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:Chaumette et al. 2020).

The generalized transport reaction catalyzed by PLM and Mat8 is:

Anions (out) Anions (in).