Regulation of the human skeletal muscle chloride channel hClC-1 by protein kinase C

The regulation of a recombinant human muscle chloride channel, hClC-1, by protein kinase C (PKC) was investigated in human embryonic kidney (HEK 293) cells.External application of 4β-phorbol esters (4β-PMA) reduced the instantaneous whole-cell current amplitude over the entire voltage range tested. This effect was abolished when the cells were intracellularly perfused with a specific protein kinase C inhibitor, chelerythine. Inactive 4α-phorbolesters did not affect the chloride currents. We conclude that the effect of 4β-phorbol esters is mediated by protein kinase C (PKC).Activation of PKC resulted in changes in macroscopic current kinetics. The time course of current deactivation determined in the presence and absence of 4β-phorbol esters could be fitted with the sum of two exponentials and a constant value. In the presence of phorbol esters, the fast time constants and the minimum value of the fraction of non-deactivating current were increased, whereas the voltage dependence of all fractional current amplitudes remained unchanged. PKC-induced phosphorylation had only small effects on the voltage dependence of the relative open probability and the maximum absolute open probability was unaffected by treatment with 4β-PMA, as shown by non-stationary noise analysis.The kinetic changes indicate that phosphorylation alters functional properties of active channels. Since the absolute open probability is not reduced, the observed macroscopic current reduction implies alterations of the ion permeation process.Phosphorylation by PKC appears to affect ion transfer and gating processes. It is postulated that the phosphorylation site may be located at the cytoplasmic vestibule face of the pore

Cellular uptake and efficacy of antisense oligonucleotides against RNAs of two Na+ channel isoforms

ABSTRACT The effects of 15-mer phosphorothioate antisense oligodeoxynucleotides (aODNs) specifically designed against the RNAs of either of two closely related Na ϩ channel isoforms, hSkM1 or hH1, were tested in human myotubes. Fluorescence (3Ј-fluorescein isothiocyanate) labeling showed that mere incubation of cultures with aODNs did not result in aODN uptake, but liposome-mediated transfer was successful and resulted in cytoplasmic and nuclear localization of ODNs. Intracellular fluorescence was stable for at least 3 days. At 5 M, the hH1-specific aODN was effective in suppressing ion channel function, but the hSkM1-specific aODN was not. Reverse transcription-polymerase chain reaction gave corresponding results on the mRNA level. However, in HEK-293 cells stably expressing hSkM1, the same hSkM1-specific aODN was able to reduce Na ϩ currents (2.4 Ϯ 0.5 nA, n ϭ 11; controls: 6.5 Ϯ 1.0 nA, n ϭ 14). We conclude that cellular uptake of aODNs and intracellular access to the RNA target are necessary, but not always sufficient conditions for an effective block of mRNA translation in intact cells. A modern strategy in pharmacology for interfering with receptor or channel proteins is to manipulate lifetime and translation efficacy of their coding mRNAs using antisense oligodeoxynucleotides (aODNs) For an effective inhibition of protein translation in vivo or in cultured cells, the aODNs have to be protected against extracellular degradation, they must also be able to pass through the lipophilic plasma membrane, and finally they must be made to escape intracellular degradation and sequestration into organelles Some of these tasks can now easily be solved, e.g., degradation is substantially slowed when phosphorothioate-protected ODNs are used instead of phosphodiester ODNs (Crooke, 1998). The accessibility of a specific target site in a given RNA can be tested in advance in vitro by means of binding assays or translation arrest assays (Schu and Brinkmeier, 1999). As for the specificity for closely related isoforms of voltage-gated Na ϩ channels, we have earlier developed highly specific 15-mer aODNs against the RNAs of the Na ϩ channels in human heart (hH1, Materials and Methods Cells and Application of Oligonucleotides. Human myotubes expressing both the tetrodotoxin (TTX)-sensitive skeletal musclespecific hSkM1 and the TTX-insensitive heart-specific hH1 Na ϩ channe

A novel alteration of muscle chloride channel gating in myotonia levior

Mutations in the voltage-dependent skeletal muscle chloride channel, ClC-1, result in dominant or recessive myotonia congenita. The Q552R mutation causes a variant of dominant myotonia with a milder phenotype, myotonia levior. To characterise the functional properties of this mutation, homodimeric mutant and heterodimeric wild-type (WT) mutant channels were expressed in tsA201 cells and studied using the whole-cell recording technique. Q552R ClC-1 mutants formed functional channels with normal ion conduction but altered gating properties. Mutant channels were activated by membrane depolarisation, with a voltage dependence of activation that was shifted by more than +90 mV compared to WT channels. Q552R channels were also activated by hyperpolarisation, and this process was dependent upon the intracellular chloride concentration ([Cl−]i). Together, these alterations resulted in a substantial reduction in the open probability at −85 mV at a physiological [Cl−]i. Heterodimeric WT-Q552R channels did not exhibit hyperpolarisation-activated gating transitions. As was the case for WT channels, activation occurred upon depolarisation, but the activation curve was shifted by 28 mV to more positive potentials. The functional properties of heterodimeric channels suggest a weakly dominant effect, a finding that correlates with the inheritance pattern and symptom profile of myotonia levior