.2+], membrane potential, and transcription of IL-6 were studied in monocyte-derived human macrophages. During inflammation or infection many cells secrete ATP. We show here that application of 10 [micro]M ATP or 10 [micro]M UTP induces oscillations in cytosolic [Ca.sup.2+] with a frequency of [approximately equal to] 12 [min.sup.-1] and oscillations in membrane potential. RT-PCR analysis showed expression of P2[Y.sub.1], P2[Y.sub.2], P2[Y.sub.11], P2[X.sub.1], P2[X.sub.4], and P2[X.sub.7] receptors, large-conductance (KCNMA1 and KCNMB1-4), and intermediate-conductance (KCNN4) [Ca.sup.2+]- activated [K.sup.+] channels. The [Ca.sup.2+] oscillations were unchanged after removal of extracellular [Ca.sup.2+], indicating that they were mainly due to movements of [Ca.sup.2+] between intracellular compartments. Comparison of the effects of different nucleotides suggests that the [Ca.sup.2+] oscillations were elicited by activation of P2[Y.sub.2] receptors coupled to phospholipase C. Patch-clamp experiments showed that ATP induced a transient depolarization, probably mediated by activation of P2[X.sub.4] receptors, followed by membrane potential oscillations due to opening of [Ca.sup.2+]- activated [K.sup.+] channels. We also found that 10 [micro]M ATP[gamma]S increased transcription of IL-6 [aproximately equal to] 40-fold within 2 h. This effect was abolished by blockade of P2Y receptors with 100 [micro]M suramin. Our results suggest that ATP released from inflamed, damaged, or metabolically impaired cells represents a 'danger signal' that plays a major role in activating the innate immune system.