Vacuolar ion channels in the liverwort Conocephalum conicum

As a liverwort Conocephalum conicum belongs to the oldest terrestrial plants¹ and is phylogenetically located between green algae and higher plants. Recent patch-clamp recordings on Conocephalum vacuoles^2,3 demonstrate ion channels very similar to higher plants and clearly different from vacuolar ion channels described in green algae. Here we summarize the features of a vacuolar cation channel and a vacuolar anion channel that both are common in terrestrial plants but are not detected in green algae, and we speculate about the molecular identity of these channels in the liverwort Conocephalum.Key words: vacuole, SV channel, anion channel, Conocephalum conicum, Embryophyta     

Vacuolar two-pore K+ channels act as vacuolar osmosensors

? Plant two-pore K(+) channels (TPKs) have been shown previously to play a role in vacuolar K(+) homeostasis. TPK activity is insensitive to membrane voltage, but regulated by cytoplasmic calcium and 14-3-3 proteins. This study reports that membrane stretch and osmotic gradients also alter the activity of TPKs from Arabidopsis, rice and barley, and that this may have a physiological relevance for osmotic homeostasis. ? Mechanosensitivity was studied using patch clamp experiments and TPKs from Arabidopsis, rice and barley. In addition, the capability of TPKs to act as osmosensors was determined. By using protoplast disruption assays and intact plant survival assays, in genotypes that differed in TPK expression, the physiological relevance of TPK-based osmosensing was tested. ? TPKs from all three species showed varying degrees of mechanosensitivity. TPK activity in channels from all three species was sensitive to trans-tonoplast osmotic gradients. TPK osmosensing is likely to proceed via the detection of small perturbations in membrane tension. Intact plant and protoplast assays showed that TPK-based osmosensing is important during exposure to rapid changes in external osmolarity. ? Vacuolar TPK channels can act as intracellular osmosensors and rapidly increase channel activity during hypo-osmotic shock to release vacuolar K(+) .     

Vacuolar ion channels: Roles in plant nutrition and signalling

Vacuoles play various roles in many physiologically relevant processes in plants. Some of the more prominent are turgor provision, the storage of minerals and nutrients, and cellular signalling. To fulfil these functions a complement of membrane transporters is present at the tonoplast. Prolific patch clamp studies have shown that amongst these, both selective and non-selective ion channels participate in turgor regulation, nutrient storage and signalling. This article reviews the physiological roles, expression patterns and structure function properties of plant vacuolar anion and cation channels that are gated by voltage and ligands.     

Voltage-gated calcium channels

The article concentrates on representatives of voltage-gated calcium ion-channels that are present in practically all cells. Considered are the activation and inactivation processes of calcium channels and their molecular mechanisms. The review represents modem classification of voltage-gated calcium channels, draws parallels with the earlier classifications and discusses calcium currents going through various calcium channels. Presented are the genetic, molecular-biological, bio-physical, physiological and pharmacological information for each type of the ten known voltage-gated calcium channels.     

Mitochondrial calcium channels

Mitochondrial Ca²⁺ handling plays an important role in energy production and various cellular signaling processes. Mitochondrial Ca²⁺ uptake is regulated by the mitochondrial Ca²⁺ uniporter (MCU), at least one non-MCU Ca²⁺ channel and possibly a mitochondrial ryanodine receptor. Two distinct mechanisms mediate Ca²⁺ outward transport, the Na⁺-dependent (mNCX) and the Na⁺-independent Ca²⁺ efflux. In recent years we gained more insight into the regulation and function of these different Ca²⁺ transport mechanisms. However, the precise physiological role and the molecular structure of all mitochondrial Ca²⁺ transporters and channels still has to be determined.     

Voltage-gated calcium channels

The article concentrates on representatives of voltage-gated calcium ion channels that are present in practically all cells. Regarded is the molecular arrangement of a voltage-gated calcium channel that consists of pore forming trans-membrane alpha1 subunit and auxiliary alpha2delta-, beta-, and gamma-subunits. Under discussion are the structure and functions of each subunit. The principles of subunits interaction are considered. The research represents modern classification of voltage-gated calcium channels, draws parallels with the earlier classifications and discusses calcium currents going through various calcium channels. Considered are the problems of regulating the activity of voltage-gated channels by proteinkinases. The issues of blockers and activators of voltage-gated calcium channels are brought up. The article gives a detailed analysis of the mechanisms of voltage-gated calcium channels selectivity. The molecular organization of the selectivity filter is considered. Presented are the basic theories of permeability of voltage-gated calcium channels.     

Voltage-dependent calcium channels

Voltage-activated calcium channels can be divided into two subgroups based on their activation threshold, low-voltage-activated (LVA) and high-voltage-activated (HVA). Auxiliary subunits of the HVA calcium channels contribute significantly to biophysical properties of the channels. We have cloned and characterized members of two families of auxiliary subunits: alpha2delta and gamma. Two new alpha2delta subunits, alpha2delta-2 and alpha2delta-3, regulate all classes of HVA calcium channels. While the ubiquitous alpha2delta-2 modulates both neuronal and non-neuronal channels with similar efficiency, the alpha2delta-3 subunit regulates Ca(v)2.3 channels more effectively. Furthermore, alpha2delta-2 may modulate the LVA Ca(v)3.1 channel. Four new gamma subunits, gamma-2, gamma-3, gamma-4 and gamma-5, were characterized. The gamma-2 subunit modulated both the non-neuronal Ca(v)1.2 channel and the neuronal Ca(v)2.1 channel. The gamma-4 subunit affected only the Ca(v)2.1 channel. The gamma-5 subunit may be a regulatory subunit of the LVA Ca(v)3.1 channel. The Ca(v)1.2 channel is a major target for treatment of cardiovascular diseases. We have mapped the interaction site for clinically important channel blockers - dihydropyridines (DHPs) - and analysed the underlying inhibition mechanism. High-affinity inhibition is characterized by interaction with inactivated state of the channel. Its structural determinants are amino acids of the IVS6 segment, with smaller contribution of the IS6 segment, which contributes to voltage-dependence of DHP inhibition. Removal of amino acids responsible for the high-affinity inhibition revealed a low-affinity open channel block, in which amino acids of the IIIS5 and IIIS6 segments take part. Experiments with a permanently charged DHP suggested that there is another low-affinity interaction site on the alpha(1) subunit. We have cloned and characterized murine neuronal LVA Ca(v)3.1 channel. The channel has high sensitivity to the organic blocker mibefradil, moderate sensitivity to phenytoin, and low sensitivity to ethosuximide, amiloride and valproat. The channel is insensitive to tetrodotoxin and DHPs. The inorganic blockers Ni2+ and Cd2+ are moderately effective compared to La3+. The current through the Ca(v)3.1 channel inactivates faster with Ba2+ compared to Ca2+. Molecular determinants of fast inactivation are located in amino side of the intracellular carboxy terminus. The voltage dependence of charge movement is very shallow compared to the voltage dependence of current activation. Transfer of 30 % of charge correlates with activation of 70 % of measurable macroscopic current. Prolonged depolarization does not immobilize charge movement of the Ca(v)3.1 channel.     

Voltage-gated calcium channels

Voltage-gated calcium (Ca(2+)) channels are key transducers of membrane potential changes into intracellular Ca(2+) transients that initiate many physiological events. There are ten members of the voltage-gated Ca(2+) channel family in mammals, and they serve distinct roles in cellular signal transduction. The Ca(V)1 subfamily initiates contraction, secretion, regulation of gene expression, integration of synaptic input in neurons, and synaptic transmission at ribbon synapses in specialized sensory cells. The Ca(V)2 subfamily is primarily responsible for initiation of synaptic transmission at fast synapses. The Ca(V)3 subfamily is important for repetitive firing of action potentials in rhythmically firing cells such as cardiac myocytes and thalamic neurons. This article presents the molecular relationships and physiological functions of these Ca(2+) channel proteins and provides information on their molecular, genetic, physiological, and pharmacological properties.     

Inactivation of calcium channels

Rapid progress in our understanding of the properties and functions of voltage-gated calcium channels had produced the need for an update to our previous review of calcium inactivation. The major elements of change included in this review are: 1. The existence of multiple forms of voltage-sensitive Ca+ channels, with distinctive single channel properties, thus necessitating a reappraisal of properties deduced from macroscopic current recordings, particularly of the processes of activation and inactivation. 2. The differences in biochemical properties between channel types are reflected in their differences in divalent selectivity, their requirement for metabolic maintenance and their mechanism of inactivation. These properties appear to divide the channels into two categories which may relate to their molecular structures. Further subgroupings, based upon the voltage thresholds, have also been observed. 3. Molecular properties of one class of channels have been elucidated, which correlate with the observed biochemistry of channel modulation and inactivation. 4. An enzymatic process underlying the mechanism of Ca2+-dependent inactivation has been elucidated and may serve as a model for other modulatory systems. The interweaving of the properties of these Ca2+ channels, with their spatial distributions and their influence upon other channel types, acts to transduce and integrate information within cells.     

Capacitative calcium entry channels

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.     

Cell proliferation, calcium influx and calcium channels

Both increases in the basal cytosolic calcium concentration ([Ca²⁺]_cyt) and [Ca²⁺]_cyt transients play major roles in cell cycle progression, cell proliferation and division. Calcium transients are observed at various stages of cell cycle and more specifically during late G₁ phase, before and during mitosis. These calcium transients are mainly due to calcium release and reuptake by the endoplasmic reticulum (ER) and are observed over periods of hours in oocytes and mammalian cells. Calcium entry sustains the ER Ca²⁺ load and thereby helps to maintain these calcium transients for such a long period. Calcium influx also controls cell growth and proliferation in several cell types. Various calcium channels are involved in this process and the tight relation between the expression and activity of cyclins and calcium channels also suggests that calcium entry may be needed only at particular stages of the cell cycle. Consistent with this idea, the expression of l-type and T-type calcium channels and SOCE amplitude fluctuate along the cell cycle. But, as calcium influx regulates several other transduction pathways, the presence of a specific connection to trigger activation of proliferation and cell division in mammalian cells will be discussed in this review.     

Voltage-gated calcium channels and disease

Voltage-gated calcium channels are a family of integral membrane calcium-selective proteins found in all excitable and many nonexcitable cells. Calcium influx affects membrane electrical properties by depolarizing cells and generally increasing excitability. Calcium entry further regulates multiple intracellular signaling pathways as well as the biochemical factors that mediate physiological functions such as neurotransmitter release and muscle contraction. Small changes in the biophysical properties or expression of calcium channels can result in pathophysiological changes leading to serious chronic disorders. In humans, mutations in calcium channel genes have been linked to a number of serious neurological, retinal, cardiac, and muscular disorders.     

Disease-causing mutations of calcium channels

Many cellular functions are directly or indirectly regulated by the free cytosolic calcium concentration. Thus, calcium levels must be very tightly regulated in time and space. Intracellular calcium ions are essential second messengers and play a role in many functions including, action potential generation, neurotransmitter and hormone release, muscle contraction, neurite outgrowth, synaptogenesis, calcium-dependent gene expression, synaptic plasticity and cell death. Calcium ions that control cell activity can be supplied to the cell cytosol from two major sources: the extracellular space or intracellular stores. Voltage-gated and ligand-gated channels are the primary way in which Ca2+ ions enter from the extracellular space. The sarcoplasm reticulum (SR) in muscle and the endoplasmic reticulum in non-muscle cells are the main intracellular Ca2+ stores: the ryanodine receptor (RyR) and inositol-triphosphate receptor channels are the major contributors of calcium release from internal stores. Mutations of genes encoding calcium have been implicated in the etiology of a diverse group of nerve and muscle diseases. These mutations have been identified in humans, mice and other organisms. In this review, we will summarize calcium channelopathies of humans and mice. Of the ten calcium channel α1 subunits cloned and sequenced (see ref. 1), disease-causing mutations have been found in CaV1.4 and CaV2.1 in the nervous system, and CaV1.1 and CaV1.2 in muscle. Mutations in calcium channel auxiliary subunits (α2δ, β and γ) have also been associated with both human and/or mouse neurological diseases. The disease-causing mutations may provide new insight into the cell biological roles of calcium channels as well as into relationships between structure and function. In addition, understanding how the mutations affect the physiology of the cell could lead to advances in disease treatment by relieving symptoms or slowing the progression of the disease. However, due to the multifaceted functions of calcium in the cell, the correlation between molecular mutation, physiological alterations and disease etiology is neither straightforward nor easily understood. Since calcium is an important intracellular signaling molecule, altered calcium channel function can give rise to widespread changes in cellular function. Indeed, serious diseases result from mutations that cause trivial alterations of calcium currents analyzed in vitro.     

电压依赖性钙通道

电压依赖性钙通道分为L、N、T三种类型,L型通道是由四个结构相似的亚区组成的跨膜蛋白亲水孔道,每个亚区又由六个α螺旋构成,其中一个为带正电的α螺旋,可能构成电压敏感装置。通道内有两个钙亲和位点,对钙的选择通透由亲和位点和离子间静电作用造成。钙通道的调制表现为对通道单位电流值、通道开放数目、电压依赖性,以及时间依赖性单独或复合的影响,通道蛋白磷酸化介导了许多生物活性物质对通道功能的调制。