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Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel.

ABSTRACT: The activation of eukaryotic SLO K(+) channels by intracellular cues, mediated by a cytoplasmic structure called the gating ring, is central to their physiological roles. SLO3 channels are exclusively expressed in mammalian sperm, where variations of intracellular pH are critical to cellular function. Previous studies primarily focused on the mouse SLO3 orthologue and revealed that, in murine sperm, SLO3 mediates a voltage- and alkalization-activated K(+) current essential to male fertility. Here we investigate the activation of the human SLO3 channel by intracellular pH at the functional and structural level. By using electrophysiology in a heterologous system, we show that human SLO3 opens upon intracellular pH increase and that its expression and functional properties are modulated by LRRC52, a testis-specific accessory subunit. We next present the crystal structure of the human SLO3 gating ring. Comparison with the known structures of the corresponding domain from SLO1, a Ca(2+)-activated homologue, suggests that the SLO3 gating ring structure may represent an open state. Together, these results present insights into the function of a protein expected to be critical for human reproduction and provide a framework to study the mechanism of pH gating in SLO3 channels.

SUBMITTER: Leonetti MD

PROVIDER: S-EPMC3511096 | biostudies-literature | 2012 Nov

REPOSITORIES: biostudies-literature

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Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel.

Leonetti Manuel D MD Yuan Peng P Hsiung Yichun Y Mackinnon Roderick R

Proceedings of the National Academy of Sciences of the United States of America 20121105 47

The activation of eukaryotic SLO K(+) channels by intracellular cues, mediated by a cytoplasmic structure called the gating ring, is central to their physiological roles. SLO3 channels are exclusively expressed in mammalian sperm, where variations of intracellular pH are critical to cellular function. Previous studies primarily focused on the mouse SLO3 orthologue and revealed that, in murine sperm, SLO3 mediates a voltage- and alkalization-activated K(+) current essential to male fertility. Her ...[more]

PMID: 23129643

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Project description:Marine coccolithophorid phytoplankton are major producers of biogenic calcite, playing a significant role in the global carbon cycle. Predicting the impacts of ocean acidification on coccolithophore calcification has received much recent attention and requires improved knowledge of cellular calcification mechanisms. Uniquely amongst calcifying organisms, coccolithophores produce calcified scales (coccoliths) in an intracellular compartment and secrete them to the cell surface, requiring large transcellular ionic fluxes to support calcification. In particular, intracellular calcite precipitation using HCO?? as the substrate generates equimolar quantities of H+ that must be rapidly removed to prevent cytoplasmic acidification. We have used electrophysiological approaches to identify a plasma membrane voltage-gated H+ conductance in Coccolithus pelagicus ssp braarudii with remarkably similar biophysical and functional properties to those found in metazoans. We show that both C. pelagicus and Emiliania huxleyi possess homologues of metazoan H(v)1 H+ channels, which function as voltage-gated H+ channels when expressed in heterologous systems. Homologues of the coccolithophore H+ channels were also identified in a diversity of eukaryotes, suggesting a wide range of cellular roles for the H(v)1 class of proteins. Using single cell imaging, we demonstrate that the coccolithophore H+ conductance mediates rapid H+ efflux and plays an important role in pH homeostasis in calcifying cells. The results demonstrate a novel cellular role for voltage gated H+ channels and provide mechanistic insight into biomineralisation by establishing a direct link between pH homeostasis and calcification. As the coccolithophore H+ conductance is dependent on the trans-membrane H+ electrochemical gradient, this mechanism will be directly impacted by, and may underlie adaptation to, ocean acidification. The presence of this H+ efflux pathway suggests that there is no obligate use of H+ derived from calcification for intracellular CO? generation. Furthermore, the presence of H(v)1 class ion channels in a wide range of extant eukaryote groups indicates they evolved in an early common ancestor.

Project description:The mouse Slo3 gene (KCNMA3) encodes a K(+) channel that is regulated by changes in cytosolic pH. Like Slo1 subunits responsible for the Ca(2+) and voltage-activated BK-type channel, the Slo3 alpha subunit contains a pore module with homology to voltage-gated K(+) channels and also an extensive cytosolic C terminus thought to be responsible for ligand dependence. For the Slo3 K(+) channel, increases in cytosolic pH promote channel activation, but very little is known about many fundamental properties of Slo3 currents. Here we define the dependence of macroscopic conductance on voltage and pH and, in particular, examine Slo3 conductance activated at negative potentials. Using this information, the ability of a Horrigan-Aldrich-type of general allosteric model to account for Slo3 gating is examined. Finally, the pH and voltage dependence of Slo3 activation and deactivation kinetics is reported. The results indicate that Slo3 differs from Slo1 in several important ways. The limiting conductance activated at the most positive potentials exhibits a pH-dependent maximum, suggesting differences in the limiting open probability at different pH. Furthermore, over a 600 mV range of voltages (-300 to +300 mV), Slo3 conductance shifts only about two to three orders of magnitude, and the limiting conductance at negative potentials is relatively voltage independent compared to Slo1. Within the context of the Horrigan-Aldrich model, these results indicate that the intrinsic voltage dependence (z(L)) of the Slo3 closed-open equilibrium and the coupling (D) between voltage sensor movement are less than in Slo1. The kinetic behavior of Slo3 currents also differs markedly from Slo1. Both activation and deactivation are best described by two exponential components, both of which are only weakly voltage dependent. Qualitatively, the properties of the two kinetic components in the activation time course suggest that increases in pH increase the fraction of more rapidly opening channels.

Dataset Information

Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel.

Publications

Functional and structural analysis of the human SLO3 pH- and voltage-gated K+ channel.

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