1.A.27 The Phospholemman (PLM) Family The PLM family, a member of the Dysadherin or FXYD superfamily (Garty and Karlish 2006), 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). Four of the seven members of the FXYD protein family have been identified as specific regulators of the Na,K-ATPase, and FXYD3 decreases its apparent affinity for Na⁺ and K⁺ (Crambert et al. 2005). Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity, particularly for one isoform, was observed for phospholemman-deficient mice (Jia et al. 2005). 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.3.1). FXYDs appear to be a vertebrate innovation and an important site of hormonal action (Pirkmajer and Chibalin 2019). Members of the FXYD family (FXYD1-12) regulate the Na⁺-K⁺-ATPase; phospholamban, sarcolipin, myoregulin, and DWORF 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 or FXYD 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. FXYD proteins and sodium pump regulatory mechanisms have been reviewed (Yap et al. 2021). 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 an 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 K_m for Na⁺ and increasing the V_max, 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 the FXYD 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. Mutations in ATP1A3 (TC# 3.A.3.1.1) and FXYD genes (this family) can cause childhood-onset schizophrenia (Chaumette et al. 2020). The generalized transport reaction catalyzed by PLM and Mat8 is: Anions (out) Anions (in).
This family belongs to the Phospholemman/SIMP/Viroporin (PSV) Superfamily.
References:
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
Barlow, I.L., E. Mackay, E. Wheater, A. Goel, S. Lim, S. Zimmerman, I. Woods, D.A. Prober, and J. Rihel. (2023). The zebrafish mutant implicates sodium homeostasis in sleep regulation. Elife 12:.
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.
Chaumette, B., V. Ferrafiat, A. Ambalavanan, A. Goldenberg, A. Dionne-Laporte, D. Spiegelman, P.A. Dion, P. Gerardin, C. Laurent, D. Cohen, J. Rapoport, and G.A. Rouleau. (2020). Missense variants in ATP1A3 and FXYD gene family are associated with childhood-onset schizophrenia. Mol Psychiatry 25: 821-830.
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.
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.
Crambert, G., C. Li, D. Claeys, and K. Geering. (2005). FXYD3 (Mat-8), a new regulator of Na,K-ATPase. Mol. Biol. Cell 16: 2363-2371.
Crambert, G., C. Li, L.K. Swee, and K. Geering. (2004). FXYD7, mapping of functional sites involved in endoplasmic reticulum export, association with and regulation of Na,K-ATPase. J. Biol. Chem. 279: 30888-30895.
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.
Essandoh, K., J.M. Philippe, P.M. Jenkins, and M.J. Brody. (2020). Palmitoylation: A Fatty Regulator of Myocardial Electrophysiology. Front Physiol 11: 108.
Foskett, J.K. (1998). ClC and CFTR chloride channel gating. Annu. Rev. Physiol. 60: 689-717.
Garty, H. and S.J. Karlish. (2006). Role of FXYD proteins in ion transport. Annu. Rev. Physiol. 68: 431-459.
Goldschmidt, I., F. Grahammer, R. Warth, A. Schulz-Baldes, H. Garty, R. Greger, and M. Bleich. (2004). Kidney and colon electrolyte transport in CHIF knockout mice. Cell Physiol Biochem 14: 113-120.
Hou, W., J. Cai, P. Shen, S. Zhang, S. Xiao, P. You, Y. Tong, K. Li, Z. Qi, and H. Luo. (2023). Identification of FXYD6 as the novel biomarker for glioma based on differential expression and DNA methylation. Cancer Med 12: 22170-22184.
Howie, J., K.J. Wypijewski, F. Plain, L.B. Tulloch, N.J. Fraser, and W. Fuller. (2018). Greasing the wheels or a spanner in the works? Regulation of the cardiac sodium pump by palmitoylation. Crit. Rev. Biochem. Mol. Biol. 1-17. [Epub: Ahead of Print]
Jespersen, T., M. Grunnet, H.B. Rasmussen, N.B. Jørgensen, H.S. Jensen, K. Angelo, S.P. Olesen, and D.A. Klaerke. (2006). The corticosteroid hormone induced factor: a new modulator of KCNQ1 channels? Biochem. Biophys. Res. Commun. 341: 979-988.
Jia, L.G., C. Donnet, R.C. Bogaev, R.J. Blatt, C.E. McKinney, K.H. Day, S.S. Berr, L.R. Jones, J.R. Moorman, K.J. Sweadner, and A.L. Tucker. (2005). Hypertrophy, increased ejection fraction, and reduced Na-K-ATPase activity in phospholemman-deficient mice. Am. J. Physiol. Heart Circ Physiol 288: H1982-1988.
Kadowaki, K., K. Sugimoto, F. Yamaguchi, T. Song, Y. Watanabe, K. Singh, and M. Tokuda. (2004). Phosphohippolin expression in the rat central nervous system. Brain Res Mol Brain Res 125: 105-112.
Kayed, H., J. Kleeff, A. Kolb, K. Ketterer, S. Keleg, K. Felix, T. Giese, R. Penzel, H. Zentgraf, M.W. Büchler, M. Korc, and H. Friess. (2006). FXYD3 is overexpressed in pancreatic ductal adenocarcinoma and influences pancreatic cancer cell growth. Int J Cancer 118: 43-54.
Kirk, K. and K. Strange (1998). Functional properties and physiological roles of organic solute channels. Annu. Rev. Physiol. 60: 719-739.
Li, C., G. Crambert, D. Thuillard, S. Roy, D. Schaer, and K. Geering. (2005). Role of the transmembrane domain of FXYD7 in structural and functional interactions with Na,K-ATPase. J. Biol. Chem. 280: 42738-42743.
Li, M., T. Nishimura, Y. Takeuchi, T. Hongu, Y. Wang, D. Shiokawa, K. Wang, H. Hirose, A. Sasahara, M. Yano, S. Ishikawa, M. Inokuchi, T. Ota, M. Tanabe, K.I. Tada, T. Akiyama, X. Cheng, C.C. Liu, T. Yamashita, S. Sugano, Y. Uchida, T. Chiba, H. Asahara, M. Nakagawa, S. Sato, Y. Miyagi, T. Shimamura, L.A.E. Nagai, A. Kanai, M. Katoh, S. Nomura, R. Nakato, Y. Suzuki, A. Tojo, D.C. Voon, S. Ogawa, K. Okamoto, T. Foukakis, and N. Gotoh. (2023). FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters. J Clin Invest 133:.
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.
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.
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.
Pavlovic, D., W. Fuller, and M.J. Shattock. (2013). Novel regulation of cardiac Na pump via phospholemman. J Mol. Cell Cardiol 61: 83-93.
Pirkmajer, S. and A.V. Chibalin. (2019). Hormonal regulation of Na-K-ATPase from the evolutionary perspective. Curr Top Membr 83: 315-351.
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]
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.
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.
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.
Yap, J.Q., J. Seflova, R. Sweazey, P. Artigas, and S.L. Robia. (2021). FXYD proteins and sodium pump regulatory mechanisms. J Gen Physiol 153:.
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.
Examples:
TC#	Name	Organismal Type	Example
1.A.27.1.1	Phospholemman (PLM; FXYD1) forms anion channels and regulates L-type Ca²⁺ channels as well as several other cation transport systems in cardiac myocytes (Zhang et al. 2015), most importantly the Na⁺,K⁺-ATPase (Pavlovic et al. 2013). Palmitoylation of the mammalian Na⁺ pump's accessory subunit PLM by the cell surface palmitoyl acyl transferase DHHC5 leads to pump inhibition, possibly by altering the relationship between the pump's catalytic α-subunit and specifically bound membrane lipids (Howie et al. 2018). PLM is also regulated by phosphorylation and glutathionylation (Pavlovic et al. 2013). and phosphorylation couteracts the inhibitory effect of palmitoylation (Cheung et al. 2013). The human ortholog has UniProt acc #O00168 and is 89% identical to the dog protein, with all of the difference occurring in the first 20 aas. Palmitoylation affects the regulation of cardiac electrophysiology, by modifying the sodium-calcium exchanger, phospholemman and the cardiac sodium pump, as well as the voltage-gated sodium channel (Essandoh et al. 2020). The conserved FXYD motif is found in this protein as residues 29-32.	Animals	PLM of Canis familiaris

1.A.27.1.2	Cl^- conductance inducer protein, Mat-8, of 88 aas and 1 TMS.	Animals	Mat-8 of Mus musculus

1.A.27.1.3	FXYD6 regulator of Na,K-ATPase in the ear and taste buds, phosphohippolin, of 95 aas and 1 TMS (Delprat et al., 2007; Shindo et al., 2011). It is expressed in the central nervous system (Kadowaki et al. 2004) and is the novel biomarker for glioma (Hou et al. 2023).	Animals	*FXYD6 of Homo sapiens* (Q9H0Q3)**

1.A.27.1.4	The sterol (dexamethasone, aldosterone) and low NaCl diet-inducible FXYD domain-containing ion transport regulator 4 precursor (Channel inducing factor, CHIF). It is an IsK-like MinK homologue (Attali et al., 1995). It regulates the Na⁺,K⁺-ATPase and the KCNQ1 channel protein as well as other ICNQ channels, opening them at all membrane potentials (Jespersen et al. 2006). CHIF as an indirect modulator of several different ion transport mechanisms, consistent with regulation of the Na⁺-K⁺-ATPase as the common denominator (Goldschmidt et al. 2004).	Animals	CHIF of Rattus norvegicus (Q63113)

1.A.27.1.5	FXYD3 (FXYD-3; Mat-8; PLML) with two splice variants, one of 87 aas with 2 TMSs (an N-terminal leader sequence and a central very hydrophobic TMS) and the other of 116 aas and 2 TMSs (Bibert et al. 2006). Both FXYD3 variants co-immunoprecipitate with the Na,K-ATPase. They both associate stably with Na,K-ATPase isozymes but not with the H,K-ATPase or Ca-ATPase. The short human FXYD3 has 72% sequence identity with mouse FXYD3, whereas long human FXYD3 is identical to the short human FXYD3 but has a 26-amino acid insertion after the transmembrane domain. Short and long human FXYD3 RNAs and proteins are differentially expressed during differentiation with long FXYD3 being mainly expressed in nondifferentiated cells while short FXYD3 is expressed in differentiated cells (Bibert et al. 2006). Overexpression of FXYD3, as it occurs in pancreatic cancer, may contribute to the proliferative activity of this malignancy (Kayed et al. 2006). FXYD3 functionally demarcates an ancestral breast cancer stem cell subpopulation with features of drug-tolerant persisters (Li et al. 2023).		FXYD3 of Homo sapiens

1.A.27.1.6	FXYD4 of 89 aas and 1 TMS.		FXYD4 of Homo sapiens

1.A.27.1.7	FXYD7 of 80 aas and 1 TMS. The TMS mediates the complex interactions with the Na,K-ATPase (Li et al. 2005). The brain-specific FXYD7 is a member of the FXYD family that associates with the alpha1-beta1 Na,K-ATPase isozyme and induces a 2-fold decrease in its apparent K⁺ affinity. In contrast to FXYD2 and FXYD4, the conserved FXYD motif in the extracytoplasmic domain is not involved in the association of FXYD7 with the Na,K-ATPase. The conserved Gly40 and Gly29, located on the same face of the TMS, were implicated in the association with and the regulation of Na,K-ATPase (Crambert et al. 2004). The C-terminal valine residue is involved in ER export of FXYD7. FXYDs are a vertebrate innovation and an important site of hormonal action (Pirkmajer and Chibalin 2019).		FXYD7 of Homo sapiens

1.A.27.1.8	Phospholemman, FXYD1 or PLM of 92 aas and 1 TMS. See 1.A.27.1.1 for details for the dog ortholog. Palmitoylation affects the regulation of cardiac electrophysiology, by modifying the sodium-calcium exchanger, phospholemman and the cardiac sodium pump, as well as the voltage-gated sodium channel (Essandoh et al. 2020). Palmitoylation of PLM inhibits the Na⁺ K⁺-ATPase while phosphorylation reverses this inhibition. The conserved FXYD motif is found in this protein at residues 29-32 (Cheung et al. 2013). Dreammist in zebrafish, a neuronal-expressed phospholemman homolog, is important for regulating sleep-wake behaviour (Barlow et al. 2023).		PLM of Homo sapiens

Examples:
TC#	Name	Organismal Type	Example
1.A.27.2.1	*γ-subunit (proteolipid) of Na⁺,K⁺-ATPase, FXYD2. Also functions as a cation-selective channel (Sha et al.* 2008).**	Animals	FXYD2 channel and γ-subunit of the Na⁺,K⁺-ATPase of Homo sapiens

1.A.27.2.2	Sodium/potassium-transporting ATPase subunit gamma isoform X1of 82 aas and 1 TMS.		γ-subunit of Pseudopodoces humilis

1.A.27.2.3	Sodium/potassium-transporting ATPase subunit gamma of 61 aas and 1 TMS.		*γ-subunit of Xenopus tropicalis* (tropical clawed frog)**

1.A.27.2.4	Sodium/potassium-transporting ATPase subunit gamma isoform X1		*Na⁺, K⁺-ATPase regulator of Mus pahari* (shrew mouse)**

1.A.27.2.5	Sodium/potassium-transporting ATPase subunit gamma isoform X1of 65 aas and 1 TMS. The 3-d structure of a 31 aa peptide including the single TMS is available (PDB# 2N23).		γ-subunit of Sus scrofa (pig)

Examples:
TC#	Name	Organismal Type	Example
1.A.27.3.1	FXYD5 regulator of Na,K⁺-ATPase and ion channel activities of 178 aas and 1 C-terminal TMS. FXYD5 interacts directly with the Na⁺,K⁺-ATPase via their TMSs to affect the V_max of the latter, and residues involved have been identified (Lubarski et al. 2007).	Animals	*FXYD5 of Homo sapiens* (178 aas; Q96DB9)**

1.A.27.3.2	FXYD domain-containing ion transport regulator 5-like isoform X2 of 89 aas and 2 TMSs.		*FXYD regulator of Ornithorhynchus anatinus* (platypus)**

1.A.27.3.3	FXYD domain-containing ion transport regulator 5-like isoform X1 of 101 aas and 2 TMSs, N- and C-terminal. TC Blast with this protein retrieves 1.G.12.2.3 with about 90 residues aligning with 29% identity and 45% similarity. These two families may be related.		FXYD domain protein of Carassius auratus (goldfish)

1.A.27.3.4	FXYD domain-containing ion transport regulator 5-like isoform X2 of 174 aas and 2 TMSs, N- and C-terminal.		*FXYD domain protein of Denticeps clupeoides* (denticle herring)**

1.A.27.3.5	FXYD domain-containing ion transport regulator 5-like isoform X2 of 170aas and 2 TMSs.		*FXYD domain protein of Rhinatrema bivittatum* (two-lined caecilian)**