Anna Petris

Biological activities in the granules isolated from the mouse submaxillary gland

Abstract Growth factors, proteins which specifically enhance the growth of target tissues, were found in the homogenate of submaxillary gland in various molecular forms, due to the association with an esteroproteolytic enzyme. Since it has not been stated if macromolecular complexes were due to a loss of cellular compartmentalization during homogenization, secretory granules were isolated from the gland homogenate and both enzymatic and hormonal activities on the various fractions of granules were determined. The results obtained indicate that growth factors and esteroprotease activity are associated in the same intracellular particles, suggesting a possible role of the enzyme in the maturation of native molecules of growth factors, as described for proinsulin-insulin conversion.

Verapamil Block of Large-Conductance Ca-Activated K Channels in Rat Aortic Myocytes

Abstract. The effects of verapamil on the large conductance Ca-activated K (BK) channel from rat aortic smooth muscle cells were examined at the single channel level. Micromolar concentrations of verapamil produced a reversible flickering block of the BK channel activity. Kinetic analysis showed that verapamil decreased markedly the time constants of the open states, without any significant change in the time constants of the closed states. The appearance of an additional closed state — specifically, a nonconducting, open-blocked state — was also observed, whose time constant would reflect the mean residence time of verapamil on the channel. These observations are indicative of a state-dependent, open-channel block mechanism. Dedicated kinetic (group) analysis confirmed the state-dependent block exerted by verapamil. D600 (gallopamil), the methoxy derivative of verapamil, was also tested and found to exert a similar type of block, but with a higher affinity than verapamil. The permanently charged and membrane impermeant verapamil analogue D890 was used to address other important features of verapamil block, such as the sidedness of action and the location of the binding site on the channel protein. D890 induced a flickering block of BK channels similar to that observed with verapamil only when applied to the internal side of the membrane, indicating that D890 binds to a site accessible from the cytoplasmic side. Finally, the voltage dependence of D890 block was assessed. The experimental data fitted with a Langmuir equation incorporating the Woodhull model for charged blockers confirms that the D890-binding site is accessed from the internal mouth of the BK channel, and locates it approximately 40% of the membrane voltage drop along the permeation pathway.

Chloramphenicol resistance in Streptomyces coelicolor A3(2): Possible involvement of a transposable element

SummaryThe transfer of a Chl element, causing resistance to chloramphenicol in Streptomyces coelicolor A3(2), was studied in NF x SCP1− superfertile crosses. When the Chl element is on the donor side (NF) its transfer to the recombinant cells was virtually total as if the element acted as a second concomitant transfer origin. When the Chl element was on the recipient side (SCP1−) it was never displaced by the immigrant chromosome even when the region facing chl+ was selected for. A fraction of the original Chl− mutants presented a requirement for arginine (ArgB−). A Chl− mutant gave rise spontaneously to ArgB− derivatives at high frequency. The same ArgB− requirement come out at high frequency among Chl− derivatives from a cross NFChl− x SCP1−Chl+ in which neither parent required arginine or produced spontaneously arginineless derivatives. It is suggested that the Chl element is a “transposable element” (Tn) presumably associated with “insertion sequences” (IS). The insertional inactivation of the Chl element may be accompanied or followed by a deletion in the adjacent ArgB gene.

Characterization of a Neuronal Delayed Rectifier K Current Permeant to Cs and Blocked by Verapamil

Abstract. We have used the patch-clamp method in the whole-cell configuration to characterize the delayed rectifier K current (IDRK) in embryonic chick dorsal root ganglion (DRG) neurons. The IDRK is activated by depolarizing pulses positive to −40 mV, and its V½ is near −20 mV. The slope factor of 10.4 mV for an e-fold change in conductance indicates an equivalent gating charge of 2.4e. Inactivation during sustained depolarizing pulses displays two distinct time constants of 200–300 msec and 6–9 sec, respectively. Outward current through the delayed rectifier K (DRK) channels could also be carried by internal Cs, which however exerts mild block when in mixtures with K, as evidenced by the anomalous mole fraction effect. The relative permeability of Cs vs. K, PCs/PK, as calculated from reversal potential measurements, is 0.25. Rb likewise permeates the DRK channel (PRb/PK= 0.67). The IDRK was effectively suppressed by external application of the Ca channel blocker Verapamil, with apparent dissociation constant of ca. 4 μm. The time course of Verapamil block, its good description by equations derived from open-channel block kinetic scheme, and the frequency-dependent effect of the blocker indicate that Verapamil can bind to the channel only when it is in the open state.

Single chloride channels in cultured rat neurones

Single-channel currents through chloride channels were recorded in cultured hippocampal neurones from rats using the patch-clamp method. The channel is active at voltages between -80 and +80 mV, and the time spent in the open state increases with depolarization (almost fourfold for 120 mV). The channel conductance is 62 pS in symmetrical 150 mM NaCl saline. In salt gradient conditions the channel was measurably permeable to Na+. Substitution of NO3- and Br- for Cl- gave higher single-channel currents, meaning a higher permeability of the channel toward the two anions than to Cl-. SO4(2-) ions were poorer charge carriers, yet contributed measurable inward current at negative voltages. Channel activity appeared independent of intracellular Ca2+ concentration. Taken together, these features would suggest for this channel a role in stabilizing resting membrane potential and in maintaining normal cell excitability.

A factor involved in chloramphenicol resistance in Streptomyces coelicolor A3(2): its transfer in the absence of the fertility factor.

An element controlling chloramphenicol resistance (chl) was detected in Streptomyces coelicolor A3(2). Strains sensitive to 1 microgram chloramphenicol ml-1 were obtained among dark scarlet variants. Transfer of the resistance factor was attempted in matings between chloramphenicol-resistant (Chl+) and chloramphenicol-sensitive (Chl-) strains, both of which lacked the SCP1 fertility factor. Transfer of chl was obtained at a much higher rate than that expected for chromosomal markers in SCP1- X SCP1- matings. However, in these particular crosses the latter was also several times higher than usual. All recombinants for chromosomal markers were Chl+. Attempts to locate the chl element failed to distinguish between a chromosomal and an extrachromosomal site. The observed increase in the recombination frequency for chromosomal markers suggests that the chl element may promote recombination.

Mechanism of verapamil block of a neuronal delayed rectifier K channel: active form of the blocker and location of its binding domain

The mechanism of verapamil block of the delayed rectifier K currents (IK(DR)) in chick dorsal root ganglion (DRG) neurons was investigated using the whole‐cell patch clamp configuration. In particular we focused on the location of the blocking site, and the active form (neutral or charged) of verapamil using the permanently charged verapamil analogue D890. Block by D890 displayed similar characteristics to that of verapamil, indicating the same state‐dependent nature of block. In contrast with verapamil, D890 was effective only when applied internally, and its block was voltage dependent (136 mV/e‐fold change of the on rate). Given that verapamil block is insensitive to voltage ( Trequattrini et al., 1998 ), these observations indicate that verapamil reaches its binding site in the uncharged form, and accesses the binding domain from the cytoplasm. In external K and saturating verapamil we recorded tail currents that did not decay monotonically but showed an initial increase (hook). As these currents can only be observed if verapamil unblock is significantly voltage dependent, it has been suggested ( DeCoursey, 1995 ) that neutral drug is protonated upon binding. We tested this hypothesis by assessing the voltage dependence of the unblock rate from the hooked tail currents for verapamil and D890. The voltage dependence of the off rate of D890, but not of verapamil, was well described by adopting the classical Woodhull (1973) model for ionic blockage of Na channels. The voltage dependence of verapamil off rate was consistent with a kinetic scheme where the bound drug can be protonated with rapid equilibrium, and both charged and neutral verapamil can unbind from the site, but with distinct kinetics and voltage dependencies.

Tityus bahiensis toxin IV-5b selectively affects Na channel inactivation in chick dorsal root ganglion neurons

A novel toxin was isolated from the venom of the Brazilian scorpion Tityus (T.) bahiensis. The N-terminal amino acid sequence of this toxin was shown to be 80% identical to the corresponding segment of T. serrulatus toxin IV-5. The new toxin was thus named toxin IV-5b. Toxin IV-5b was found to markedly slow inactivation of Na channel in dorsal root ganglion neurons from chick embryo. By contrast, Na channel activation was only negligibly delayed, and deactivation completely unaffected. Similarly unaffected by the toxin were K and Ca currents. The slowing effect of the toxin starts to appear at concentrations of c. 80 nM, and shows a KD of 143 nM. With a toxin concentration of 2.4 microM, the Na channel inactivation time constant was increased c. 3-fold with respect to the control. The slowing of inactivation was voltage dependent, and increased with depolarization.

Bimodal kinetics of a chloride channel from human fibroblasts.

Abstract. Excised patches were used to study the kinetics of a Cl channel newly identified in cultured human fibroblasts (L132). The conductance of ca. 70 pS in 150 mm symmetrical Cl, and the marked outward rectification ascribe this channel to the ICOR family. Long single-channel recordings (>30 min) revealed that the channel spontaneously switches from a kinetic mode characterized by high voltage dependence (with activity increasing with depolarization; mode 1), into a second mode (mode 2) insensitive to voltage, and characterized by a high activity in the voltage range ±120 mV. On patch excision the channel always appeared in mode 1, which was maintained for a variable time (5–20 min). In most instances the channels then switched into mode 2, and never were seen to switch back, in spite of the eight patches that cumulatively dwelled in this mode 2.33-fold as compared to mode 1. Stability plots of long recordings showed that the channel was kinetically stable in both modes, allowing standard analysis of steady-state kinetics to be performed. Open and closed time distributions of mode 1 and mode 2 revealed that the apparent number of kinetic states of the channel was the same in the two modes. The transition from mode 1 into mode 2 was not instantaneous, but required a variable time in the range 5–60 sec. During the transition the channel mean open time was intermediate between mode 1 and mode 2. The intermediate duration in the stability plot however is not to be interpreted as if the channel, during the transition, rapidly switches between mode 1 and mode 2, but represents a distinct kinetic feature of the transitional channel.