Voltage-gated Ion Channel Superfamily Research Articles

Voltage-gated sodium channel (NaV) protein dissection creates a set of functional pore-only proteins

Many voltage-gated ion channel (VGIC) superfamily members contain six-transmembrane segments in which the first four form a voltage-sensing domain (VSD) and the last two form the pore domain (PD). Studies of potassium channels from the VGIC superfamily together with identification of voltage-sensor only proteins have suggested that the VSD and the PD can fold independently. Whether such transmembrane modularity is common to other VGIC superfamily members has remained untested. Here we show, using protein dissection, that the Silicibacter pomeroyi voltage-gated sodium channel (Na(V)Sp1) PD forms a stand-alone, ion selective pore (Na(V)Sp1p) that is tetrameric, α-helical, and that forms functional, sodium-selective channels when reconstituted into lipid bilayers. Mutation of the Na(V)Sp1p selectivity filter from LESWSM to LDDWSD, a change similar to that previously shown to alter ion selectivity of the bacterial sodium channel Na(V)Bh1 (NaChBac), creates a calcium-selective pore-only channel, Ca(V)Sp1p. We further show that production of PDs can be generalized by making pore-only proteins from two other extremophile Na(V)s: one from the hydrocarbon degrader Alcanivorax borkumensis (Na(V)Ab1p), and one from the arsenite oxidizer Alkalilimnicola ehrlichei (Na(V)Ae1p). Together, our data establish a family of active pore-only ion channels that should be excellent model systems for study of the factors that govern both sodium and calcium selectivity and permeability. Further, our findings suggest that similar dissection approaches may be applicable to a wide range of VGICs and, thus, serve as a means to simplify and accelerate biophysical, structural, and drug development efforts.

Role of the Putative Glycines Hinge of Cav3.3 Channels

Voltage-gated ion channels (VGIC) are proteins that form transmembrane pores in cell membranes. Based on the crystal structure of an open potassium channel (KV) that reveals the bending of a glycine residue in the inner helix that lines the pore (M2 or S6), and the conserved pore sequences in many members of the VGIC superfamily, is thought that the opening mechanism might be quite similar within the family. In fact, this has been demonstrated for a bacterial sodium channel (NaV). However, in LVA calcium channels, the mid-S6 hinge glycine residue is present only in IS6 and IIS6, suggesting a likely different opening mechanism. Here, we explored this possibility in the CaV3.3 member of LVA channels by performing a mutational analysis of the two conserved glycines (Gly385 and Gly814), and a valine (Val1383) in IIIS6 which, according to sequence similarity, corresponds to a hinge glycine in KV and NaV channels. Substitution of Gly814 for proline (G814P) or alanine (G814A), as well as the mutant V1383G, drastically decreased whole-cell current density, with discrete effects on the voltage-dependence of activation. However, the mutant G814P displayed a 10 mV shift in the steady-state inactivation curve to hyperpolarized potentials; the current inactivation kinetics was slowed down by 50%, and the recovery was also more than two-fold slower. The substitution by proline also increased the channel closing time constant by 60%; this effect was also displayed by mutant V1383G, which in addition induced the strongest effect on the inactivation kinetics slowing (≥ 2-fold). Our preliminary results suggest that residues Gly814 and Val1383 are mostly involved in the inactivation gating and, in a lesser degree, stabilizing the open state of CaV3.3. Additional mutations, including double and triple substitutions, are currently under investigation.Supported by CONACYT Mexico J50250Q.

Two-pore Channels for Calcium Mobilization from Acidic Organelles and Cell Signaling by NAADP

Two-pore channels (TPCs) are novel members of the superfamily of voltage-gated ion channels. Their predicted structures indicate 2-fold symmetry with a total of 12 putative transmembrane (TM) α-helices. Sequence homology and membrane topology analyses suggest that TPCs may represent evolutionary intermediates from the single domain 6-TM architecture K+ and non-selective cation channels to the four-repeat 24-TM structure of voltage-gated Ca2+ and Na+ channels. Three genes (TPCN1-3) exist in most vertebrates but their functions remain elusive. We now show that TPC1 and TPC3 are expressed on the membrane of different endosome populations while TPC2 is expressed on the membrane of lysosomes. We provide functional data showing that TPC2 is a target of nicotinic acid adenine dinucleotide phosphate (NAADP), a potent Ca2+ mobilizing messenger that evokes Ca2+ release from acidic organelles rather than from the sarco/endoplasmic reticulum. Thus, microsomal membranes enriched with TPC2 exhibit similar high affinity binding with NAADP as native NAADP receptors. In response to NAADP, cells overexpressing TPC2 exhibit enhanced intracellular Ca2+ release and more efficient coupling to IP3 receptors to evoke global Ca2+ transients. These effects were blocked by disrupting lysosomal H+ gradient or RNAi-mediated silencing of TPC2 expression. Our findings provide for the first time a molecular basis for further detailed characterization of the regulatory mechanisms and physiological functions of NAADP-mediated signaling and, in addition, suggest a general role for TPCs in Ca2+ mobilization, Ca2+ homeostasis, and Ca2+ signaling from endosomal/lysosomal compartments of vertebrate cells, which are known to be important for diverse functions in many physiological systems.

Overview of Molecular Relationships in the Voltage-Gated Ion Channel Superfamily

Complex multicellular organisms require rapid and accurate transmission of information among cells and tissues and tight coordination of distant functions. In vertebrates, electrical signals and the resulting intracellular calcium transients control contraction of muscle, secretion of hormones,

The Origin and Early Evolution of Membrane Channels

The origin and early evolution of ion channels are considered from the point of view that the transmembrane segments of membrane proteins are structurally quite simple and do not require specific sequences to fold. We argue that the transport of solute species, especially ions, required an early evolution of efficient transport mechanisms, and that the emergence of simple ion channels was protobiologically plausible. We also argue that, despite their simple structure, such channels could possess properties that, at the first sight, appear to require markedly greater complexity. These properties can be subtly modulated by local modifications to the sequence rather than global changes in molecular architecture. In order to address the evolution and development of ion channels, we focus on identifying those protein domains that are commonly associated with ion channel proteins and are conserved throughout the three main domains of life (Eukarya, Bacteria, and Archaea). We discuss the potassium-sodium-calcium superfamily of voltage-gated ion channels, mechanosensitive channels, porins, and ABC-transporters and argue that these families of membrane channels have sufficiently universal architectures that they can readily adapt to the diverse functional demands arising during evolution.

Cyclic nucleotide-gated ion channels.

Cyclic nucleotide-gated (CNG) channels are nonselective cation channels first identified in retinal photoreceptors and olfactory sensory neurons (OSNs). They are opened by the direct binding of cyclic nucleotides, cAMP and cGMP. Although their activity shows very little voltage dependence, CNG channels belong to the superfamily of voltage-gated ion channels. Like their cousins the voltage-gated K+ channels, CNG channels form heterotetrameric complexes consisting of two or three different types of subunits. Six different genes encoding CNG channels, four A subunits (A1 to A4) and two B subunits (B1 and B3), give rise to three different channels in rod and cone photoreceptors and in OSNs. Important functional features of these channels, i.e., ligand sensitivity and selectivity, ion permeation, and gating, are determined by the subunit composition of the respective channel complex. The function of CNG channels has been firmly established in retinal photoreceptors and in OSNs. Studies on their presence in other sensory and nonsensory cells have produced mixed results, and their purported roles in neuronal pathfinding or synaptic plasticity are not as well understood as their role in sensory neurons. Similarly, the function of invertebrate homologs found in Caenorhabditis elegans, Drosophila, and Limulus is largely unknown, except for two subunits of C. elegans that play a role in chemosensation. CNG channels are nonselective cation channels that do not discriminate well between alkali ions and even pass divalent cations, in particular Ca2+. Ca2+ entry through CNG channels is important for both excitation and adaptation of sensory cells. CNG channel activity is modulated by Ca2+/calmodulin and by phosphorylation. Other factors may also be involved in channel regulation. Mutations in CNG channel genes give rise to retinal degeneration and color blindness. In particular, mutations in the A and B subunits of the CNG channel expressed in human cones cause various forms of complete and incomplete achromatopsia.

Dynamic selectivity filters in ion channels.

This work was supported by a Wellcome Trust (UK) International Prize Travelling Fellowship and the NIH (NS-11756).

MOLECULAR AND FUNCTIONAL DIVERSITY OF K+ CHANNELS

1. Amino acid sequences encoding K+ channels belong to several subfamilies of the voltage-gated ion channel superfamily which includes Na2+-, Ca2+-, and cyclic nucleotide gated channels. The Kv family is the largest group, and encodes delayed rectifier, A-type, and large conductance Ca2+ activated K+ channels. 2. The alpha-subunits of Kv channels form as tetramers of four independent subunits. Each subunit has six membrane spanning regions and a pore forming 'P' region. Subunits belong to subfamilies (Kv1-4, BK, Eag) comprising multiple members, each of which has distinct properties resembling each of the major types of native Kv channel when expressed as homomultimers in heterologous systems. 3. Enormous diversity of Kv channel function arises from the multiplicity of subunits, the formation of heteromultimers within subfamilies and from association with intracellular beta-subunit proteins. 4. In the absence of direct structural information, mutational analyses have provided considerable insights into the structure of the voltage-sensor, pore-forming region and the sites of action of drugs, toxins and associated proteins. 5. Another subfamily, the inwardly rectifying, or KIR, family, appears to have arisen from a deletion of the first four membrane spanning regions of ancient Kv channels, changing gating properties from outward to inward rectification. These include the G-protein gated inward rectifiers and KATP channels.

Images of purified Shaker potassium channels

Voltage-gated K+ channels play an important role in the control of neuronal excitability and synaptic plasticity. Their low abundance and extraordinary heterogeneity have rendered their purification from natural sources difficult. We have previously cloned a voltage-gated K(+)-channel gene, Shaker, from Drosophila. The Shaker K(+)-channel polypeptide resembles one of the four internal repeats of a Na(+)- or Ca(2+)-channel alpha subunit, suggesting that this example of a K+ channel contains four identical or homologous subunits. Similar K(+)-channel polypeptides have been characterized from mammals, other vertebrate and invertebrate species, and from plants. Electrophysiological studies of K+ channels expressed in Xenopus oocytes suggest that they are indeed tetramers, and heteromultimeric K+ channels have been found in the mammalian brain. Until now, however, no K+ channel, nor any other member of the superfamily of voltage-gated ion channels, has been characterized by electron microscopy or other structural analysis. We have purified Shaker K+ channels, expressed in insect Sf9 cells, to apparent homogeneity, and imaged them using the electron microscope. The physical dimensions of these molecules, as well as their biochemical characteristics, are consistent with a tetrameric subunit composition. Moreover, the Shaker channel revealed by negative staining has the appearance of a four-fold symmetric tetramer, with a large, central vestibule that presumably constitutes part of the pathway for ions. These first clear images of a voltage-gated ion channel reveal a marked four-fold symmetry. The integrity of the purified tetrameric complex indicates that the purification scheme used in this study may be further developed for future structural analysis of voltage-gated K+ channels.