Determinants of pore folding in potassium channel biogenesis

Delaney, Erin; Khanna, Pooja; Tu, Li‐Wei; Robinson, John M.; Deutsch, Carol

doi:10.1073/pnas.1324274111

Cited by 17 publications

(29 citation statements)

References 20 publications

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“…These findings indicate that the VGIC-PD fold is built from a fundamentally ancient scaffold comprising a pair of anti-parallel helices bridged by a loop. The capacity of such a structure to fold independently of higher order association is in line with long-standing ideas for a two-step folding mechanism for membrane proteins 26,27 agrees with cysteine accessibility studies suggesting that Kv channel pore domains adopt a native-like topology during folding [23][24][25] and points to a possible route for the evolutionary origins of the tetrameric VGIC pore. This structural independence of the VGIC-PD fold has important implications for VGIC biogenesis, understanding disease mutants, and may have a role in transitions between VGIC functional states.…”

Section: Introductionsupporting

confidence: 83%

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits

Arrigoni¹,

Lolicato²,

Shaya³

et al. 2021

Preprint

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Every voltage-gated ion channel (VGIC) superfamily member has an ion conducting pore consisting of four pore domain (PD) subunits that are each built from a common plan comprising an antiparallel transmembrane helix pair, a short, obliquely positioned helix (the pore helix), and selectivity filter. The extent to which this structure, the VGIC-PD fold, relies on the extensive quaternary interactions observed in PD assemblies is unclear. Here, we present crystal structures of three bacterial voltage-gated sodium channel (BacNaV) pores that adopt a surprising set of non canonical quaternary structures and yet maintain the native tertiary structure of the PD monomer. This context-independent structural robustness demonstrates that the VGIC-PD fold, the fundamental VGIC structural building block, can adopt its native-like tertiary fold independent of native quaternary interactions. In line with this observation, we find that the VGIC PD fold is not only present throughout the VGIC superfamily and other channel classes but has homologs in diverse transmembrane and soluble proteins. Characterization of the structures of two synthetic Fabs (sFabs) that recognize the VGIC PD fold shows that such sFabs can bind purified full-length channels and indicates that non-canonical quaternary PD assemblies can occur in the context of complete VGICs. Together, our data demonstrate that the VGIC-PD structure can fold independently of higher order assembly interactions and suggest that full length VGIC PDs can access previously unknown non-canonical quaternary states. These PD properties have deep implications for understanding how the complex quaternary architectures of VGIC superfamily members are achieved and point to possible evolutionary origins of this fundamental VGIC structural element.

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Section: Introductionsupporting

confidence: 83%

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits

Arrigoni¹,

Lolicato²,

Shaya³

et al. 2021

Preprint

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“…2C and Dataset S2). Reentrant loops contribute to the formation of solventaccessible pores in polytopic ion channels and transporters (26) but could also include the "helical hairpins" that have been proposed to embed MIPs into LD membranes (6, 7). The 19 HMDs predicted to contain reentrant loops included the experimentally validated membrane-embedded regions of UBXD8 (25) and UBXN4 (27), as well as the proposed membraneembedded region of ATGL (13,28).…”

Section: Resultsmentioning

confidence: 99%

Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors

Pataki

Rodrigues

Zhang

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Despite not spanning phospholipid bilayers, monotopic integral proteins (MIPs) play critical roles in organizing biochemical reactions on membrane surfaces. Defining the structural basis by which these proteins are anchored to membranes has been hampered by the paucity of unambiguously identified MIPs and a lack of computational tools that accurately distinguish monolayer-integrating motifs from bilayer-spanning transmembrane domains (TMDs). We used quantitative proteomics and statistical modeling to identify 87 high-confidence candidate MIPs in lipid droplets, including 21 proteins with predicted TMDs that cannot be accommodated in these monolayer-enveloped organelles. Systematic cysteine-scanning mutagenesis showed the predicted TMD of one candidate MIP, DHRS3, to be a partially buried amphipathic α-helix in both lipid droplet monolayers and the cytoplasmic leaflet of endoplasmic reticulum membrane bilayers. Coarse-grained molecular dynamics simulations support these observations, suggesting that this helix is most stable at the solvent-membrane interface. The simulations also predicted similar interfacial amphipathic helices when applied to seven additional MIPs from our dataset. Our findings suggest that interfacial helices may be a common motif by which MIPs are integrated into membranes, and provide high-throughput methods to identify and study MIPs.

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“…To prove that Kv1.3 activation serves as molecular trigger of GLUT4 translocation to the membrane, HEK 293 cells were transiently transfected with the cDNA coding for GLUT4 plus wildtype or a pore mutant Kv1.3 channel. W386F Kv1.3 is a construct whereby a threonine is substituted for a phenylalanine and results in the translation of a non-conducting channel (Holmes et al, 1997 ; Delaney et al, 2014 ). Whole-cell or surface-labeled GLUT4 was visualized using anti-c-myc directed against a myc extracellular, epitope tag on GLUT4 (Figure 2A ).…”

Section: Resultsmentioning

confidence: 99%

“…We now report the presence of GLUT4 and Kv1.3, centrally, co-localized in the mitral cell layer of the OB. Moreover, using a heterologous expression system (HEK 293 cells), we are able to increase the translocation of GLUT4 when the pore of Kv1.3 (Delaney et al, 2014 ) is mutated to yield a non-conducting channel. This suggests that a number of modulators of Kv1.3 that can inhibit channel conductance (src, EGF, insulin, BDNF, glp-1, Nedd4-2) (Holmes et al, 1996 ; Bowlby et al, 1997 ; Fadool et al, 1997 , 2000 ; Fadool and Levitan, 1998 ; Cook and Fadool, 2002 ; Colley et al, 2009 ; Thiebaud et al, 2016 ; Velez et al, 2016 ), could be poised to increase GLUT4 translocation and the utilization of glucose by mitral cells of the OB.…”

Section: Introductionmentioning

confidence: 99%

Mitochondrial Ultrastructure and Glucose Signaling Pathways Attributed to the Kv1.3 Ion Channel

et al. 2016

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Gene-targeted deletion of the potassium channel Kv1.3 (Kv1.3−∕−) results in “Super-smeller” mice with a sensory phenotype that includes an increased olfactory ability linked to changes in olfactory circuitry, increased abundance of olfactory cilia, and increased expression of odorant receptors and the G-protein, Golf. Kv1.3−∕− mice also have a metabolic phenotype including lower body weight and decreased adiposity, increased total energy expenditure (TEE), increased locomotor activity, and resistance to both diet- and genetic-induced obesity. We explored two cellular aspects to elucidate the mechanism by which loss of Kv1.3 channel in the olfactory bulb (OB) may enhance glucose utilization and metabolic rate. First, using in situ hybridization we find that Kv1.3 and the insulin-dependent glucose transporter type 4 (GLUT4) are co-localized to the mitral cell layer of the OB. Disruption of Kv1.3 conduction via construction of a pore mutation (W386F Kv1.3) was sufficient to independently translocate GLUT4 to the plasma membrane in HEK 293 cells. Because olfactory sensory perception and the maintenance of action potential (AP) firing frequency by mitral cells of the OB is highly energy demanding and Kv1.3 is also expressed in mitochondria, we next explored the structure of this organelle in mitral cells. We challenged wildtype (WT) and Kv1.3−∕− male mice with a moderately high-fat diet (MHF, 31.8 % kcal fat) for 4 months and then examined OB ultrastructure using transmission electron microscopy. In WT mice, mitochondria were significantly enlarged following diet-induced obesity (DIO) and there were fewer mitochondria, likely due to mitophagy. Interestingly, mitochondria were significantly smaller in Kv1.3−∕− mice compared with that of WT mice. Similar to their metabolic resistance to DIO, the Kv1.3−∕− mice had unchanged mitochondria in terms of cross sectional area and abundance following a challenge with modified diet. We are very interested to understand how targeted disruption of the Kv1.3 channel in the OB can modify TEE. Our study demonstrates that Kv1.3 regulates mitochondrial structure and alters glucose utilization; two important metabolic changes that could drive whole system changes in metabolism initiated at the OB.

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Determinants of pore folding in potassium channel biogenesis

Cited by 17 publications

References 20 publications

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits

Quaternary structure independent folding of voltage-gated ion channel pore domain subunits

Proteomic analysis of monolayer-integrated proteins on lipid droplets identifies amphipathic interfacial α-helical membrane anchors

Mitochondrial Ultrastructure and Glucose Signaling Pathways Attributed to the Kv1.3 Ion Channel

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