Satpal Singh

Complete separation of four potassium currents in drosophila

A number of voltage-activated and Ca2+ activated K+ currents are known to coexist and play a major role in a wide variety of cellular processes including neuromuscular phenomena. Separation of these currents is important for analyzing their individual functional roles and for understanding whether or not they are mediated by entirely different channels. In Drosophila, we have now been able to manipulate four different K+ currents, individually and in combination with one another, by a combined use of mutations and pharmacological agents. This allows analysis of the physiological and molecular properties of different K+ channels and of the role of individual currents in membrane excitability.

Audiograms of a South Indian bat community

Summary1.Audiograms are recorded from one non-echolocating and nine echolocating sympatrically living bat species of South India. These species areCynopterus sphinx (non-echolocating),Tadarida aegyptiaca, Taphozous melanopogon, T. kachhensis, Rhinopoma hardwickei, Pipistrellus dormeri, P. mimus, Hipposideros speoris, H. bicolor andMegaderma lyra.2.InRhinopoma hardwickei a highly sensitive frequency range was found which is narrowly tuned to the frequency band of the bats CF-echolocation call (32–35 kHz, Fig. 3). In hipposiderids a ‘filter’ narrowly tuned to the frequency of the CF-part of the CF-FM echolocation sounds (137.5 kHz inH. speoris and 151.5 kHz inH. bicolor, Fig. 5) could be recorded from deeper parts of IC.3.In the echolocating species the best frequency of the audiograms closely matched with that frequency range in the echolocation calls containing most energy.4.In bat species foraging flying prey best frequencies of audiograms and height of preferred foraging areas are inversely related, i.e. bat species hunting high above canopy have lower best frequencies than those foraging close to or within canopy (Fig. 6).5.A hypothesis is forwarded explaining how fluttering target detection by constant frequency echolocation might have evolved from long distance echolocation by pure tone signals.

Mutations in Cytochrome c Oxidase Subunit VIa Cause Neurodegeneration and Motor Dysfunction in Drosophila

Mitochondrial dysfunction is involved in many neurodegenerative disorders in humans. Here we report mutations in a gene (designated levy) that codes for subunit VIa of cytochrome c oxidase (COX). The mutations were identified by the phenotype of temperature-induced paralysis and showed the additional phenotypes of decreased COX activity, age-dependent bang-induced paralysis, progressive neurodegeneration, and reduced life span. Germ-line transformation using the levy+ gene rescued the mutant flies from all phenotypes including neurodegeneration. The data from levy mutants reveal a COX-mediated pathway in Drosophila, disruption of which leads to mitochondrial encephalomyopathic effects including neurodegeneration, motor dysfunction, and premature death. The data present the first case of a mutation in a nuclear-encoded structural subunit of COX that causes mitochondrial encephalomyopathy rather than lethality, whereas several previous attempts to identify such mutations have not been successful. The levy mutants provide a genetic model to understand the mechanisms underlying COX-mediated mitochondrial encephalomyopathies and to explore possible therapeutic interventions.

Inhibition of Delayed Rectifier Potassium Channels and Induction of Arrhythmia A NOVEL EFFECT OF CELECOXIB AND THE MECHANISM UNDERLYING IT

Selective inhibitors of cyclooxygenase-2 (COX-2), such as rofecoxib (Vioxx), celecoxib (Celebrex), and valdecoxib (Bextra), have been developed for treating arthritis and other musculoskeletal complaints. Selective inhibition of COX-2 over COX-1 results in preferential decrease in prostacyclin production over thromboxane A2 production, thus leading to less gastric effects than those seen with nonselective COX inhibitors such as acetylsalicylic acid (aspirin). Here we show a novel effect of celecoxib via a mechanism that is independent of COX-2 inhibition. The drug inhibited the delayed rectifier (Kv2) potassium channels from Drosophila, rats, and humans and led to pronounced arrhythmia in Drosophila heart and arrhythmic beating of rat heart cells in culture. These effects occurred despite the genomic absence of cyclooxygenases in Drosophila and the failure of acetylsalicylic acid, a potent inhibitor of both COX-1 and COX-2, to inhibit rat Kv2.1 channels. A genetically null mutant of Drosophila Shab (Kv2) channels reproduced the cardiac effect of celecoxib, and the drug was unable to further enhance the effect of the mutation. These observations reveal an unanticipated effect of celecoxib on Drosophila hearts and on heart cells from rats, implicating the inhibition of Kv2 channels as the mechanism underlying this effect.

Selective blockade of the delayed rectifier potassium current by tacrine in Drosophila.

Tetrahydroaminoacridine (tacrine) is an anticholinesterase agent used in the treatment of Alzheimers disease. Its effectiveness against dementia is attributed to its inhibition of acetylcholine breakdown in the synaptic cleft. Tacrine has also been shown to block ionic currents, including many types of potassium (K+) currents, calcium currents, and sodium currents. However, the physiologic significance of this blockade, especially with respect to its effectiveness against Alzheimers disease, is not clear because of relatively high (several hundred micromolar to millimolar) concentrations of tacrine employed in many studies of channel blockade, and because it blocks several types of currents. A complete mutational and pharmacologic resolution of ionic currents in the larval muscles of Drosophila allowed us to examine the selectivity of tacrines effects at very low concentrations. At concentrations as low as 10 microM, tacrine selectively blocked the delayed rectifier K+ current without affecting the three other K+ currents or the calcium channel current in these cells. It also increased the duration of the action potentials significantly. An interesting aspect of tacrines selectivity is that the current blocked by it is the quinidine-sensitive delayed rectifier K+ current rather than the 4-aminopyridine (4-AP)-sensitive transient K+ current. This is in contrast to the generally emphasized structural relationship between tacrine and 4-AP. Since tacrine is structurally related to quinidine as well, these observations suggest a structural basis for the selectivity of tacrine, 4-AP, and quinidine for specific K+ channels. Furthermore, the data are consistent with the possibility of increased neurotransmitter release, due to prolonged presynaptic action potentials, acting synergistically with the anticholinesterase activity of tacrine to increase its therapeutic effectiveness.

Mutational and gene dosage analysis of calcium-activated potassium channels in Drosophila: correlation of micro- and macroscopic currents.

In Drosophila, two Ca2(+)-activated K+ currents, ICF and ICS, have previously been distinguished in conventional voltage clamp experiments. The slowpoke (slo) mutation eliminates ICF specifically. We report that in patch clamp recordings a single-channel Ca2(+)-activated K+ current is readily distinguished from other channel activities in normal larval muscle membrane, whereas no such current is observed in slo muscles. This single-channel current thus correlates with the macroscopic ICF. No obvious differences in amplitude or properties were detected between normal (+/+) and heterozygous (slo/+) ICF channels in whole-cell voltage clamp recordings or single-channel patch clamp recordings. These results are consistent with the hypothesis that slo is a structural gene for the ICF channels only under certain conditions. The selective effect of the slo mutation may reflect a defect in a regulatory mechanism that is specific for the functioning of the ICF channel protein.

Modulation of L-type Calcium Channels in Drosophila via a Pituitary Adenylyl Cyclase-activating Polypeptide (PACAP)-mediated Pathway

Modulation of calcium channels plays an important role in many cellular processes. Previous studies have shown that the L-type Ca2+ channels in Drosophila larval muscles are modulated via a cAMP-protein kinase A (PKA)-mediated pathway. This raises questions on the identity of the steps prior to cAMP, particularly the endogenous signal that may initiate this modulatory cascade. We now present data suggesting the possible role of a neuropeptide, pituitary adenylyl cyclase-activating polypeptide (PACAP), in this modulation. Mutations in the amnesiac (amn) gene, which encodes a polypeptide homologous to human PACAP-38, reduced the L-type current in larval muscles. Conditional expression of a wild-type copy of the amn gene rescued the current from this reduction. Bath application of human PACAP-38 also rescued the current. PACAP-38 did not rescue the mutant current in the presence of PACAP-6–38, an antagonist at type-I PACAP receptor. 2′,5′-dideoxyadenosine, an inhibitor of adenylyl cyclase, prevented PACAP-38 from rescuing the amn current. In addition, 2′,5′-dideoxyadenosine reduced the wild-type current to the level seen in amn, whereas it failed to further reduce the current observed in amn muscles. H-89, an inhibitor of PKA, suppressed the effect of PACAP-38 on the current. The above data suggest that PACAP, the type-I PACAP receptors, and adenylyl cyclase play a role in the modulation of L-type Ca2+ channels via cAMP-PKA pathway. The data also provide support for functional homology between human PACAP-38 and the amn gene product in Drosophila.

Mutational Analysis of the Shab-encoded Delayed Rectifier K+ Channels in Drosophila

K+ currents inDrosophila muscles have been resolved into two voltage-activated currents (I A andI K) and two Ca2+-activated currents (I CF and I CS). Mutations that affect IA (Shaker) andI CF (slowpoke) have helped greatly in the analysis of these currents and their role in membrane excitability. Lack of mutations that specifically affect channels for the delayed rectifier current (I K) has made their genetic and functional identity difficult to elucidate. With the help of mutations in the Shab K+ channel gene, we show that this gene encodes the delayed rectifier K+channels in Drosophila. Three mutant alleles with a temperature-sensitive paralytic phenotype were analyzed. Analysis of the ionic currents from mutant larval body wall muscles showed a specific effect on delayed rectifier K+ current (I K). Two of the mutant alleles contain missense mutations, one in the amino-terminal region of the channel protein and the other in the pore region of the channel. The third allele contains two deletions in the amino-terminal region and is a null allele. These observations identity the channels that carry the delayed rectifier current and provide an in vivophysiological role for the Shab-encoded K+channels in Drosophila. The availability of mutations that affect I K opens up possibilities for studyingI K and its role in larval muscle excitability.

Modulation of dihydropyridine-sensitive calcium channels in Drosophila by a cAMP-mediated pathway.

Drosophila has proved to be a valuable system for studying the structure and function of ion channels. However, relatively little is known about the regulation of ion channels, particularly that of Ca2+ channels, in Drosophila. Physiological and pharmacological differences between invertebrate and mammalian L-type Ca2+ channels raise questions on the extent of conservation of Ca2+ channel modulatory pathways. We have examined the role of cyclic adenosine monophosphate (cAMP) cascade in modulating the dihydropyridine (DHP)-sensitive Ca2+ channels in the larval muscles of Drosophila, using mutations and drugs that disrupt specific steps in this pathway. The L-type (DHP-sensitive) Ca2+ channel current was increased in the dunce mutants, which have high cAMP concentration owing to cAMP-specific phosphodiesterase (PDE) disruption. The current was decreased in the rutabaga mutants, where adenylyl cyclase (AC) activity is altered thereby decreasing the cAMP concentration. The dunce effect was mimicked by 8-Br-cAMP, a cAMP analog, and IBMX, a PDE inhibitor. The rutabaga effect was rescued by forskolin, an AC activator. H-89, an inhibitor of protein kinase-A (PKA), reduced the current and inhibited the effect of 8-Br-cAMP. The data suggest modulation of L-type Ca2+ channels of Drosophila via a cAMP-PKA mediated pathway. While there are differences in L-type channels, as well as in components of cAMP cascade, between Drosophila and vertebrates, main features of the modulatory pathway have been conserved. The data also raise questions on the likely role of DHP-sensitive Ca2+ channel modulation in synaptic plasticity, and learning and memory, processes disrupted by the dnc and the rut mutations.

Auditory spatial sensitivity of inferior collicular neurons of echolocating bats

The sensitivity of 94 inferior collicular (IC) neurons of Eptesicus fuscus and Myotis lucifugus to spatial location of the acoustic stimulus were studied under free-field stimulus conditions. The best frequency (BF) and minimum threshold (MT) of each neuron were determined with sound delivered in front of the bat. Then the variation in discharge rate of the neuron was measured with a BF sound broadcast from a moving loudspeaker at different angular positions along the horizontal, vertical or diagonal plane of the frontal auditory space. A wide range of stimulus intensities above the MT of the neuron was used. Neurons were classified into 3 classes on the basis of their spatial sensitivity: (1) omnisensitive neurons (15%) were broadly tuned to sound delivered in the frontal auditory space and their responses did not show any correlation with sound location; (2) stimulus intensity-dependent neurons (28%) varied their discharge rates with sound location and intensity so that the peak of their spatial response profiles also varied with stimulus intensity; and (3) stimulus intensity-independent neurons (57%) varied their discharge rates only with sound location over a wide range of stimulus intensities so that their peak discharge always appeared at the same or a small range of angle. In most cases, the medial limbs of the spatial sensitivity curve for these neurons were extremely sharp and congruent. By moving the loudspeaker along the horizontal, vertical and diagonal planes, it was possible to approximate the boundary of the spatial response area of a neuron. Most IC neurons responded to sound delivered within 20 degrees ipsilateral, 60 degrees contralateral, 45 degrees up and 40 degrees down of the frontal auditory space, confirming previous similar studies. In general, an increasing stimulus repetition rate appeared to sharpen the spatial sensitivity curve of a neuron. Conversely, an increasing moving velocity of the stimulus decreased its response. The possible role of these 3 classes of neurons in echolocation and neural mechanisms underlying the spatial sensitivity of these neurons is discussed.