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1.A.27 The Phospholemman (PLM) Family

The PLM family, a member of the Dysadherin superfamily, includes mammalian phospholemmans of 8-10 kDa size. They span the membrane once with their N-termini outside. These proteins induce a hyperpolarization-activated chloride current in Xenopus oocytes. They are found in muscle and many body tissues and are targets of protein kinases A and C. Other possible members include the chloride-conductance inducer protein, Mat8, and Na+/K+-ATPase γ-subunit 'proteolipids.' These proteins are smaller, but of the same orientation in the membrane (see below).

PLM forms anion-selective channels when reconstituted in planar lipid bilayers. These channels display a linear current-voltage relationship, have a unitary conductance and are open most of the time at voltages between -70 and +70 mV. The PLM channel is permeable to both organic and inorganic anions including chloride, taurine, lactate, glutamate, isethionate, and gluconate. These channel proteins resemble cardiac γ-subunits of the Na+, K+-ATPase (FXYD) (TC #3.A.3).

Members of the FXYD family (FXYD1-12) regulate the Na+-K+-ATPase, phospholamban, sarcolipin, myoregulin, and DWORF, which regulate the sarco-endoplasmic reticulum Ca2+-ATPase (SERCA) (Pirkmajer et al. 2017). FXYD5, a member of the FXYD family of single spanning type I membrane proteins (also called FXYD-containing ion transporter regulator 5) interacts with and regulates ion channels and the Na+,K+-ATPase (Lubarski et al., 2005). The same is true for at least 5 other FXYD proteins including FXYD1. These proteins are members of the dysadherin superfamily of ion channels and ion channel regulators (Lifshitz et al., 2006). The cytoplasmic tail of PLM interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger (Wang et al., 2006).  PLM coordinately regulates the cardiac Na+/Ca2+ exchanger and the Na+,K+-ATPase (Cheung et al. 2013).  For this reason, TC Family 1.A.27 could also be listed under subfamily 8.A.

A mutation in the human FXYD2 polypeptide (Na-K-ATPase gamma subunit) that changes a conserved transmembrane glycine to arginine is linked to dominant renal hypomagnesemia. Xenopus laevis oocytes injected with wild-type FXYD2 or the mutant G41R cRNAs expressed large nonselective ion currents. However, in contrast to the wild-type FXYD2 currents, inward rectifying cation currents were induced by hyperpolarization pulses in oocytes expressing the G41R mutant. Injection of EDTA into the oocyte removed inward rectification in the oocytes expressing the mutant, but did not alter the nonlinear current-voltage relationship of the wild-type FXYD2 pseudo-steady-state currents. Extracellular divalent ions, Ca2+ and Ba2+, and trivalent cations, La3+, blocked both the wild-type and mutant FXYD2 currents. Site-directed mutagenesis of G41 demonstrated that a positive charge at this site is required for the inward rectification. When the wild-type FXYD2 was expressed in Madin-Darby canine kidney cells, the cells in the presence of a large apical-to-basolateral Mg2+ gradient and at negative potentials had an increase in transepithelial current compared with cells expressing the G41R mutant or control transfected cells. Moreover, this current was inhibited by extracellular Ba2+ at the basolateral surface. These results suggest that FXYD2 can mediate basolateral extrusion of magnesium from cultured renal epithelial cells (Sha et al. 2008).

PLM is a 72-amino acid protein consisting of the signature PFXYD motif in the extracellular N terminus, a single transmembrane (TM) domain, and a C-terminal cytoplasmic tail containing three phosphorylation sites. In the heart, PLM co-localizes and co-immunoprecipitates with the Na+-K+-ATPase, the Na+/Ca2+ exchanger, and L-type Ca2+ channel. The TM domain of PLM interacts with TM9 of the α-subunit of Na+-K+-ATPase, while its cytoplasmic tail interacts with two small regions (spanning residues 248-252 and 300-304) of the proximal intracellular loop of the Na+/Ca2+ exchanger. Under stress, catecholamine stimulation phosphorylates PLM at serine(68), resulting in relief of inhibition of the Na+-K+-ATPase by decreasing the Km for Na+ and increasing the Vmax, and simultaneously inhibiting the Na+/Ca2+ exchanger. Enhanced Na+-K+-ATPase activity lowers the intracellular Na+, thereby minimizing Ca2+ overload and risks of arrhythmias. Inhibition of Na+/Ca2+ exchanger reduces Ca2+ efflux, thereby preserving contractility. Thus, the coordinated actions of PLM during stress serve to minimize arrhythmogenesis and maintain inotropy.

Many members of this superfamily have been characterized. FXYD5 is of 178 aas and has N-terminal (residues 7-24) and C-terminal (residues 146-162) hydrophobic regions. These proteins display a short region (130-167) with striking sequence similarity (50% identity) to established members of the PLM family. Since the region of sequence similarity includes a transmembrane domain, these auxiliary proteins may have anion-selective channel activity.

The generalized transport reaction catalyzed by PLM and Mat8 is:

Anions (out) Anions (in).

References associated with 1.A.27 family:

Attali B., H. Latter, N. Rachamim, H. Garty. (1995). A corticosteroid-induced gene expressing an 'IsK-like' K+ channel activity in Xenopus oocytes. Proc. Natl. Acad. Sci. U.S.A. 92: 6092-6096 7597086
Bibert, S., S. Roy, D. Schaer, E. Felley-Bosco, and K. Geering. (2006). Structural and functional properties of two human FXYD3 (Mat-8) isoforms. J. Biol. Chem. 281: 39142-39151. 17077088
Chen, L.S.K., C.F. Lo, R. Numann and M. Cuddy (1997). Characterization of the human and rat phospholemman (PLM) cDNAs and localization of the human PLM gene to chromosome 19q13.1. Genomics 41: 435-443. 9169143
Cheung, J.Y., X.Q. Zhang, J. Song, E. Gao, T.O. Chan, J.E. Rabinowitz, W.J. Koch, A.M. Feldman, and J. Wang. (2013). Coordinated Regulation of Cardiac Na+/Ca (2+) Exchanger and Na (+)-K (+)-ATPase by Phospholemman (FXYD1). Adv Exp Med Biol 961: 175-190. 23224879
Delprat, B., J.L. Puel, and K. Geering. (2007). Dynamic expression of FXYD6 in the inner ear suggests a role of the protein in endolymph homeostasis and neuronal activity. Dev Dyn 236: 2534-2540. 17676640
Foskett, J.K. (1998). ClC and CFTR chloride channel gating. Annu. Rev. Physiol. 60: 689-717. 9558482
Kirk, K. and K. Strange (1998). Functional properties and physiological roles of organic solute channels. Annu. Rev. Physiol. 60: 719-739. 9558483
Lifshitz, Y., M. Lindzen, H. Garty, and S.J. Karlish. (2006). Functional interactions of phospholemman (PLM) (FXYD1) with Na+,K+-ATPase. Purification of alpha1/beta1/PLM complexes expressed in Pichia pastoris. J. Biol. Chem. 281: 15790-15799. 16608841
Lubarski, I., K. Pihakaski-Maunsbach, S.J. Karlish, A.B. Maunsbach, and H. Garty. (2005). Interaction with the Na,K-ATPase and tissue distribution of FXYD5 (related to ion channel). J. Biol. Chem. 280: 37717-37724. 16148001
Lubarski, I., S.J. Karlish, and H. Garty. (2007). Structural and functional interactions between FXYD5 and the Na+-K+-ATPase. Am. J. Physiol. Renal Physiol 293: F1818-1826. 17881459
Pirkmajer, S., H. Kirchner, L. Lundell, P.V. Zelenin, J.R. Zierath, K.S. Makarova, Y.I. Wolf, and A.V. Chibalin. (2017). Early vertebrate origin and diversification of small transmembrane regulators of cellular ion transport. J. Physiol. [Epub: Ahead of Print] 28436536
Sha, Q., W. Pearson, L.C. Burcea, D.A. Wigfall, P.H. Schlesinger, C.G. Nichols, and R.W. Mercer. (2008). Human FXYD2 G41R mutation responsible for renal hypomagnesemia behaves as an inward-rectifying cation channel. Am. J. Physiol. Renal Physiol 295: F91-99. 18448590
Shindo, Y., K. Morishita, E. Kotake, H. Miura, P. Carninci, J. Kawai, Y. Hayashizaki, A. Hino, T. Kanda, and Y. Kusakabe. (2011). FXYD6, a Na,K-ATPase regulator, is expressed in type II taste cells. Biosci. Biotechnol. Biochem. 75: 1061-1066. 21670532
Wang, J., X.Q. Zhang, B.A. Ahlers, L.L. Carl, J. Song, L.I. Rothblum, R.C. Stahl, D.J. Carey, and J.Y. Cheung. (2006). Cytoplasmic tail of phospholemman interacts with the intracellular loop of the cardiac Na+/Ca2+ exchanger. J. Biol. Chem. 281: 32004-32014. 16921169
Zhang XQ., Wang J., Song J., Rabinowitz J., Chen X., Houser SR., Peterson BZ., Tucker AL., Feldman AM. and Cheung JY. (2015). Regulation of L-type calcium channel by phospholemman in cardiac myocytes. J Mol Cell Cardiol. 84:104-11. 25918050