Person:

Clapham, David

When cells swell in hypo-osmotic solutions, chloride-selective ion channels ((Cl_{swell})) activate to reduce intracellular osmolality and prevent catastrophic cell rupture. Despite intensive efforts to assign a molecular identity to the mammalian (Cl_{swell}) channel, it remains unknown. In an unbiased genome-wide RNA interference (RNAi) screen of Drosophila cells stably expressing an anion-sensitive fluorescent indicator, we identify Bestrophin 1 (dBest1) as the Drosophila (Cl_{swell}) channel. Of the 23 screen hits with mammalian homologs and predicted transmembrane domains, only RNAi specifically targeting dBest1 eliminated the (Cl_{swell}) current ((I_{Clswell})). We further demonstrate the essential contribution of dBest1 to Drosophila (I_{Clswell}) with the introduction of a human Bestrophin disease-associated mutation (W94C). Overexpression of the W94C construct in Drosophila cells significantly reduced the endogenous (I_{Clswell}). We confirm that exogenous expression of dBest1 alone in human embryonic kidney (HEK293) cells creates a clearly identifiable Drosophila–like (I_{Clswell}). In contrast, activation of mouse Bestrophin 2 (mBest2), the closest mammalian ortholog of dBest1, is swell-insensitive. The first 64 residues of dBest1 conferred swell activation to mBest2. The chimera, however, maintains mBest2-like pore properties, strongly indicating that the Bestrophin protein forms the (Cl_{swell}) channel itself rather than functioning as an essential auxiliary subunit. dBest1 is an anion channel clearly responsive to swell; this activation depends upon its N-terminus.

Phagocytic cell NADPH oxidase (NOX) generates reactive oxygen species (ROS) as part of innate immunity. Unfortunately, ischemia can also induce this pathway and inflict damage on native cells. Here we show that NOX–mediated damage can be inhibited by suppression of the voltage-gated proton channel, Hv1. Hv1 is required for full NOX activity since it compensates for loss of NOX–exported charge. We show that Hv1 is required for NOX–dependent ROS generation in brain microglia in situ and in vivo. Mouse and human brain microglia, but not neurons or astrocytes, express large Hv1-mediated currents. Mice lacking Hv1 were protected from NOX–mediated neuronal death and brain damage 24 hours after stroke. These results demonstrate that Hv1–dependent ROS production is responsible for a significant fraction of brain damage at early time points after ischemic stroke and provide a rationale for Hv1 as a therapeutic target for the treatment of ischemic stroke.

Native PKD2-L1 channel subunits are present in primary cilia and other restricted cellular spaces. Here we investigate the mechanism for the channel's unusual regulation by external calcium, and rationalize this behavior to its specialized function. We report that the human PKD2-L1 selectivity filter is partially selective to calcium ions (Ca2+) moving into the cell, but blocked by high internal Ca2+concentrations, a unique feature of this transient receptor potential (TRP) channel family member. Surprisingly, we find that the C-terminal EF-hands and coiled-coil domains do not contribute to PKD2-L1 Ca2+-induced potentiation and inactivation. We propose a model in which prolonged channel activity results in calcium accumulation, triggering outward-moving Ca2+ ions to block PKD2-L1 in a high-affinity interaction with the innermost acidic residue (D523) of the selectivity filter and subsequent long-term channel inactivation. This response rectifies Ca2+ flow, enabling Ca2+ to enter but not leave small compartments such as the cilium. DOI: http://dx.doi.org/10.7554/eLife.13413.001

Human sperm cells rely on an unusual type of potassium ion channel.

During passage through the female reproductive tract, mammalian sperm undergo a maturation process termed capacitation that renders sperm competent to produce fertilization. Capacitation involves a sequence of changes in biochemical and electrical properties, the onset of a hyperactivated swimming behavior, and development of the ability to undergo successful fusion and penetration with an egg. In mouse sperm, the development of hyperactivated motility is dependent on cytosolic alkalization that then results in an increase in cytosolic Ca2+. The elevation of Ca2+ is thought to be primarily driven by the concerted interplay of two alkalization-activated currents, a K+ current (KSPER) composed of pore-forming subunits encoded by the Kcnu1 gene (also termed Slo3) and a Ca2+ current arising from a family of CATSPER subunits. After deletion of any of four CATSPER subunit genes (CATSPER1–4), the major remaining current in mouse sperm is alkalization-activated KSPER current. After genetic deletion of the Slo3 gene, KSPER current is abolished, but there remains a small voltage-activated K+ current hypothesized to reflect monovalent flux through CATSPER. Here, we address two questions. First, does the residual outward K+ current present in the Slo3 −/− sperm arise from CATSPER? Second, can any additional membrane K+ currents be detected in mouse sperm by patch-clamp methods other than CATSPER and KSPER? Here, using mice bred to lack both SLO3 and CATSPER1 subunits, we show conclusively that the voltage-activated outward current present in Slo3 −/− sperm is abolished when CATSPER is also deleted. Any leak currents that may play a role in setting the resting membrane potential in noncapacitated sperm are likely smaller than the pipette leak current and thus cannot be resolved within the limitation of the patch-clamp technique. Together, KSPER and CATSPER appear to be the sole ion channels present in mouse sperm that regulate membrane potential and Ca2+ influx in response to alkalization.

Voltage-gated sodium channels have essential roles in electrical signalling. Prokaryotic sodium channels are tetramers consisting of transmembrane (TM) voltage-sensing and pore domains, and a cytoplasmic carboxy-terminal domain. Previous crystal structures of bacterial sodium channels revealed the nature of their TM domains but not their C-terminal domains (CTDs). Here, using electron paramagnetic resonance (EPR) spectroscopy combined with molecular dynamics, we show that the CTD of the NavMs channel from Magnetococcus marinus includes a flexible region linking the TM domains to a four-helix coiled-coil bundle. A 2.9 Å resolution crystal structure of the NavMs pore indicates the position of the CTD, which is consistent with the EPR-derived structure. Functional analyses demonstrate that the coiled-coil domain couples inactivation with channel opening, and is enabled by negatively charged residues in the linker region. A mechanism for gating is proposed based on the structure, whereby splaying of the bottom of the pore is possible without requiring unravelling of the coiled-coil.

Entry and extrusion of cations are essential processes in living cells. In alkaliphilic prokaryotes, high external pH activates voltage-gated sodium channels (Nav), which allows Na+ to enter and be used as substrate for cation/proton antiporters responsible for cytoplasmic pH homeostasis. Here, we describe a new member of the prokaryotic voltage-gated Na+ channel family (NsvBa; Non-selective voltage-gated, Bacillus alcalophilus) that is nonselective among Na+, Ca2+ and K+ ions. Mutations in NsvBa can convert the nonselective filter into one that discriminates for Na+ or divalent cations. Gain-of-function experiments demonstrate the portability of ion selectivity with filter mutations to other Bacillus Nav channels. Increasing pH and temperature shifts their activation threshold towards their native resting membrane potential. Furthermore, we find drugs that target Bacillus Nav channels also block the growth of the bacteria. This work identifies some of the adaptations to achieve ion discrimination and gating in Bacillus Nav channels. DOI: http://dx.doi.org/10.7554/eLife.04387.001

Parasympathetic regulation of sinoatrial node (SAN) pacemaker activity modulates multiple ion channels to temper heart rate. The functional role of the G-protein–activated K+ current (IKACh) in the control of SAN pacemaking and heart rate is not completely understood. We have investigated the functional consequences of loss of IKACh in cholinergic regulation of pacemaker activity of SAN cells and in heart rate control under physiological situations mimicking the fight or flight response. We used knockout mice with loss of function of the Girk4 (Kir3.4) gene (Girk4−/− mice), which codes for an integral subunit of the cardiac IKACh channel. SAN pacemaker cells from Girk4−/− mice completely lacked IKACh. Loss of IKACh strongly reduced cholinergic regulation of pacemaker activity of SAN cells and isolated intact hearts. Telemetric recordings of electrocardiograms of freely moving mice showed that heart rate measured over a 24-h recording period was moderately increased (10%) in Girk4−/− animals. Although the relative extent of heart rate regulation of Girk4−/− mice was similar to that of wild-type animals, recovery of resting heart rate after stress, physical exercise, or pharmacological β-adrenergic stimulation of SAN pacemaking was significantly delayed in Girk4−/− animals. We conclude that IKACh plays a critical role in the kinetics of heart rate recovery to resting levels after sympathetic stimulation or after direct β-adrenergic stimulation of pacemaker activity. Our study thus uncovers a novel role for IKACh in SAN physiology and heart rate regulation.

Massive endocytosis is initiated by a series of steps that involve a sudden influx of calcium ions, changes in mitochondria, and modification of surface proteins by lipids. A better understanding of this process could lead to new approaches to reducing the tissue damage that is caused by heart attacks.

Summary Primary cilia are solitary nonmotile extensions of the centriole found on nearly all nucleated eukaryotic cells between cell divisions. Only ∼200-300 nm in diameter and a few microns long, they are separated from the cytoplasm by the ciliary neck and basal body. Often called sensory cilia, they are hypothesized to receive chemical and mechanical stimuli and initiate specific cellular signal transduction pathways. When activated by a ligand, Hedgehog (Hh) pathway proteins, such as Gli2 and Smoothened (Smo), translocate from the cell into the cilium1,2. Mutations in primary ciliary proteins are associated with severe developmental defects3. The ionic conditions, permeability of the primary cilia membrane, and effectiveness of the diffusion barriers between the cilia and cell body are unknown. Here we show that cilia are a unique calcium compartment regulated by a heteromeric TRP channel, PKD1-L1/PKD2-L1. In contrast to the hypothesis that polycystin (PKD) channels initiate changes in ciliary calcium that are conducted into the cytoplasm4, we show that changes in ciliary calcium concentration ([Ca2+]cilia) occur without substantially altering global cytoplasmic calcium ([Ca2+]cyto). PKD1-L1/PKD2-L1 acts as a ciliary calcium channel controlling [Ca2+]cilia and thereby modifying Smo-activated Gli2 translocation and Gli1 expression.