During hyperpolarizing pulses, defolliculated Xenopus oocytes have time-and voltage-dependent inward chloride currents. The currents vary greatly in amplitude from batch to batch; activate slowly and, in general, do not decay; have a selectivity sequence of I-> NO~ > Br-> C1-> propionate > acetate; are insensitive to Ca 2+ and pH; are blocked by Ba 2+ and some chloride channel blockers; and have a gating valence of ~ 1.3 charges. In contrast to hyperpolarization-activated chloride currents induced after expression of phospholemman (Palmer, C.

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The genetic abnormality in myotonic muscular dystrophy, multiple CTG repeats lie upstream of a gene that encodes a novel protein kinase, myotonic dystrophy protein kinase (DMPK). Phospholemman (PLM), a major membrane substrate for phosphorylation by protein kinases A and C, induces Cl currents (I Cl(PLM) ) when expressed in Xenopus oocytes. To test the idea that PLM is a substrate for DMPK, we measured in vitro phosphorylation of purified PLM by DMPK. To assess the functional effects of PLM phosphorylation we compared I Cl(PLM) in Xenopus oocytes expressing PLM alone to currents in oocytes co-expressing DMPK, and examined the effect of DMPK on oocyte membrane PLM expression. We found that PLM is indeed a good substrate for DMPK in vitro. Co-expression of DMPK with PLM in oocytes resulted in a reduction in I Cl(PLM) . This was most likely a specific effect of phosphorylation of PLM by DMPK, as the effect was not present in oocytes expressing a phos(؊) PLM mutant in which all potential phosphorylation had been disabled by Ser 3 Ala substitution. The biophysical characteristics of I Cl(PLM) were not changed by DMPK or by the phos(؊) mutation. Co-expression of DMPK reduced the expression of PLM in oocyte membranes, suggesting a possible mechanism for the observed reduction in I Cl(PLM) amplitude. These data show that PLM is a substrate for phosphorylation by DMPK and provide functional evidence for modulation of PLM function by phosphorylation.Phosphorylation of membrane proteins by protein kinases is an important mechanism for receptor-mediated signal transduction in the regulation of cellular function. For instance, stimulation of ␤-adrenergic receptors activates protein kinase A (PKA) 1 , whereas stimulation of ␣1-adrenergic receptors activates protein kinase C (PKC). Though the end result of these kinases on cellular physiology can be very different, they share at least one membrane substrate, a 72-amino acid peptide with a single transmembrane domain called phospholemman (PLM) (1). Several lines of evidence suggest a role for PLM in ion transport and regulation of cell volume. In particular, expression of PLM in Xenopus oocytes leads to the appearance of a hyperpolarization-activated noninactivating Cl current (I Cl(PLM) ) (2-4). We have shown previously that the amplitude of I Cl(PLM) correlates with the level of PLM expression, and that co-expression of PKA increases both current amplitude and PLM expression (5). The highly conserved (6) cytoplasmic domain contains four potential phosphorylation sites, and loci for phosphorylation by PKA and PKC have been identified (1,7,8). PLM is also a substrate for NIMA ("never in mitosis") kinase, a product of the cell cycle regulatory gene nimA, which is necessary for cells to enter mitosis. PLM is, thus, a substrate for multiple kinases and may have a role as an integrator of adrenergic inputs.Myotonic muscular dystrophy, the commonest muscular dystrophy in adults, is an autosomal dominant-inherited multisystem disorder with prominent effects on skeletal and cardiac mu...

In myotonic muscular dystrophy, abnormal muscle Na currents underlie myotonic discharges. Since the myotonic muscular dystrophy gene encodes a product, human myotonin protein kinase, with structural similarity to protein kinases, we tested the idea that human myotonin protein kinase modulates skeletal muscle Na channels. Coexpression of human myotonin protein kinase with rat skeletal muscle Na channels in Xenopus oocytes reduced the amplitude of Na currents and accelerated current decay. The effect required the presence of a potential phosphorylation site in the inactivation mechanism of the channel. The mutation responsible for human disease, trinucleotide repeats in the 3' untranslated region, did not prevent the effect. The consequence of an abnormal amount of the kinase would be altered muscle cell excitability, consistent with the clinical finding of myotonia in myotonic dystrophy. (J. Clin. Invest. 1995. 95:2379-2384

Phospholemman (PLM), the major sarcolemmal substrate for phosphorylation by cAMP-dependent kinase (PKA) protein kinase C (PKC) and NIMA kinase in muscle, induces hyperpolarization-activated anion currents in Xenopus oocytes, most probably by enhancing endogenous oocyte currents. PLM peptides from the cytoplasmic tail are phosphorylated by PKA at S68, by NIMA kinase at S63, and by PKC at both S63 and S68. We have confirmed the phosphorylation sites in the intact protein, and we have investigated the role of phosphorylation in the regulatory activity of PLM using oocyte expression experiments. We found: (1) the cytoplasmic domain is not essential for inducing currents in oocytes; (2) co-expression of PKA increased the amplitude of oocyte currents and the amount of PLM in the oocyte membrane largely, but not exclusively, through phosphorylation of S68; (3) co-expression of PKA had no effect on a PLM mutant in which all putative phosphorylation sites had been inactivated by serine to alanine mutation (SSST 62, 63, 68, 69 AAAA); (4) co-expression of PKC had no effect in this system; (5) co-expression of NIMA kinase increased current amplitude and membrane protein level, but did not require PLM phosphorylation. These findings point to a role for phosphorylation in the function of PLM.

Phospholemman (PLM) is a 72-amino-acid phosphoprotein that is a major substrate for cAMP-dependent protein kinase, protein kinase C, and NIMA kinase. In lipid bilayers, PLM forms ion channels selective for Cl-, K+, and taurine. Effluxes of these abundant intracellular osmolytes play an important role in the control of dynamic cell volume changes in many cell types. We measured swelling-activated ion currents and regulatory volume decrease (RVD) in human embryonic kidney cells stably overexpressing canine cardiac PLM. In response to swelling, two clonal cell lines overexpressing PLM had increased swelling-activated ion current densities and faster and more extensive RVD. A third clonal cell line overexpressing mutant PLM showed reduced ion current densities and a diminished RVD response. These results suggest a role for PLM in the regulation of cell volume, perhaps as a modulator of an endogenous swelling-activated signal transduction pathway or possibly by participating directly in swelling-induced osmolyte efflux.

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1 Voltage-gated Na channels, which are potential targets for general anaesthetics, are substrates for PKC, which phosphorylates a conserved site in the channel inactivation gate. We investigated the idea that PKC modulates the eect of volatile anaesthetics on Na channels via phosphorylation of this inactivation gate site. 2 Na currents through rat skeletal muscle Na channel a-subunits expressed in Xenopus oocytes were measured by two-microelectrode voltage clamp in the presence of the volatile anaesthetic agent halothane (2-bromo-2-chloro-1,1,1-tri¯uroethane). PKC activity was modulated by co-expression of a constitutively active PKC a-isozyme. 3 Halothane (0.4 mM) had no eect on Na currents. With co-expression of PKC, however, halothane dose-dependently enhanced the rate of Na current decay and caused a small, but statistically signi®cant reduction in Na current amplitude. 4 The enhancement of Na current decay was absent in a Na channel mutant in which the inactivation gate phosphorylation site was disabled. Eects of halothane on amplitude were independent of this mutation. 5 Co-expression of a PKC a-isozyme permits an eect of halothane to hasten current decay and reduce current amplitude, at least in part through interaction with the inactivation gate phosphorylation site. We speculate that the interaction between halothane and Na channels is direct, and facilitated by PKC activity and by phosphorylation of a site in the channel inactivation gate.