PubMed Health⌕ Search

Biomedical subjects

R D Hinrichsen

Publications and source records attributed to R D Hinrichsen.

21 records · Page 2Linked to original sources

A mutation that alters properties of the calcium channel in Paramecium tetraurelia.

The membrane properties of a new mutant of Paramecium tetraurelia, dancer, were compared under voltage clamp with those of the wild type. The Ca2+ current was isolated and examined using CsCl-filled electrodes and tetraethylammonium in the bath solution to block K+ channels. The amplitude of the Ca2+ transient was not altered by the mutation. However, the Ca2+ current in the mutant inactivated more slowly and less extensively: hence a larger sustained Ca2+ current remained in the mutant. A change in the time course of the deactivation of the Ba2+ current was observed in the mutant. This mutational change is not likely to be the consequence of the Ca2+-channel inactivation because it is seen in the Ba2+ solution where there is little inactivation of the current. Other measured properties of the Ca2+ channel, the voltage-dependent K+ current, and the resting properties of the membrane were normal in the mutant. The Ca2+-activated K+ current and the Ca2+-activated Na+ current were larger in the mutant than in the wild type, consistent with a greater elevation of free intracellular Ca2+ during depolarization in the mutant. It is likely that the mutation causes an alteration in the Ca2+-channel structure or in its immediate environment and thereby affects the inactivation and deactivation processes of the Ca2+ channel. As would be expected from the greater Ca2+ current, the mutant tends to generate all-or-none Ca action potentials as opposed to the graded action potentials in the wild type.

Action Potentials↗

Mutant analysis shows that the Ca2+-induced K+ current shuts off one type of excitation in Paramecium.

Two mutants of Paramecium tetraurelia, called "pantophobiacs," were found to lack most of the slow Ca2+-induced K+ outward current. Passive properties, the transient Ca2+ inward current, and the fast depolarization-induced K+ outward current remain normal. The mutant defect reduces the ability to shut off a normal, excited state of the membrane and results in repeated, long backward swimming instead of the wild-type jerks in response to a variety of ions, to heat, and to touch.

Action Potentials↗

Synthesis and evaluation of nuclear targeting peptide-antisense oligodeoxynucleotide conjugates.

An endogenous nuclear enzyme, RNase H, is an important component in determining the efficacy of antisense oligodeoxynucleotides (ODNs). In an effort to improve the potency of antisense ODNs, conjugates with three different nuclear targeting signal peptides were prepared. These short peptide sequences have been shown to facilitate transport of macromolecules into the nucleus of cells. Efficient chemistry for the synthesis of ODN-peptide conjugates is described. Reaction of 5'-aminohexyl-modified ODNs with iodoacetic anhydride gave pure iodoacetamide ODNs (IA-ODNs) in good yield. These electrophilic intermediates were reacted with thiol-containing peptides to give ODN-peptides in excellent yield and purity. The ODN-peptides were further characterized by proteolysis with trypsin. Thermal denaturation studies with ssDNA targets showed little effect of the 5'-peptide modifications on the hybridization properties of the ODN. The effect of the nuclear signal peptides on antisense potency was evaluated in the freshwater ciliate Paramecium. A 3'-hexanol-modified 24-mer antisense ODN, complementary to the mRNA for calmodulin, alters regulation of membrane ion channels and swimming behavior of these cells. A 2'-O-methyl analog of this ODN was inactive, thus providing evidence that this activity in Paramecium is mediated by RNase H. Antisense ODN-nuclear signal peptide conjugates were transfected into the cells by electroporation. Surprisingly, these conjugates showed no antisense effects in comparison to a 5'-unmodified control ODN. Random peptides or amino acids conjugated to the 5'-terminus did not decrease antisense activity.

Amino Acid Sequence↗