Distinct modulating effects of TipE-homologs 2–4 on Drosophila sodium channel splice variants

The Drosophila melanogaster TipE protein is thought to be an insect sodium channel auxiliary subunit functionally analogous to the β subunits of mammalian sodium channels. Besides TipE, four TipE-homologous proteins (TEH1–4) have been identified. It has been reported that TipE and TEH1 have both common and distinct effects on the gating properties of splice variants of the Drosophila sodium channel, DmNa_v. However, limited information is available on the effects of TEH2, TEH3 and TEH4 on the function of DmNa_v channel variants. In this study, we found that TEH2 increased the amplitude of peak current, but did not alter the gating properties of three examined DmNa_v splice variants expressed in Xenopus oocytes. In contrast, TEH4 had no effect on peak current, yet altered the gating properties of all three channel variants. Furthermore, TEH4 enhanced persistent current and slowed sodium current decay. The effects of TEH3 on DmNa_v variants are similar to those of TEH4, but the data were collected from a small portion of oocytes because co-expression of TEH3 with DmNa_v variants generated a large leak current in the majority of oocytes examined. In addition, TEH3 and TEH4 enhanced the expression of endogenous currents in oocytes. Taken together, our results reveal distinct roles of TEH proteins in modulating the function of sodium channels and suggest that TEH proteins might provide an important layer of regulation of membrane excitability in vivo. Our results also raise an intriguing possibility of TEH3/TEH4 as auxiliary subunits of other voltage-gated ion channels besides sodium channels.     

Four novel sequences in Drosophila melanogaster homologous to the auxiliary Para sodium channel subunit TipE

TipE is an auxiliary subunit of the Drosophila Para sodium channel. Here we describe four sequences, TEH1-4, homologous to TipE in the Drosophila melanogaster genome, harboring all typical structures of both TipE and the beta-Subunit family of big-conductance Ca(2+)-activated potassium channels: short cytosolic N- and C-terminal stretches, two transmembrane domains, and a large extracellular loop with two disulfide bonds. Whereas TEH1 and TEH2 lack the TipE-specific extension in the extracellular loop, both TEH3 and TEH4 possess two extracellular EGF-like domains. A CNS-specific expression was found for TEH1, while TEH2-4 were more widely expressed. The genes for TEH2-4 are localized close to the tipE gene on chromosome 3L. Coexpression of TEH subunits with Para in Xenopus oocytes showed a strong (30-fold, TEH1), medium (5- to 10-fold, TEH2 and TEH3), or no (TEH4) increase in sodium current amplitude, while TipE increased the current 20-fold. In addition, steady-state inactivation and the recovery from fast inactivation were altered by coexpression of Para with TEH1. We conclude that members of the TEH-family are auxiliary subunits for Para sodium channels and possibly other ion channels.     

Differential Effects of TipE and a TipE-Homologous Protein on Modulation of Gating Properties of Sodium Channels from Drosophila melanogaster

β subunits of mammalian sodium channels play important roles in modulating the expression and gating of mammalian sodium channels. However, there are no orthologs of β subunits in insects. Instead, an unrelated protein, TipE in Drosophila melanogaster and its orthologs in other insects, is thought to be a sodium channel auxiliary subunit. In addition, there are four TipE-homologous genes (TEH1-4) in D. melanogaster and three to four orthologs in other insect species. TipE and TEH1-3 have been shown to enhance the peak current of various insect sodium channels expressed in Xenopus oocytes. However, limited information is available on how these proteins modulate the gating of sodium channels, particularly sodium channel variants generated by alternative splicing and RNA editing. In this study, we compared the effects of TEH1 and TipE on the function of three Drosophila sodium channel splice variants, DmNa_v9-1, DmNa_v22, and DmNa_v26, in Xenopus oocytes. Both TipE and TEH1 enhanced the amplitude of sodium current and accelerated current decay of all three sodium channels tested. Strikingly, TEH1 caused hyperpolarizing shifts in the voltage-dependence of activation, fast inactivation and slow inactivation of all three variants. In contrast, TipE did not alter these gating properties except for a hyperpolarizing shift in the voltage-dependence of fast inactivation of DmNa_v26. Further analysis of the gating kinetics of DmNa_v9-1 revealed that TEH1 accelerated the entry of sodium channels into the fast inactivated state and slowed the recovery from both fast- and slow-inactivated states, thereby, enhancing both fast and slow inactivation. These results highlight the differential effects of TipE and TEH1 on the gating of insect sodium channels and suggest that TEH1 may play a broader role than TipE in regulating sodium channel function and neuronal excitability in vivo.     

昆虫钠离子通道的研究进展

昆虫只有一个或两个电压门控钠离子通道α亚基基因,但两种转录后修饰(选择性剪切和RNA编辑)实现了昆虫钠离子通道的功能多样性.昆虫β辅助亚基TipE和TEH1-4在钠离子通道表达和调控中也起着重要作用.电压门控钠离子通道在动作电位的产生和传递中至关重要,是多种天然和人工合成神经毒素及杀虫剂的作用靶标,包括广泛使用的拟除虫...     

Molecular properties of sodium and calcium channels

Voltage-gated sodium and calcium channels are responsible for inward movement of sodium and calcium during electrical signals in cell membranes. Their principal subunits are members of a gene family and can function as voltage-gated ion channels by themselves. They are expressed in association with one or more auxiliary subunits which increase functional expression and modify the functional properties of the principal subunits. Structural elements which are required for voltage-dependent activation, selective ion conductance, and inactivation have been identified, and their mechanisms of action are being explored through mutagenesis, expression in heterologous cells, and functional analysis. These experiments reveal that these two channels are built on a common structural theme with variations appropriate for functional specialization of each channel type.     

Molecular and functional characterization of a novel sodium channel TipE-like auxiliary subunit from the American cockroach Periplaneta americana

In Drosophila melanogaster, the functions of voltage-gated sodium (Na_v) channels are modulated by TipE and its orthologs. Here, we describe a novel TipE homolog of the American cockroach, Periplaneta americana, called PaTipE. Like DmTipE, PaTipE mRNAs are ubiquitously expressed. Surprisingly, PaTipE mRNA was undetectable in neurosecretory cells identified as dorsal unpaired median neurons. Phylogenetic analysis placed this new sequence in TipE clade, indicating an independent evolution from a common ancestor. Contrary to previous reports, our data indicate that the auxiliary subunits of insect Na_v channels are very distant from the mammalian BKCa auxiliary subunits. To decipher the functional roles of PaTipE, we characterized the gating properties of DmNa_v1-1 channels co-expressed with DmTipE or PaTipE, in Xenopus oocytes. Compared to DmTipE, PaTipE increased Na⁺ currents by a 4.2-fold. The voltage-dependence of steady-state fast inactivation of DmNa_v1-1/PaTipE channels was shifted by 5.8 mV to more negative potentials than that of DmNa_v1-1/DmTipE channels. DmNa_v1-1/PaTipE channels recovered 3.2-fold slower from the fast-inactivated state than DmNa_v1-1/DmTipE channels. In conclusion, this study supports that the insect Na_v auxiliary subunits share functional features with their mammalian counterparts, although structurally and phylogenetically distant.     

The second hydrophobic domain contributes to the kinetic properties of epithelial sodium channels

The epithelial sodium channel (ENaC) is the prototype of a new class of ion channels known as the ENaC/Deg family. The hallmarks of ENaC are a high selectivity for Na(+), block by amiloride, small conductance, and slow kinetics that are voltage-independent. We have investigated the contribution of the second hydrophobic domain of each of the homologous subunits alpha, beta, and gamma to the kinetic properties of ENaC. Chimeric subunits were constructed between alpha and beta subunits (alpha-beta) and between gamma and beta subunits (gamma-beta). Chimeric and wild-type subunits were expressed in various combinations in Xenopus oocytes. Analysis of whole-cell and unitary currents made it possible to correlate functional properties with specific sequences in the subunits. Functional channels were generated without the second transmembrane domain from alpha subunits, indicating that it is not essential to form functional pores. The open probability and kinetics varied with the different channels and were influenced by the second hydrophobic domains. Amiloride affinity, Li(+)/Na(+) selectivity, and single channel conductance were also affected by this segment.     

In vivo functional role of the Drosophila hyperkinetic beta subunit in gating and inactivation of Shaker K+ channels.

The physiological roles of the beta, or auxiliary, subunits of voltage-gated ion channels, including Na+, Ca2+, and K+ channels, have not been demonstrated directly in vivo. Drosophila Hyperkinetic (Hk) mutations alter a gene encoding a homolog of the mammalian K+ channel beta subunit, providing a unique opportunity to delineate the in vivo function of auxiliary subunits in K+ channels. We found that the Hk beta subunit modulates a wide range of the Shaker (Sh) K+ current properties, including its amplitude, activation and inactivation, temperature dependence, and drug sensitivity. Characterizations of the existing mutants in identified muscle cells enabled an analysis of potential mechanisms of subunit interactions and their functional consequences. The results are consistent with the idea that via hydrophobic interaction, Hk beta subunits modulate Sh channel conformation in the cytoplasmic pore region. The modulatory effects of the Hk beta subunit appeared to be specific to the Sh alpha subunit because other voltage- and Ca(2+)-activated K+ currents were not affected by Hk mutations. The mutant effects were especially pronounced near the voltage threshold of IA activation, which can disrupt the maintenance of the quiescent state and lead to the striking neuromuscular and behavioral hyperexcitability previously reported.     

Sodium channel beta subunits mediate homophilic cell adhesion and recruit ankyrin to points of cell-cell contact

Sodium channels isolated from mammalian brain are composed of alpha, beta1, and beta2 subunits. The auxiliary beta subunits do not form the ion conducting pore, yet play important roles in channel modulation and plasma membrane expression. beta1 and beta2 are transmembrane proteins with one extracellular V-set immunoglobulin (Ig) protein domain. It has been shown recently that beta1 and beta2 interact with the extracellular matrix proteins tenascin-C and tenascin-R. In the present study we show that rat brain beta1 and beta2, but not alphaIIA, subunits interact in a trans-homophilic fashion, resulting in recruitment of the cytoskeletal protein ankyrin to sites of cell-cell contact in transfected Drosophila S2 cells. Whereas alphaIIA subunits expressed alone do not cause cellular aggregation, beta subunits co-expressed with alphaIIA retain the ability to adhere and recruit ankyrin. Truncated beta subunits lacking cytoplasmic domains interact homophilically to produce cell aggregation but do not recruit ankyrin. Thus, the cytoplasmic domains of beta1 and beta2 are required for cytoskeletal interactions. It is hypothesized that sodium channel beta subunits serve as a critical communication link between the extracellular and intracellular environments of the neuron and may play a role in sodium channel placement at nodes of Ranvier.     

Redox-sensitive extracellular gates formed by auxiliary beta subunits of calcium-activated potassium channels

An important step to understanding ion channels is identifying the structural components that act as the gates to ion movement. Here we describe a new channel gating mechanism, produced by the beta3 auxiliary subunits of Ca2+-activated, large-conductance BK-type K+ channels when expressed with their pore-forming alpha subunits. BK beta subunits have a cysteine-rich extracellular segment connecting two transmembrane segments, with small cytosolic N and C termini. The extracellular segments of the beta3 subunits form gates to block ion permeation, providing a mechanism by which current can be rapidly diminished upon cellular repolarization. Furthermore, this gating mechanism is abolished by reduction of extracellular disulfide linkages, suggesting that endogenous mechanisms may regulate this gating behavior. The results indicate that auxiliary beta subunits of BK channels reside sufficiently close to the ion permeation pathway defined by the alpha subunits to influence or block access of small molecules to the permeation pathway.     

Cloning and functional characterization of a putative sodium channel auxiliary subunit gene from the house fly (Musca domestica)

The functional expression of cloned Drosophila melanogaster and house fly (Musca domestica) voltage-sensitive sodium channels in Xenopus oocytes is enhanced, and the inactivation kinetics of the expressed channels are accelerated, by coexpression with the tipE protein, a putative sodium channel auxiliary subunit encoded by the tipE gene of D. melanogaster. These results predict the existence of a tipE ortholog in the house fly. Using a PCR-based homology probing approach, we isolated cDNA clones encoding an ortholog of tipE (designated Vssc beta) from adult house fly heads. Clones comprising 3444 bp of cDNA sequence contained a 1317 bp open-reading frame encoding a 438 amino acid protein. The predicted Vssc beta protein exhibited 72% amino acid sequence identity to the entire D. melanogaster tipE protein sequence and 97% identity within the two hydrophobic segments identified as probable transmembrane domains. Coexpression of Vssc beta with the house fly sodium channel alpha subunit (Vssc1) in oocytes enhanced the level of sodium current expression five-fold and accelerated the rate of sodium current inactivation 2.2-fold. Both of these effects were significantly larger in magnitude than the corresponding effects of the D. melanogaster tipE protein on the expression and kinetics of Vssc1 sodium channels. These results identify a second example of a putative sodium channel auxiliary subunit from an insect having functional but not structural homology to vertebrate sodium channel beta subunits.     

Lysine-rich extracellular rings formed by hbeta2 subunits confer the outward rectification of BK channels

The auxiliary beta subunits of large-conductance Ca(2+)-activated K(+) (BK) channels greatly contribute to the diversity of BK (mSlo1 alpha) channels, which is fundamental to the adequate function in many tissues. Here we describe a functional element of the extracellular segment of hbeta2 auxiliary subunits that acts as the positively charged rings to modify the BK channel conductance. Four consecutive lysines of the hbeta2 extracellular loop, which reside sufficiently close to the extracellular entryway of the pore, constitute three positively charged rings. These rings can decrease the extracellular K(+) concentration and prevent the Charybdotoxin (ChTX) from approaching the extracellular entrance of channels through electrostatic mechanism, leading to the reduction of K(+) inflow or the outward rectification of BK channels. Our results demonstrate that the lysine rings formed by the hbeta2 auxiliary subunits influences the inward current of BK channels, providing a mechanism by which current can be rapidly diminished during cellular repolarization. Furthermore, this study will be helpful to understand the functional diversity of BK channels contributed by different auxiliary beta subunits.     

MEC-2 and MEC-6 in the Caenorhabditis elegans sensory mechanotransduction complex: auxiliary subunits that enable channel activity

The ion channel formed by the homologous proteins MEC-4 and MEC-10 forms the core of a sensory mechanotransduction channel in Caenorhabditis elegans. Although the products of other mec genes are key players in the biophysics of transduction, the mechanism by which they contribute to the properties of the channel is unknown. Here, we investigate the role of two auxiliary channel subunits, MEC-2 (stomatin-like) and MEC-6 (paraoxonase-like), by coexpressing them with constitutively active MEC-4/MEC-10 heteromeric channels in Xenopus oocytes. This work extends prior work demonstrating that MEC-2 and MEC-6 synergistically increase macroscopic current. We use single-channel recordings and biochemistry to show that these auxiliary subunits alter function by increasing the number of channels in an active state rather than by dramatically affecting either single-channel properties or surface expression. We also use two-electrode voltage clamp and outside-out macropatch recording to examine the effects of divalent cations and proteases, known regulators of channel family members. Finally, we examine the role of cholesterol binding in the mechanism of MEC-2 action by measuring whole-cell and single-channel currents in MEC-2 mutants deficient in cholesterol binding. We suggest that MEC-2 and MEC-6 play essential roles in modulating both the local membrane environment of MEC-4/MEC-10 channels and the availability of such channels to be gated by force in vivo.     

The intracellular segment of the sodium channel beta 1 subunit is required for its efficient association with the channel alpha subunit

Sodium channels consist of a pore-forming alpha subunit and auxiliary beta 1 and beta 2 subunits. The subunit beta 1 alters the kinetics and voltage-dependence of sodium channels expressed in Xenopus oocytes or mammalian cells. Functional modulation in oocytes depends on specific regions in the N-terminal extracellular domain of beta 1, but does not require the intracellular C-terminal domain. Functional modulation is qualitatively different in mammalian cells, and thus could involve different molecular mechanisms. As a first step toward testing this hypothesis, we examined modulation of brain Na(V)1.2a sodium channel alpha subunits expressed in Chinese hamster lung cells by a mutant beta1 construct with 34 amino acids deleted from the C-terminus. This deletion mutation did not modulate sodium channel function in this cell system. Co-immunoprecipitation data suggest that this loss of functional modulation was caused by inefficient association of the mutant beta 1 with alpha, despite high levels of expression of the mutant protein. In Xenopus oocytes, injection of approximately 10,000 times more mutant beta 1 RNA was required to achieve the level of functional modulation observed with injection of full-length beta 1. Together, these findings suggest that the C-terminal cytoplasmic domain of beta 1 is an important determinant of beta1 binding to the sodium channel alpha subunit in both mammalian cells and Xenopus oocytes.     

Hydrophobic properties of the beta 1 and beta 2 subunits of the rat brain sodium channel

Voltage-sensitive sodium channels purified from rat brain in functional form consist of a stoichiometric complex of three glycoprotein subunits, alpha of 260 kDa, beta 1 of 36 kDa, and beta 2 of 33 kDa. The alpha and beta 2 subunits are linked by disulfide bonds. The hydrophobic properties of these three subunits were examined by covalent labeling with the photoreactive hydrophobic probe 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) which labels transmembrane segments in integral membrane proteins. All three subunits of the sodium channel were labeled by [125I]TID when the purified protein was solubilized in mixed micelles of Triton X-100 and phosphatidylcholine (4:1). The half-time for photolabeling was approximately 7 min consistent with the half-time of 9 min for photolysis of TID under our conditions. Comparable amounts of TID per mg of protein were incorporated into each subunit. Purified sodium channels reconstituted in phosphatidylcholine vesicles were also labeled by TID with comparable incorporation per mg of protein into all three subunits. The efficiency of photolabeling of the three subunits was reduced from 39 to 44% by a 2-fold expansion of the hydrophobic phase of the reaction mixture but was unaffected by a 2-fold expansion of the aqueous phase, confirming that the photolabeling reaction took place in the lipid phase of the vesicle bilayer. The hydrophobic properties of the sodium channel subunits were examined further using phase separation in the nonionic detergent Triton X-114. Under conditions in which beta 1 is dissociated from alpha, the beta 1 subunit was preferentially extracted into the Triton X-114 phase, and the disulfide-linked alpha beta 2 complex was retained in the aqueous phase. When the disulfide bonds between the alpha and beta 2 subunits were reduced with dithioerythritol, the beta 2 subunit was also preferentially extracted into the Triton X-100 phase leaving the free alpha subunit in the aqueous phase. A preparative method for isolation of the beta 1 and beta 2 subunits was developed based on this technique. Considered together, the results of our hydrophobic labeling and phase separation experiments indicate that the alpha, beta 1, and beta 2 subunits all have substantial hydrophobic domains that may interact with the hydrocarbon phase of phospholipid bilayer membranes. Since the alpha subunit is known to be a transmembrane protein with many potential membrane-spanning segments, we conclude that the beta 1 and beta 2 subunits are likely to also be integral membrane proteins with one or more membrane-spanning segments.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phylogenetic analyses of potassium channel auxiliary subunits

Results of recent genome-sequencing projects together with advances in biochemical, molecular genetic and physiological experimentation have allowed discovery of many transport auxiliary subunits. These subunits facilitate the proper movement of substrates across cell membranes. Mutations of any of these subunits can cause catastrophic effects to the transport mechanism and cause certain genetic diseases. Auxiliary subunits of ion channels are of particular interest because of their potential to diversify the transport properties of the principal subunits. Furthermore, ion channel auxiliary subunits may function in the capacity of enhancing surface expression, allowing gating, and providing chaperone-like activities. As a result of their evolutionary histories, these proteins can be grouped exclusively by phylogenetic techniques. Many of these families are found to be restricted to a single kingdom of life while others seem to be ubiquitous. Here we report the results of systematic analyses of three families of ion channel auxiliary subunits. Some subunits contain one or more transmembrane segments while others exist only in the cytoplasm. We have also observed potential horizontal transfer across kingdoms with these auxiliary subunits. In this report, we present tabulated results of homology searches, partial multiple alignments, secondary structure analyses, and phylogenetic trees for these families.     

Membrane interaction of TNF is not sufficient to trigger increase in membrane conductance in mammalian cells

The Shaker type voltage-gated potassium (K+) channel consists of four pore-forming Kv alpha subunits. The channel expression and kinetic properties can be modulated by auxiliary hydrophilic Kv beta subunits via formation of heteromultimeric Kv alpha-Kv beta complexes. Because each (Kv alpha)4 could recruit more than one Kv beta subunit and different Kv beta subunits could potentially interact, the stoichiometry of alpha-beta and beta-beta complexes is therefore critical for understanding the functional regulation of Shaker type potassium channels. We expressed and purified Kv beta 2 subunit in Sf9 insect cells. The purified Kv beta 2, examined by atomic force and electron microscopy techniques, is found predominately as a square-shaped tetrameric complex with side dimensions of 100 x 100 A2 and height of 51 A. Thus, Kv beta 2 is capable of forming a tetramer in the absence of pore-forming alpha subunits. The center of the Kv beta 2 complex was observed to be the most heavily stained region, suggesting that this region could be part of an extended tubular structure connecting the inner mouth of the ion permeation pathway to the cytoplasmic environment.     

Structural determinants for functional coupling between the beta and alpha subunits in the Ca2+-activated K+ (BK) channel

High conductance, calcium- and voltage-activated potassium (BK, MaxiK) channels are widely expressed in mammals. In some tissues, the biophysical properties of BK channels are highly affected by coexpression of regulatory (beta) subunits. The most remarkable effects of beta1 and beta2 subunits are an increase of the calcium sensitivity and the slow down of channel kinetics. However, the detailed characteristics of channels formed by alpha and beta1 or beta2 are dissimilar, the most remarkable difference being a reduction of the voltage sensitivity in the presence of beta1 but not beta2. Here we reveal the molecular regions in these beta subunits that determine their differential functional coupling with the pore-forming alpha-subunit. We made chimeric constructs between beta1 and beta2 subunits, and BK channels formed by alpha and chimeric beta subunits were expressed in Xenopus laevis oocytes. The electrophysiological characteristics of the resulting channels were determined using the patch clamp technique. Chimeric exchange of the different regions of the beta1 and beta2 subunits demonstrates that the NH3 and COOH termini are the most relevant regions in defining the behavior of either subunit. This strongly suggests that the intracellular domains are crucial for the fine tuning of the effects of these beta subunits. Moreover, the intracellular domains of beta1 are responsible for the reduction of the BK channel voltage dependence. This agrees with previous studies that suggested the intracellular regions of the alpha-subunit to be the target of the modulation by the beta1-subunit.