Structural and functional determinants in the S5-P region of HCN-encoded pacemaker channels revealed by cysteine-scanning substitutions

2008 ◽  
Vol 294 (1) ◽  
pp. C136-C144 ◽  
Author(s):  
Ka-Wing Au ◽  
Chung-Wah Siu ◽  
Chu-Pak Lau ◽  
Hung-Fat Tse ◽  
Ronald A. Li
Keyword(s):  
Structure Function ◽  
Side Chain ◽  
Hcn Channels ◽  
Spontaneous Generation ◽  
Loss Of Function ◽  
Pacemaker Channels ◽  
Cysteine Scanning ◽  
Reactive Compound ◽  
Membrane Pacemaker ◽  
Function Relationship

Hyperpolarization-activated cyclic nucleotide-modulated (HCN) channels are responsible for the membrane pacemaker current that underlies the spontaneous generation of bioelectrical rhythms. However, their structure-function relationship is poorly understood. Previously, we identified several pore residues that influence HCN gating properties and proposed a pore-to-gate mechanism. Here, we systematically introduced cysteine-scanning substitutions into the descending portion of the P loop (residues 339–345) of HCN1-R (where R is resistance to sulfhydryl-reactive agents) channels, in which all endogenous cysteines except C303 have been removed or replaced. F339C, K340C, A341C, M342C, S343C, and M345C did not produce functional currents. Interestingly, the loss of function phenotype of F339C could be rescued by the reducing agent dithiothreitol (DTT). H344C but not HCN1-R and DTT-treated F339C channels were sensitive to blockade by divalent Cd2+ (current with 100 μM Cd2+/control current at −140 mV = 67.6 ± 2.9%, 109.3 ± 3.1%, and 103.8 ± 1.7%, respectively). Externally applied methanethiosulfate ethylammonium, a covalent sulfhydryl-reactive compound, irreversibly modified H344C by reducing the current at −140 mV (to 43.7 ± 6.5%), causing a hyperpolarizing steady-state activation shift (change in half-activation voltage: ∼6 mV) and decelerated gating kinetics (by up to 3-fold). Based on these results, we conclude that pore residues 339–345 are important determinants of the structure-function properties of HCN channels and that the side chain of H344 is externally accessible.

scholarly journals Phosphate-binding loop and Rab GTPase function: mutations at Ser29 and Ala30 of Rab5 lead to loss-of-function as well as gain-of-function phenotype

2001 ◽  
Vol 355 (3) ◽  
pp. 681-689 ◽  
Author(s):  
Guangpu LI ◽  
Zhimin LIANG
Keyword(s):  
Structure Function ◽  
Binding Domain ◽  
Loss Of Function ◽  
Phosphate Binding ◽  
Gain Of Function ◽  
Gtp Binding ◽  
Early Endosomes ◽  
Structure Function Relationship ◽  
Function Relationship ◽  
Binding Loop

Ras-like GTPases contain a structurally conserved GTP-binding domain. An important element of the GTP-binding domain is the phosphate-binding loop, which contains two Gly residues (Gly12 and Gly13) in Ras. Because the two Gly residues are crucial for normal Ras function, it is intriguing that they are not conserved in other Ras-like GTPases, including the Rab GTPases; for example, the equivalent residues in Rab5 are Ser29 and Ala30. The present study builds on earlier biochemical characterizations of the Rab5 mutants containing substitutions at Ala30 and provides a comprehensive analysis of the structure–function relationship of the Rab5 phosphate-binding loop. We have generated 19 new mutants containing amino acid substitutions at Ser29 and determined whether these Ser29 mutants, as well as the Ala30 mutants, remain able to stimulate the endocytosis of horseradish peroxidase in baby hamster kidney cells. A total of 11 mutants lose the activity of stimulating endocytosis. Of these 11 mutants, 9 are defective in membrane association. In contrast, 27 mutants remain able to stimulate endocytosis. Five of them induce a novel cellular phenotype: cell rounding and detachment from culture dishes. They also induce super-large early endosomes such as the constitutively activated Rab5:Q79L mutant. Biochemical results suggest that the constitutive activation of Rab5 requires an increased nucleotide exchange rate and/or decreased GTPase activity. This study establishes functional significance for the phosphate-binding loop of Rab5 and shows that mutations in this region lead to either a loss-of-function or a gain-of-function phenotype, indicating a structure–function relationship distinct from that of Ras.

scholarly journals Random Mutagenesis of the Proton-coupled Folate Transporter (SLC46A1), Clustering of Mutations, and the Bases for Associated Losses of Function

2011 ◽  
Vol 286 (27) ◽  
pp. 24150-24158 ◽  
Author(s):  
Rongbao Zhao ◽  
Daniel Sanghoon Shin ◽  
Ndeye Diop-Bove ◽  
Channa Gila Ovits ◽  
I. David Goldman
Keyword(s):  
Structure Function ◽  
Random Mutagenesis ◽  
Autosomal Recessive Disorder ◽  
Base Substitution ◽  
Loss Of Function ◽  
Insertion And Deletion ◽  
Substitution Mutations ◽  
Function Relationship ◽  
Reduced Rate ◽  
Relationship Of

Loss-of-function mutations in the proton-coupled folate transporter (PCFT, SLC46A1) result in the autosomal recessive disorder, hereditary folate malabsorption (HFM). Identification and characterization of HFM mutations provide a wealth of information on the structure-function relationship of this transporter. In the current study, PCR-based random mutagenesis was employed to generate unbiased loss-of-function mutations of PCFT, simulating the spectrum of alterations that might occur in the human disorder. A total of 26 mutations were generated and 4 were identical to HFM mutations. Eleven were base deletion or insertion mutations that led to a frameshift and, along with similar HFM mutations, are predominantly localized to two narrow regions of the pcft gene at the 5′-end. Base substitution mutations identified in the current study and HFM patients were largely distributed across the pcft gene. Elimination of the ATG initiation codon by a one-base substitution (G > A) did not result in a complete lack of translation at the same codon consistent with rare non-ATG translation initiation. Among six missense mutants evaluated, three mutant PCFTs were not detected at the plasma membrane, one mutation resulted in decreased binding to folate substrate, and one had a reduced rate of conformational change associated with substrate translocation. The remaining PCFT mutant had defects in both processes. These results broaden understanding of the regions of the pcft gene prone to base insertion and deletion and inform further approaches to the analysis of the structure-function of PCFT.

Mouse models to study von Willebrand factor structure-function relationships in vivo

2009 ◽  
Vol 29 (01) ◽  
pp. 17-20 ◽  
Author(s):  
I. Marx ◽  
I. Badirou ◽  
R. Pendu ◽  
O. Christophe ◽  
C. V. Denis
Keyword(s):  
Structure Function ◽  
Transient Expression ◽  
Von Willebrand Factor ◽  
Large Size ◽  
Mouse Hepatocytes ◽  
Function Relationship ◽  
Von Willebrand ◽  
Willebrand Factor

SummaryVon Willebrand factor (VWF) structure-function relationship has been studied only through in vitro approaches. The VWF-deficient mouse model has been extremely useful to examine the in vivo function of VWF but does not allow a more subtle analysis of the relative importance of its different domains. However, considering the large size of VWF and its capacity to interact with various ligands in order to support platelet adhesion and aggregation, the necessity to evaluate independently these interactions appeared increasingly crucial. A recently developed technique, known as hydrodynamic injection, which allows transient expression of a transgene by mouse hepatocytes, proved very useful in this regard. Indeed, transient expression of various VWF mutants in VWF-deficient mice contributed to improve our knowledge about the role of VWF interaction with subendothelial collagens and with platelets receptors in VWF roles in haemostasis and thrombosis. These findings can provide new leads in the development of anti-thrombotic therapies.

scholarly journals Network-based approaches to quantify multicellular development

2017 ◽  
Vol 14 (135) ◽  
pp. 20170484 ◽  
Author(s):  
Matthew D. B. Jackson ◽  
Salva Duran-Nebreda ◽  
George W. Bassel
Keyword(s):  
Structure Function ◽  
Spatial Relations ◽  
Higher Order ◽  
Cellular Interaction ◽  
Cellular Organization ◽  
Multicellular Development ◽  
Cell Organization ◽  
Function Relationship ◽  
And Function ◽  
The Relationship

Multicellularity and cellular cooperation confer novel functions on organs following a structure–function relationship. How regulated cell migration, division and differentiation events generate cellular arrangements has been investigated, providing insight into the regulation of genetically encoded patterning processes. Much less is known about the higher-order properties of cellular organization within organs, and how their functional coordination through global spatial relations shape and constrain organ function. Key questions to be addressed include: why are cells organized in the way they are? What is the significance of the patterns of cellular organization selected for by evolution? What other configurations are possible? These may be addressed through a combination of global cellular interaction mapping and network science to uncover the relationship between organ structure and function. Using this approach, global cellular organization can be discretized and analysed, providing a quantitative framework to explore developmental processes. Each of the local and global properties of integrated multicellular systems can be analysed and compared across different tissues and models in discrete terms. Advances in high-resolution microscopy and image analysis continue to make cellular interaction mapping possible in an increasing variety of biological systems and tissues, broadening the further potential application of this approach. Understanding the higher-order properties of complex cellular assemblies provides the opportunity to explore the evolution and constraints of cell organization, establishing structure–function relationships that can guide future organ design.

Sign in / Sign up

Export Citation Format

Share Document