Fife, a Drosophila Piccolo-RIM homolog, promotes active zone organization and neurotransmitter release.

Neuronal communication depends on the precisely orchestrated release of neurotransmitter at specialized sites called active zones (AZs). A small number of scaffolding and cytoskeletal proteins comprising the cytomatrix of the active zone (CAZ) are thought to organize the architecture and functional properties of AZs. The majority of CAZ proteins are evolutionarily conserved, underscoring the fundamental similarities in neurotransmission at all synapses. However, core CAZ proteins Piccolo and Bassoon have long been believed exclusive to vertebrates, raising intriguing questions about the conservation of the molecular mechanisms that regulate presynaptic properties. Here, we present the identification of a piccolo-rim-related gene in invertebrates, together with molecular phylogenetic analyses that indicate the encoded proteins may represent Piccolo orthologs. In accordance, we find that the Drosophila homolog, Fife, is neuronal and localizes to presynaptic AZs. To investigate the in vivo function of Fife, we generated a deletion of the fife locus. We find that evoked neurotransmitter release is substantially decreased in fife mutants and loss of fife results in motor deficits. Through morphological analysis of fife synapses, we identify underlying AZ abnormalities including pervasive presynaptic membrane detachments and reduced synaptic vesicle clustering. Our data demonstrate the conservation of a Piccolo-related protein in invertebrates and identify critical roles for Fife in regulating AZ structure and function. These findings suggest the CAZ is more conserved than previously thought, and open the door to a more complete understanding of how CAZ proteins regulate presynaptic structure and function through genetic studies in simpler model systems.

Small GTPase determinants for the Golgi processing and plasmalemmal expression of human ether-a-go-go related (hERG) K+ channels.

The pro-arrhythmic Long QT syndrome (LQT) is linked to 10 different genes (LQT1-10). Approximately 40% of genotype-positive LQT patients have LQT2, which is characterized by mutations in the human ether-a-go-go related gene (hERG). hERG encodes the voltage-gated K(+) channel alpha-subunits that form the pore of the rapidly activating delayed rectifier K(+) current in the heart. The purpose of this study was to elucidate the mechanisms that regulate the intracellular transport or trafficking of hERG, because trafficking is impaired for about 90% of LQT2 missense mutations. Protein trafficking is regulated by small GTPases. To identify the small GTPases that are critical for hERG trafficking, we coexpressed hERG and dominant negative (DN) GTPase mutations in HEK293 cells. The GTPases Sar1 and ARF1 regulate the endoplasmic reticulum (ER) export of proteins in COPII and COPI vesicles, respectively. Expression of DN Sar1 inhibited the Golgi processing of hERG, decreased hERG current (I(hERG)) by 85% (n > or = 8 cells per group, *, p < 0.01), and reduced the plasmalemmal staining of hERG. The coexpression of DN ARF1 had relatively small effects on hERG trafficking. Surprisingly, the coexpression of DN Rab11B, which regulates the endosomal recycling, inhibited the Golgi processing of hERG, decreased I(hERG) by 79% (n > or = 8 cells per group; *, p < 0.01), and reduced the plasmalemmal staining of hERG. These data suggest that hERG undergoes ER export in COPII vesicles and endosomal recycling prior to being processed in the Golgi. We conclude that hERG trafficking involves a pathway between the ER and endosomal compartments that influences expression in the plasmalemma.

Kv11.1 (ERG1) K+ channels localize in cholesterol and sphingolipid enriched membranes and are modulated by membrane cholesterol.

The localization of ion channels to specific membrane microdomains can impact the functional properties of channels and their role in cellular physiology. We determined the membrane localization of human Kv11.1 (hERG1) alpha-subunit protein, which underlies the rapidly activating, delayed rectifier K(+) current (I(Kr)) in the heart. Immunocytochemistry and membrane fractionation using discontinuous sucrose density gradients of adult canine ventricular tissue showed that Kv11.1 channel protein localized to both the cell surface and T-tubular sarcolemma. Furthermore, density gradient membrane fractionation using detergent (Triton X-100) and non-detergent (OptiPrep) methods from canine ventricular myocytes or HEK293 cells demonstrated that Kv11.1 protein, along with MiRP1 and Kv7.1 (KCNQ1) proteins, localize in cholesterol and sphingolipid enriched membrane fractions. In HEK293 cells, Kv11.1 channels, but not long QT-associated mutant G601S-Kv11.1 channels, also localized to cholesterol and sphingolipid enriched membrane fractions. Depletion of membrane cholesterol from HEK293 cells expressing Kv11.1 channels using methyl-beta-cyclodextrin (MbetaCD) caused a positive shift of the voltage dependence of activation and an acceleration of deactivation kinetics of Kv11.1 current (I(Kv11.1)). Cholesterol loading of HEK293 cells reduced the steep voltage dependence of I(Kv11.1) activation and accelerated the inactivation kinetics of I(Kv11.1). Incubation of neonatal mouse myocytes in MbetaCD also accelerated the deactivation kinetics of I(Kr). We conclude that Kv11.1 protein localizes in cholesterol and sphingolipid enriched membranes and that membrane cholesterol can modulate I(Kv11.1) and I(Kr).

Intragenic suppression of trafficking-defective KCNH2 channels associated with long QT syndrome.

Mutations in the KCNH2 or human ether-a-go-go-related gene-encoded K(+) channel reduce functional KCNH2 current (I(KCNH2)) to cause long QT syndrome (LQT2) by multiple mechanisms, including defects in intracellular transport (trafficking). Trafficking-deficient, or class 2, LQT2 mutations reduce the Golgi processing and surface membrane expression of KCNH2 channel proteins. Drugs that associate with pore-S6 intracellular drug binding domain of KCNH2 channel proteins to cause high-affinity block of I(KCNH2) also can increase the processing of class 2 LQT2 channel proteins through the secretory pathway. We used a strategy of intragenic suppression to test the hypothesis that amino acid substitutions in the putative drug binding domain at residue Y652 could compensate for protein folding abnormalities caused by class 2 LQT2 mutations. We found that the Y652C substitution, and to lesser extent the Y652S substitution, resulted in intragenic suppression of the class 2 LQT2 G601S phenotype; these substitutions increased Golgi processing of G601S channel proteins. The Y652C substitution also caused intragenic suppression of the class 2 LQT2 V612L and F640V phenotypes but not the LQT2 N470D or F805C phenotypes. These are the first findings to demonstrate that a single amino acid substitution in the putative KCNH2 drug binding domain can cause intragenic suppression of several LQT2 mutations.