Magnesium binding to DM-nitrophen and its effect on the photorelease of calcium.

The effect of Mg(2+) on the process of Ca(2+) release from the caged Ca(2+) compound DM-nitrophen (NP) was studied in vitro by steady light UV photolysis of NP in the presence of Ca(2+) and Mg(2+). Ca(2+) release during photolysis and its relaxation/recovery after photolysis were monitored with the Ca(2+)-sensitive dye fura-2. Mg(2+) speeds the photorelease of Ca(2+) during photolysis and slows the relaxation of Ca(2+) to new steady-state levels after photolysis. Within the context of a model describing NP photolysis, we determined the on and off rates of Mg(2+) binding to unphotolyzed NP (k(on) = 6.0 x 10(4) M(-1) s(-1); k(off) = 1.5 x 10(-1) s(-1)). Furthermore, to fully account for the slow postphotolysis kinetics of Ca(2+) in the presence of Mg(2+) we were forced to add an additional photoproduct to the standard model of NP photolysis. The additional photoproduct is calculated to have a Ca(2+) affinity of 13.3 microM and is hypothesized to be produced by the photolysis of free or Mg(2+)-bound NP; photolysis of Ca(2+)-bound NP produces the previously documented 3 mM Ca(2+) affinity photoproduct.

Enhanced closed-state inactivation in a mutant Shaker K+ channel.

Many mutations that shift the voltage dependence of activation in Shaker channels cause a parallel shift of inactivation. The I2 mutation (L382I in the Shaker B sequence) is an exception, causing a 45 mV activation shift with only a 9 mV shift of inactivation midpoint relative to the wildtype (WT) channel. We compare the behavior of WT and I2 Shaker 29-4 channels in macropatch recordings from Xenopus oocytes. The behavior of WT channels can be described by both simple and detailed kinetic models which assume that inactivation proceeds only from the open state. The behavior of I2 channels requires that they inactivate from closed states as well, a property characteristic of voltage-gated sodium channels. A detailed "multiple-state inactivation" model is presented that describes both activation and inactivation of I2 channels. The results are consistent with the view that residue L382 is associated with the receptor for the inactivation particles in Shaker channels.

Olfactory physiology in the Drosophila antenna and maxillary palp: acj6 distinguishes two classes of odorant pathways.

This article provides characterization of the electrical response to odorants in the Drosophila antenna and provides physiological evidence that a second organ, the maxillary palp, also has olfactory function in Drosophila. The acj6 mutation, previously isolated by virtue of defective olfactory behavior, affects olfactory physiology in the maxillary palp as well as in the antenna. Interestingly, abnormal chemosensory jump 6 (acj6) reduces response in the maxillary palp to all odorants tested except benzaldehyde (odor of almond), as if response to benzaldehyde is mediated through a different type of odorant pathway from the other odorants. In other experiments, different parts of the antenna are shown to differ with respect to odorant sensitivity. Evidence is also provided that antennal response to odorants varies with age, and that odorants differ in their age dependence.

acj6: a gene affecting olfactory physiology and behavior in Drosophila.

Mutations affecting olfactory behavior provide material for use in molecular studies of olfaction in Drosophila melanogaster. Using the electroantennogram (EAG), a measure of antennal physiology, we have found an adult antennal defect in the olfactory behavioral mutant abnormal chemosensory jump 6 (acj6). The acj6 EAG defect was mapped to a single locus and the same mutation was found to be responsible for both reduction in EAG amplitude and diminished behavioral response, as if reduced antennal responsiveness to odorant is responsible for abnormal chemosensory behavior in the mutant. acj6 larval olfactory behavior is also abnormal; the mutation seems to alter cellular processes necessary for olfaction at both developmental stages. The acj6 mutation exhibits specificity in that visual system function appears normal in larvae and adults. These experiments provide evidence that the acj6 gene encodes a product required for olfactory signal transduction.

Development of the fast sodium current in early embryonic chick heart cells.

Single ventricle cells were dissociated from the hearts of two-, three-, four- or seven-day-old chick embryos, and were maintained in vitro for an additional 6 to 28 hr. Rounded 13 to 18 micron cells with input capacitance of 5 to 10 pF were selected for analysis of fast sodium current (INa). Voltage command protocols designed to investigate the magnitude, voltage dependence, and kinetics of INa were applied with patch electrodes in the whole-cell clamp configuration. INa was present in over half of the 2d, and all 3d, 4d and 7d cells selected. The current showed no systematic differences in activation kinetics, voltage dependence, or tetrodotoxin (TTX) sensitivity with age or culture conditions. Between the 2d and 7d stages, the rate of current inactivation doubled and channel density increased about eightfold. At all stages tested, INa was blocked by TTX at a half-effective concentration of 0.5 to 1.0 nM. We conclude that the lack of Na dependence of the action potential upstroke on the second day of development results from the relatively depolarized level of the diastolic potential, and failure to activate the small available excitatory Na current. The change from Ca to Na dependence of the upstroke during the third to the seventh day of incubation results partly from the negative shift of the diastolic potential during this period, and in part from the increase in available Na conductance.

Some limitations of the cell-attached patch clamp technique: a two-electrode analysis.

With two independent patch electrodes sealed to small clusters of electrically coupled chick embryo cardiac cells, we have measured four parameters: true seal and patch resistance, channel conductance, and membrane potential. One electrode was in the cell-attached mode, and recorded current flowing in parallel through the membrane patch and seal. The second electrode, sealed on a different cell in the cluster, was in the whole cell recording configuration, and served to record or control the membrane potential of the cluster. We fit the four measured parameters to a simple electrical model to reveal errors not usually recognized in the patch-clamp technique. Among these are the following: (1) The apparent seal resistance, determined by changing the potential in a patch electrode, may be a poor estimate of true seal resistance, since it includes the parallel combination of seal- and patch-resistance. (2) Patch resistance may be influenced by the electrode filling solution, and is often much lower than is usually assumed. (3) With a small cell preparation that has an input resistance in the gigaohm range, measurements of single-channel conductance using a cell-attached patch electrode may be inaccurate because cell membrane potential does not remain constant as electrode potential is varied.

Channel currents during spontaneous action potentials in embryonic chick heart cells. The action potential patch clamp.

Single-channel currents were recorded with the cell-attached patch-clamp technique from small clusters (2-20 cells) of spontaneously beating 7-d embryo ventricle cells. Because the preparation was rhythmically active, the trans-patch potential varied with the action potential (AP). The total current through the patch membrane was the patch action current (AC). ACs and APs could be recorded simultaneously, with two electrodes, or sequentially with one electrode. Channel activity, which varied depending on the number and type of channels in the patch, was present during normal cell firing. This method can reveal the kinetics and magnitudes of the specific currents that contributed to the AP, under conditions that reflect not only the time and voltage dependence of the channels, but also environmental factors that may influence channel behavior during the AP.