Inhibition of Phosphorylation of Na⁺,K⁺-ATPase by Mutations Causing Familial Hemiplegic Migraine

Background: Familial hemiplegic migraine type II (FHM2) is caused by mutations in the Na⁺,K⁺-ATPase α2-isoform.

Results: Several FHM2 mutations inhibit phosphorylation or dephosphorylation.

Conclusion: These mutations cause FHM2 by local and long range effects on the catalytic site and not by reducing the affinity for external K⁺.

Significance: Insights into the pathophysiological mechanism of FHM2 and the molecular mechanism of the Na⁺,K⁺-ATPase have been obtained.

Abstract

The neurological disorder familial hemiplegic migraine type II (FHM2) is caused by mutations in the α2-isoform of the Na⁺,K⁺-ATPase. We have studied the partial reaction steps of the Na⁺,K⁺-pump cycle in nine FHM2 mutants retaining overall activity at a level still compatible with cell growth. Although it is believed that the pathophysiology of FHM2 results from reduced extracellular K⁺ clearance and/or changes in Na⁺ gradient-dependent transport processes in neuroglia, a reduced affinity for K⁺ or Na⁺ is not a general finding with the FHM2 mutants. Six of the FHM2 mutations markedly affect the maximal rate of phosphorylation from ATP leading to inhibition by intracellular K⁺, thereby likely compromising pump function under physiological conditions. In mutants R593W, V628M, and M731T, the defective phosphorylation is caused by local perturbations within the Rossmann fold, possibly interfering with the bending of the P-domain during phosphoryl transfer. In mutants V138A, T345A, and R834Q, long range effects reaching from as far away as the M2 transmembrane helix perturb the function of the catalytic site. Mutant E700K exhibits a reduced rate of E₂P dephosphorylation without effect on phosphorylation from ATP. An extremely reduced vanadate affinity of this mutant indicates that the slow dephosphorylation reflects a destabilization of the phosphoryl transition state. This seems to be caused by insertion of the lysine between two other positively charged residues of the Rossmann fold. In mutants R202Q and T263M, effects on the A-domain structure are responsible for a reduced rate of the E₁P to E₂P transition.

Introduction

Hemiplegic migraine is a severe subtype of migraine with aura associated with transient motor weakness and sensory as well as speech difficulties. It is autosomal dominantly inherited (familial hemiplegic migraine). Besides mutations in neuronal voltage-gated Ca²⁺ and Na⁺ channels (FHM1 and FHM3), familial hemiplegic migraine has been associated with mutations in the α2-isoform of the Na⁺,K⁺-ATPase (FHM2) (1). The Na⁺ and K⁺ gradients created across the cell membrane by the Na⁺,K⁺-ATPase are of vital importance for cellular function and activities, including generation of action potentials and secondary active transport of ions, nutrients, and neurotransmitters. In the mammalian brain, three different isoforms of the catalytic α-subunit are expressed, α1, α2, and α3. The α2-isoform is mainly distributed in glial cells, whereas the α3-isoform is found in neurons and is absent in glial cells (2). So far, we know of more than 50 Na⁺,K⁺-ATPase α2 mutations associated with hemiplegic migraine. It has been suggested that the pathophysiology results from an impaired clearance of extracellular K⁺, producing a wide cortical depolarization (3, 4). Moreover, the gradients of both Na⁺ and K⁺ are important for the reuptake of the excitatory transmitter glutamate from the synaptic cleft via the glutamate transporter, and impaired Na⁺ transport would also lead to a rise of intracellular Ca²⁺ via the Na⁺/Ca²⁺ exchanger with secondary effects on Ca²⁺ signaling (1, 5, 6). Cell survival studies of transfected cells, in which the wild type has been inhibited by ouabain, have demonstrated the inability of certain FHM2 mutants to sustain cell growth in accordance with the theory of haploinsufficiency, i.e. only the wild type allele encodes a functional enzyme (1, 7). Other FHM2 mutants have nevertheless been found to retain transport function (4, 8–11), and more functional mutants exist, as indicated by the present results. A crucial question is therefore whether and how their function deviates from that of the wild type. One study has reported a 2-fold reduced apparent K⁺ affinity for activation of the ATPase reaction of mutant T345A, which was suggested to give rise to a reduced rate of external K⁺ clearance in vivo (4).

To fully understand the functional implications of the FHM2 mutations and the structural basis, it is necessary to reveal the mutational effects on the individual partial reaction steps of the pump cycle and the interaction with Na⁺ and K⁺ and to try to relate the observed effects to known structural features. We present here the functional consequences of nine Na⁺,K⁺-ATPase α2 mutations, including T345A, which all have been found in patients exhibiting the characteristic symptoms of hemiplegic migraine (12–17). The α-subunit carrying the mutations consists of 10 transmembrane helices M1–M10 (harboring the Na⁺- and K⁺-binding sites) and a cytoplasmic “head” made up of three subdomains as follows: A (“actuator”), N (“nucleotide binding”), and P (“phosphorylation”) (18, 19). Generally, FHM2 mutations are widely scattered over the Na⁺,K⁺-ATPase protein, but with a preference for the P-domain (7). Four of the mutations investigated here are located in the P-domain (R593W, V628M, E700K, and M731T), two are in the A-domain (R202Q and T263M), one is in M2 (V138A), another is in M4 near the interface to the P-domain (T345A), and the last is in the L6–7 loop between M6 and M7 close to the P-domain (R834Q) (Fig. 1). We assessed their functional impact in a panel of analyses of the partial reaction steps (cf. Scheme 1), using both steady-state and transient kinetic measurements, the latter allowing determination of the rates of the rapid phosphorylation and dephosphorylation reactions. The observed effects are analyzed in the context of information derived from the recently determined crystal structures of the Na⁺,K⁺-ATPase (18, 19).

FIGURE 1.

Na⁺,K⁺-ATPase structure with indication of the FHM2 mutations studied. The structure shown has Protein Data Bank code 2ZXE (E₂[K₂] with bound MgF₄²⁻ as phosphate analog (19)). Color codes for P-, A-, N-, and M-domain are gray, green, yellow, and cyan, respectively. The bound K⁺ ions are shown as purple spheres and MgF₄²⁻ (MgF) in the catalytic site as one green sphere with four cyan spheres. The mutated residues (numbered according to the human α2-isoform) are highlighted as sticks colored according to the elements (carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow).

SCHEME 1.

Model of the reaction cycle of Na⁺,K⁺-ATPase (“Post-Albers scheme”). Ligand binding and dissociation steps and conformational changes between E₁/E₁P and E₂/E₂P forms are indicated. Na⁺ binds from the cytoplasmic side to the E₁ form and activates phosphorylation from ATP bound with high affinity to E₁. The E₁P form is ADP-sensitive, i.e. able to donate the phosphoryl group back to ADP, forming ATP. The E₂P form is K⁺-sensitive, i.e. hydrolysis of E₂P is activated by binding of K⁺ from the extracellular side. Occluded ions are shown in brackets. Subscripts c and e indicate the cytoplasmic and extracellular sides, respectively. The ATP molecule binding with low affinity to E₂ is shown boxed. Because ATP binds with higher affinity to E₁ than to E₂, an increase in the apparent affinity for ATP upon mutation reflects a shift of the E₁-E₂ distribution toward the E₁ form, whereas a decrease in the affinity for ATP reflects a shift toward the E₂ form.

EXPERIMENTAL PROCEDURES

FHM2 mutations were introduced into full-length cDNA encoding an ouabain-resistant version of the human α2-isoform (4). Mutants and wild type were expressed in COS-1 cells under ouabain selection pressure (20). The plasma membrane fraction was isolated and made leaky to allow access of incubation media from both sides of the membrane, and Na⁺,K⁺-ATPase activity was studied at 37 °C by following the liberation of P_i (20). The catalytic turnover rate was calculated by relating the ATPase activity to the active site concentration determined by phosphorylation at 0 °C in the presence of 150 mm Na⁺ and oligomycin (21, 22). Measurements of phosphorylation and dephosphorylation were performed using either a manual mixing technique at 0 °C or a QFM-5 quench-flow module (Bio-Logic Instruments, Claix, France), allowing transient kinetic studies at 25 °C (21, 22). To eliminate the contribution of the endogenous Na⁺,K⁺-ATPase, ouabain was included in the reaction media (21). Data processing was performed using SigmaPlot (SPSS, Inc.) for linear regression analysis (21), and the results are reported as average values ± S.E. (shown by error bars in figures when larger than the size of the symbols). The number of independent determinations is indicated by n in Tables 1 and 2. Structural figures were prepared using PyMOL.

TABLE 1.

ATPase activity parameters

	Ouabain-affinitya	Turnover rateb	K0.5(K+)c	K0.5(VO43−)c	K0.5(ATP)c
	μm	min−1	μm	μm	μm
Wild type α2	297 ± 29	6843 ± 380	716 ± 19	27 ± 2	104 ± 6
	(n = 3)	(n = 6)	(n = 5)	(n = 6)	(n = 8)
V138A	693 ± 38	3768 ± 157	763 ± 18	290 ± 18	47 ± 2
	(n = 3)	(n = 6)	(n = 4)	(n = 4)	(n = 7)
R202Q	288 ± 40	5420 ± 277	748 ± 33	113 ± 6	67 ± 4
	(n = 3)	(n = 10)	(n = 5)	(n = 8)	(n = 4)
T263M	1323 ± 95	3233 ± 168	465 ± 19	73 ± 4	21 ± 1
	(n = 3)	(n = 6)	(n = 5)	(n = 8)	(n = 4)
T345A	657 ± 48	3419 ± 50	747 ± 20	283 ± 21	31 ± 2
	(n = 4)	(n = 6)	(n = 4)	(n = 5)	(n = 5)
R593W	2409 ± 518	1029 ± 56	263 ± 35	>1000	19 ± 1
	(n = 4)	(n = 6)	(n = 7)	(n = 6)	(n = 6)
V628M	1282 ± 206	1885 ± 63	273 ± 15	>1000	71 ± 6
	(n = 3)	(n = 6)	(n = 4)	(n = 5)	(n = 5)
E700K	146 ± 23	3669 ± 229	1145 ± 36	>1000	28 ± 1
	(n = 3)	(n = 7)	(n = 3)	(n = 6)	(n = 3)
M731T	2708 ± 597	1238 ± 84	216 ± 9	778 ± 14	26 ± 1
	(n = 3)	(n = 6)	(n = 5)	(n = 3)	(n = 3)
R834Q	2134 ± 534	1236 ± 46	332 ± 21	>1000	24 ± 2
	(n = 3)	(n = 6)	(n = 5)	(n = 4)	(n = 5)

^a The rate of ATP hydrolysis was determined at 37 °C in the presence of 130 mm NaCl, 20 mm KCl, 3 mm ATP, 3 mm MgCl₂, 30 mm histidine buffer (pH 7.4), 1 mm EGTA, and various concentrations of ouabain. The data points were fitted using a function with the ouabain-inhibited enzyme represented by the sum of two hyperbolic components corresponding to the exogenous enzyme (affinity constant K₁) and the endogenous COS cell enzyme (affinity constant K₂ ≤1 μm), respectively (24): V/V_max = 1 − a₁[ouabain]/(K₁ + [ouabain]) − a₂[ouabain]/(K₂ + [ouabain]). The values determined for K₁ are indicated in the table.

^b The Na⁺,K⁺-ATPase activity was determined at 37 °C in the presence of 30 mm histidine buffer (pH 7.4), 130 mm NaCl, 3 mm ATP, 3 mm MgCl₂, 1 mm EGTA, ouabain to inhibit the endogenous enzyme, and 20 mm KCl. The catalytic turnover rate was calculated as the ratio between the Na⁺,K⁺-ATPase activity and the active site concentration (maximum phosphorylation from [γ-³²P]ATP measured at 0 °C in the presence of 150 mm NaCl and oligomycin) (21, 22).

^c K_0.5 values for K⁺ activation, vanadate inhibition, and ATP activation were extracted from the data in Figs. 2, 8 and 9, respectively.

TABLE 2.

Phosphorylation parameters

	K0.5(Na+)a	EP/EPoligob	kphos, 2 μmATPc	Vmaxc	Kmc	E1Pd	Rate of E2P dephosphorylatione
							1 mm K+	20 mm K+
	μm	%	s−1	s−1	μm	%	s−1	s−1
Wild type α2	538 ± 15	76 ± 2	22 ± 1	83 ± 10	6.7 ± 0.8	44 ± 4	43 ± 2	132 ± 5
	(n = 8)	(n = 5)	(n = 3)	(n = 8)	(n = 8)	(n = 9)	(n = 6)	(n = 3)
V138A	807 ± 25	56 ± 3	12 ± 1	45 ± 8	5.1 ± 1.0	48 ± 9	32 ± 2	108 ± 4
	(n = 4)	(n = 6)	(n = 5)	(n = 13)	(n = 13)	(n = 5)	(n = 3)	(n = 3)
R202Q	541 ± 13	76 ± 3	17 ± 1	ND	ND	61 ± 2	59 ± 4	NDb
	(n = 7)	(n = 5)	(n = 3)			(n = 6)	(n = 3)
T263M	633 ± 20	85 ± 5	23 ± 1	ND	ND	96 ± 2	NDa	NDb
	(n = 6)	(n = 6)	(n = 3)			(n = 4)
T345A	814 ± 34	59 ± 3	13 ± 1	39 ± 2	4.5 ± 0.2	33 ± 14	43 ± 2	NDb
	(n = 7)	(n = 3)	(n = 3)	(n = 9)	(n = 9)	(n = 4)	(n = 3)
R593W	694 ± 23	16 ± 2	10 ± 1	28 ± 10	5.1 ± 2.0	71 ± 6	36 ± 2	NDb
	(n = 7)	(n = 3)	(n = 3)	(n = 10)	(n = 10)	(n = 10)	(n = 3)
V628M	890 ± 29	18 ± 2	12 ± 1	45 ± 11	5.9 ± 1.5	74 ± 6	41 ± 2	NDb
	(n = 7)	(n = 4)	(n = 3)	(n = 7)	(n = 7)	(n = 4)	(n = 3)
E700K	621 ± 19	85 ± 2	20 ± 1	69 ± 3	5.3 ± 0.2	59 ± 5	17 ± 1	42 ± 3
	(n = 6)	(n = 4)	(n = 2)	(n = 7)	(n = 7)	(n = 5)	(n = 3)	(n = 3)
M731T	218 ± 10	30 ± 1	5 ± 1	14 ± 3	3.0 ± 0.7	66 ± 4	41 ± 2	NDb
	(n = 8)	(n = 5)	(n = 4)	(n = 11)	(n = 11)	(n = 5)	(n = 5)
R834Q	2496 ± 89	21 ± 4	15 ± 1	35 ± 13	5.9 ± 2.3	56 ± 4	41 ± 2	NDb
	(n = 7)	(n = 5)	(n = 3)	(n = 9)	(n = 9)	(n = 6)	(n = 6)

^a Extracted from the data in Fig. 4.

^b Ratio between phosphorylation levels without (EP) and with oligomycin (EP_oligo). Phosphorylation was carried out for 10 s at 0 °C in 20 mm Tris (pH 7.5), 3 mm MgCl₂, 2 μm [γ-³²P]ATP, 10 μm ouabain, 150 mm NaCl, without and with 20 μg of oligomycin/ml.

^c Extracted from the data in Figs. 5 and 6. The kinetic parameters were not determined (ND) for R202Q and T263M, because the phosphorylation rate at 2 μm ATP was close to wild type.

^d Extracted from the data in Fig. 7.

^e Extracted from the data in Fig. 3. For T263M, the E₂P dephosphorylation rate constant was not determined (ND), because the high extent of accumulation of E₁P, even at 25 °C, precluded such a determination. The dephosphorylation rate at 20 mm K⁺ was determined only when the dephosphorylation at 1 mm K⁺ was slower than that of the wild type.

RESULTS

Overall Function

The nine FHM2 mutations were introduced into the ouabain-resistant version of the human α2-isoform, and the mutants were expressed in mammalian COS-1 cells and subjected to ouabain-selection pressure (20), taking advantage of the >100-fold difference between the ouabain affinities of the exogenous Na⁺,K⁺-ATPase and the endogenous COS cell enzyme. Like the wild type, all mutants were able to confer ouabain resistance in growth medium containing 5 μm ouabain, indicating that the rate of transport of Na⁺ and K⁺ is sufficiently high to sustain cell growth. Determination of the ouabain dependence of Na⁺,K⁺-ATPase activity showed 2–9-fold reduction of the apparent affinity for ouabain for mutants V138A, T263M, T345A, R593W, V628M, M731T, and R834Q, wild type-like apparent affinity for R202Q, and 2-fold increased apparent affinity for E700K (Table 1). Hence, all the functional studies described below were carried out in the presence of ouabain to selectively inhibit the endogenous Na⁺,K⁺-ATPase (K_0.5 ≤ 1 μm).

The catalytic turnover rate determined in the presence of 130 mm Na⁺, 20 mm K⁺, and a saturating concentration of MgATP was found reduced in all mutants, most severely for R593W, V628M, M731T, and R834Q, displaying less than one-third the turnover rate of the wild type. T263M, T345A, E700K, and V138A displayed a turnover rate around 50%, whereas R202Q showed ∼20% reduction (Table 1).

K⁺ Interaction

A critical issue is whether the affinity for external K⁺ is lowered, thereby resulting in reduced ability to clear K⁺ from the extracellular space. The interaction with K⁺ was investigated by determining the K⁺ dependence of ATPase activity (Fig. 2). In the wild type, K⁺ at a submillimolar concentration activates ATP hydrolysis by binding at extracellularly facing sites of the E₂P form, thereby stimulating dephosphorylation (cf. Scheme 1, Reaction 6). The majority of the mutants displayed a K_0.5 value for K⁺ activation similar to or lower (i.e. corresponding to higher affinity) than that determined for the wild type, with E700K being the only exception, exhibiting a slight 1.6-fold increase in the K_0.5 value for K⁺ activation relative to wild type (Fig. 2 and Table 1). K⁺ concentrations above 15 mm inhibited the wild type somewhat, which is explained by K⁺ binding in competition with Na⁺ at the cytoplasmically facing sites of the enzyme in E₁ conformation, leading to conversion of E₁ back to the K⁺-occluded E₂ state (cf. Scheme 1, Reaction 1) (23, 24). Mutants V138A, T345A, R593W, V628M, M731T, and R834Q exhibited a more distinct inhibitory phase than the wild type, whereas no inhibition was observed for E700K at the K⁺ concentrations studied (Fig. 2).

FIGURE 2.

K⁺ dependence of Na⁺,K⁺-ATPase activity. ATPase activity was measured at 37 °C in 40 mm NaCl, 3 mm ATP, 3 mm MgCl₂, 30 mm histidine (pH 7.4), 1 mm EGTA, 10 μm ouabain, and K⁺ concentrations as indicated. K_0.5 values for K⁺ activation are listed in Table 1.

Because the variable inhibitory phase in the K⁺ dependence of ATPase activity inevitably will influence the apparent affinity for K⁺ activation corresponding to the rising phase, the activation by extracellular K⁺ was also examined more directly by determining the rate of E₂P dephosphorylation (Fig. 3 and Table 2). At a nonsaturating K⁺ concentration of 1 mm (Fig. 3, open symbols), T345A, R593W, V628M, M731T, and R834Q displayed a dephosphorylation rate constant almost identical to that of the wild type, indicating that these mutations did not weaken the interaction with extracellular K⁺. R202Q exhibited a higher rate constant of dephosphorylation than wild type. By contrast, the dephosphorylation rate constant was reduced for V138A and E700K relative to wild type (1.3- and 2.5-fold, respectively). To determine whether this was related to a reduction in affinity for external K⁺, similar experiments were performed at a saturating K⁺ concentration of 20 mm (Fig. 3, filled symbols). The calculated ratios between the dephosphorylation rate constants at 1 and 20 mm K⁺ are 0.29 ± 0.02, 0.42 ± 0.04, and 0.32 ± 0.02 for V138A, E700K, and wild type, respectively. Hence, at 1 mm K⁺, the saturation of the external sites is similar to wild type for V138A and is actually higher for E700K, indicating that the reduced dephosphorylation rate is caused by a lower V_max of phosphoenzyme hydrolysis rather than a lower affinity for K⁺.

FIGURE 3.

Rapid kinetic measurements of K⁺-induced E₂P dephosphorylation. Phosphorylation was performed for 5 s at 25 °C in the presence of 2 μm [γ-³²P]ATP in 20 mm Tris (pH 7.5), 20 mm NaCl, 3 mm MgCl₂, 1 mm EGTA, 130 mm choline chloride, and 10 μm ouabain, followed by dephosphorylation for the indicated times in the same medium containing in addition 1 mm nonradioactive ATP and 1 mm K⁺ (open symbols) or 20 mm K⁺ (filled symbols). Each line represents the best fit of a mono-exponential decay function, giving the indicated rate constants (also listed in Table 2 with statistics). The wild type data are represented in each panel by dotted red and blue lines.

Na⁺ Interaction

Phosphorylation from ATP is triggered when three Na⁺ ions have bound to intracellularly facing high affinity sites of the E₁ form and become occluded in the E₁[Na₃] form (Scheme 1, Reaction 2) (25). To assess the Na⁺ affinity of the E₁ form of the mutants, we studied the Na⁺ dependence of phosphorylation from ATP in the absence of K⁺ and presence of oligomycin to support occlusion (Fig. 4). The mutants displayed affinities similar to that obtained for the wild type; only R834Q exhibited a more than 2-fold reduced apparent affinity for Na⁺ relative to wild type.

FIGURE 4.

Na⁺ dependence of phosphorylation. Phosphorylation was carried out for 10 s at 0 °C in 20 mm Tris (pH 7.5), 3 mm MgCl₂, 2 μm [γ-³²P]ATP, 10 μm ouabain, 20 μg of oligomycin/ml, NaCl to obtain the indicated concentrations of Na⁺, and various concentrations of N-methyl-d-glucamine to maintain the ionic strength. Oligomycin was added to optimize phosphorylation and prevent dephosphorylation, thereby minimizing effects on apparent Na⁺ affinity of variation of the phosphorylation and dephosphorylation rates. Each line represents the best fit of the equation EP = EP_max·[Na⁺]ⁿ/(K_0.5ⁿ + [Na⁺]ⁿ), giving the indicated K_0.5 values (also listed in Table 2 with statistics). The data corresponding to wild type are represented in each panel by a dotted blue line.

Rate of Phosphorylation

Because oligomycin stabilizes the Na⁺-occluded E₁[Na₃] form, thereby promoting phosphorylation (25, 26) and blocking the E₁P-E₂P transition (Scheme 1, Reaction 4), the presence of oligomycin enables the phosphoenzyme to build up a maximum level. Table 2 lists the level of phosphoenzyme accumulated at 150 mm NaCl in the absence of oligomycin relative to the maximum level obtained in the presence of oligomycin (EP/EP_oligo). Mutants R593W, V628M, M731T, and R834Q displayed a markedly reduced EP/EP_oligo ratio, and V138A and T345A showed a slight reduction relative to wild type, whereas T263M and E700K displayed higher EP/EP_oligo ratios than the wild type. Such effects can be due to changes in either the rate of phosphorylation or the rate of dephosphorylation. In the presence of oligomycin, the level of phosphoenzyme built up was sufficiently high to allow rapid kinetic studies of the rate of phosphorylation at a millisecond time scale at 25 °C, and thus the time course of phosphorylation in the presence of 2 μm ATP was determined (Fig. 5). Mutants V138A, T345A, R593W, V628M, M731T, and R834Q all displayed a reduced rate constant relative to wild type, most pronounced for M731T, suggesting that either the phosphorylation reaction or the binding of ATP is affected in these mutants. To determine the maximal rate of phosphorylation and the apparent affinity of these mutants for ATP we performed the analysis at varying ATP concentrations. E700K was included, because the dephosphorylation studies (Fig. 3) indicated that the catalytic site might be defective in this mutant. In Fig. 6, the results are depicted as double-reciprocal plots of the initial phosphorylation rate per ATPase molecule as a function of the concentration of ATP. The linear dependence allows extraction of the maximal phosphorylation rate per ATPase molecule, V_max, and the apparent affinity for ATP (the Michaelis constant K_m). M731T exhibited a most severe 6-fold reduction of V_max, relative to wild type. Mutants V138A, T345A, R593W, V628M, and R834Q showed 2–3-fold reduction, whereas E700K was wild type-like (Table 2). None of the mutations increased the K_m value. A significant reduction of K_m is noted for M731T (Fig. 6 and Table 2), likely reflecting the marked reduction of V_max, which leads to increased accumulation of nonphosphorylated enzyme with ATP bound. According to the equation K_m = (k₋₁ + k₂)/k₁, where k₁ and k₋₁ are the respective rate constants for binding and dissociation of ATP, and k₂ equals V_max (measured in units of rate per ATPase molecule), the K_m value will decrease with k₂, unless k₋₁ is much higher than the k₂ value, which is not the case here, because Na⁺,K⁺-ATPase binds the nucleotide rather tightly (27). In an analogous way, the accumulation of E₁ explains the increased apparent affinity for Na⁺ in M731T (Table 2).

FIGURE 5.

Rapid kinetic measurements of phosphorylation rate. Phosphorylation was performed for the indicated times at 25 °C in the presence of 2 μm [γ-³²P]ATP, 100 mm NaCl, 40 mm Tris (pH 7.5), 3 mm MgCl₂, 1 mm EGTA, 10 μm ouabain, and 20 μg/ml oligomycin. Each line represents the best fit of a mono-exponential “rise to max function,” giving the indicated rate constants. The wild type data are represented in each panel by a dotted blue line.

FIGURE 6.

ATP dependence of phosphorylation rate. Initial rates of phosphorylation, determined as in Fig. 5 at 1, 2, and 5 μm [γ-³²P]ATP, are shown as a function of ATP concentration in double-reciprocal plots. For V_max and K_m values, see Table 2.

E₁P-E₂P Distribution

The phosphorylated form of the Na⁺,K⁺-ATPase exists in E₁P and E₂P states that can be distinguished by their different sensitivities to K⁺ and ADP (Scheme 1). To estimate the extent of each phosphoenzyme pool present at steady state, the dephosphorylation time course was examined following addition of ADP to the phosphoenzyme. A bi-exponential function could be fitted to the data, permitting extraction of a rapid and a slow decay component, reflecting the initial amounts of E₁P and E₂P, respectively (Fig. 7). Table 2 lists the E₁P fraction. Notably, mutant T263M displayed a marked increase in the level of E₁P (to 96% versus 44% for the wild type). The other mutants displayed a less pronounced shift of the E₁P-E₂P distribution toward the E₁P form or were wild type-like.

FIGURE 7.

Distribution of the phosphoenzyme between E₁P and E₂P. Phosphorylation was carried out for 10 s at 0 °C in the presence of 2 μm [γ-³²P]ATP, 20 mm NaCl, 130 mm choline chloride, 20 mm Tris (pH 7.5), 3 mm MgCl₂, 1 mm EGTA, and 10 μm ouabain. Dephosphorylation was followed by addition of a chase solution producing final concentrations of 2.5 mm ADP and 1 mm unlabeled ATP. The dephosphorylation reaction was terminated by acid quenching after the indicated time intervals. A bi-exponential decay function was fitted to the data. The rate constant corresponding to the rapid phase, reflecting the ADP reaction with E₁P, was set to 2 s⁻¹. The fraction of E₁P phosphoenzyme obtained from this analysis is indicated in the panel (also listed in Table 2 with statistics). The wild type data are represented in each panel by a dotted line.

Vanadate and ATP Affinities

Vanadate is an inhibitor that acts as analog of the phosphoryl group in the transition state during E₂P hydrolysis and binds exclusively to the enzyme in the E₂ conformation (28). The properties of the E₂ state were investigated by determining the apparent affinity for vanadate inhibition of ATP hydrolysis (Fig. 8). All the mutants exhibited a reduced apparent affinity for vanadate (Table 1). For R593W, V628M, E700K, M731T, and R834Q, the affinity was extremely low (≥30-fold reduced relative to wild type); in fact, no significant inhibition was seen for E700K within the vanadate concentration range examined (Fig. 8, right panel). A reduced sensitivity to vanadate inhibition can in principle arise from either a lowering of the intrinsic binding affinity of E₂ (possibly reflecting destabilization of the transition state of E₂P hydrolysis) or from a shift of the E₁-E₂ distribution away from the vanadate binding E₂ form in favor of E₁. To examine the mutational effects on the E₁-E₂ distribution, the ATP dependence of ATPase activity was also studied (Fig. 9). Because ATP binds with higher affinity to E₁ than to E₂, an increase in the apparent affinity for ATP upon mutation reflects a shift of the E₁-E₂ distribution toward the E₁ form, whereas a decrease in the affinity for ATP reflects a shift toward the E₂ form. The results summarized in Table 1 show a significant increase of the apparent ATP affinity for all the mutants (2–5-fold), indicating a shift toward the E₁ form. However, for R593W, V628M, E700K, M731T, and R834Q, the effect on ATP affinity was much less spectacular than the reduction of vanadate affinity, indicating that the latter arises not only from a shift in E₁-E₂ distribution but also from a change in the intrinsic affinity of the E₂ form for vanadate.

FIGURE 8.

Vanadate dependence of Na⁺,K⁺-ATPase activity. ATPase activity was measured at 37 °C in 130 mm NaCl, 20 mm KCl, 3 mm MgCl₂, 3 mm ATP, 30 mm histidine (pH 7.4), 1 mm EGTA, 10 μm ouabain, and vanadate concentrations as indicated. Each line represents the best fit of the equation V = V_max·(1 − [vanadate]ⁿ/(K_0.5ⁿ + [vanadate]ⁿ)), with n ranging between 0.9 and 1.1, giving the K_0.5 values indicated in Table 1.

FIGURE 9.

ATP dependence of Na⁺,K⁺-ATPase activity. ATPase activity was measured at 37 °C in the presence of 130 mm NaCl, 20 mm KCl, 3 mm MgCl₂, 30 mm histidine (pH 7.4), 1 mm EGTA, 10 μm ouabain, and the indicated concentrations of ATP. K_0.5 values for activation are listed in Table 1.

DISCUSSION

The nine FHM2 mutations studied here, which are found in various regions of the Na⁺,K⁺-ATPase α2-isoform, lead to a functionally altered but active enzyme capable of sustaining cell viability. Our results do not lend support to the hypothesis that a reduced affinity for external K⁺, causing a selective disturbance of K⁺ clearance, is an obligatory part of the mechanism underlying the disease (4). Neither can a defective Na⁺ interaction generally account for the pathophysiology of the disease, as only R834Q exhibited a significant reduction of the Na⁺ affinity. This is contrary to the neurological disorder rapid-onset dystonia parkinsonism, caused by mutation of the α3-isoform of Na⁺,K⁺-ATPase, where all mutants characterized so far display markedly reduced Na⁺ affinity (21, 29, 30). At saturating Na⁺ and K⁺ concentrations, the FHM2 mutants studied here display a reduced catalytic turnover rate relative to wild type. A reduced turnover rate has previously been reported for mutants T263M, T345A, M731T, and R834Q, but the responsible change(s) in partial reaction step(s) were not elucidated (4, 9, 10). Our rapid kinetic studies of the phosphorylation from ATP identify a reduced V_max of phosphorylation as a major factor contributing to the reduction of the catalytic turnover rate of mutants V138A, T345A, R593W, V628M, M731T, and R834Q. These mutants displayed decreases in the V_max of phosphorylation of 2–6-fold (Fig. 6 and Table 2). To be able to carry out transient kinetic measurements, oligomycin was added to promote phosphorylation by stabilizing the Na⁺-occluded E₁[Na₃] form and blocking the E₁P[Na₃] → E₂P transition. In the absence of oligomycin, the relative level of phosphoenzyme at steady state was in fact very low for R593W, V628M, M731T, and R834Q (Table 2, “EP/EP_oligo”), suggesting that under physiological conditions without oligomycin the phosphorylation rate is even more dramatically affected than judged from the kinetic data obtained with oligomycin.

The low phosphorylation rate also accounts for the enhanced K⁺ inhibition of ATPase activity at high K⁺ concentrations seen particularly for R593W, V628M, M731T, and R834Q (Fig. 2). Hence, the low phosphorylation rate leads to increased availability of E₁ at steady state and consequently to enhanced K⁺ competition with Na⁺ at the intracellular E₁ sites. In the living cell with cytoplasmic concentrations of ∼150 mm K⁺ and only ∼10 mm Na⁺, the enhanced K⁺ competition with Na⁺ may be highly relevant as a factor contributing to compromise pump function.

For R593W, V628M, E700K, M731T, and R834Q, the apparent affinity for vanadate was lowered to an extent that could not be accounted for by the observed shift of the conformational equilibrium toward E₁ (Fig. 8 and Table 1). Because vanadate binds at the phosphorylation site of the E₂ form as an analog of the phosphoryl group in the transition state between E₂ and E₂P, the extraordinary low affinity for vanadate might be related to a perturbation of the phosphorylation site in E₂ and/or the transition state. For the mutants showing both a reduced V_max of phosphorylation from ATP in E₁ and an extraordinary low affinity for vanadate (R593W, V628M, M731T, and R834Q), the underlying structural changes in E₁ and E₂ forms might be similar. By contrast E700K did not show a reduced phosphorylation rate with ATP, and for this mutant the phosphorylation site therefore seems not to be perturbed in E₁. Besides its extraordinary lack of sensitivity to vanadate, E700K was characterized by a relatively low maximal rate of dephosphorylation of E₂P (42 s⁻¹ versus 132 s⁻¹ for wild type), which probably causes the reduced catalytic turnover rate of E700K. Both the disruption of vanadate binding and the reduced dephosphorylation rate can be explained by assuming that the transition state of E₂P dephosphorylation is destabilized. It is furthermore of note that E700K exhibited an increased apparent affinity for ouabain, and even though E700K did not show reduced affinity for K⁺ activation of dephosphorylation (Fig. 3 and Table 2), a slight 1.6-fold reduction of apparent K⁺ affinity in activation of ATPase activity was noted (Fig. 2 and Table 1). Both of these effects might result from accumulation at steady state of the E₂P[Na₂] phosphoenzyme form, which is an intermediate in the E₁P[Na₃] → E₂P transition (31) exhibiting a particularly high affinity for cardiotonic steroids (“E*P” cf. Refs 32 and 33). Thus, the evidence indicates that mutation E700K is most disturbing in the part of the pump cycle involving E₂P and E₂P-like states.

From the crystal structure it is known how the residues studied here are positioned in the E₂[K₂] state with bound MgF₄²⁻ as a phosphate analog (18, 19), and although crystal structures are static snapshots without the flexibility of the native enzyme, they are useful as a basis for analysis of the mechanisms underlying the observed mutational effects. Arg⁵⁹³, Val⁶²⁸, Glu⁷⁰⁰, and Met⁷³¹ are located in the Rossmann fold, which is a central feature of the P-domain, consisting of a seven-stranded parallel β-sheet associated with seven short α-helices (P1–P7) as indicated in Fig. 10. Asp³⁷⁴ (phosphorylation site) and residues involved in Mg²⁺ binding, Thr³⁷⁶, Asp⁷¹⁴, and Asp⁷¹⁸, are located centrally in the Rossmann fold (Fig. 10). The phosphorylation of Asp³⁷⁴ is triggered by an approach between the N- and P-domains associated with a bending of the P-domain. This conformational change is the result of transmission of the effect of Mg²⁺ binding to the P7 helix of the Rossmann fold (34, 35), meaning that the loop between β6 and P7, where Met⁷³¹ is located (Figs. 10 and 11), has to be strained when Mg²⁺ binds to Asp⁷¹⁴. The role of Met⁷³¹ may thus be to reduce the freedom of movement of this loop, thereby ensuring the strain. In fact the side chain of Met⁷³¹ is flanked on one side by Asp⁷¹⁴, Thr³⁷⁸, and Arg⁵⁹³ (Fig. 10B), and the introduction of the threonine side chain in M731T likely pushes Asp⁷¹⁴, thus preventing a proper bending of the P-domain. It is interesting to note that M731T and R593W are the mutations giving the largest reduction of phosphorylation rate observed in this study (cf. Table 2). Considering that the Met⁷³¹ and Arg⁵⁹³ side chains are located within a distance of only 4–5 Å from each other in the crystal structure (Fig. 10, A and B), it is possible that the bulky tryptophan in R593W actually interferes by disturbing Met⁷³¹. Furthermore, Arg⁵⁹³ is within hydrogen bonding distance of the backbone carbonyls of Gly³⁷⁷ and Thr³⁷⁸, which are located in the same loop as Asp³⁷⁴ and Thr³⁷⁶ (Fig. 10, A and B). The clash resulting from insertion of the tryptophan might therefore also disturb phosphorylation by shifting the positions of Asp³⁷⁴ and Thr³⁷⁶. It is noteworthy that mutations T376M and T378N have been found in familial hemiplegic migraine patients and were reported as loss of function mutations based on cell survival studies (14, 36–38).

FIGURE 10.

Location of the FHM2 mutations with relation to the Rossmann fold in the Na⁺,K⁺-ATPase structure. The structure used is the same as in Fig. 1. Side chains corresponding to mutated residues and interaction partners (numbered according to the human α2-isoform) are highlighted as sticks, as are key residues in the catalytic site as follows: Thr³⁷⁶, Asp⁷¹⁴, and Asp⁷¹⁸ (Mg²⁺-binding residues), and Asp³⁷⁴ (phosphorylation site). Atoms are colored according to the elements (carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow). The catalytic Mg²⁺ ion is shown as a purple sphere. A, Rossmann fold of the P-domain viewed from the cytosol. P1–P7 helices with interposed β-strands (blue arrows with yellow numbers 1–7) are indicated. Note the proximity of Arg⁵⁹³ and Met⁷³¹ to Gly³⁷⁷ and Thr³⁷⁸, and the position of Glu⁷⁰⁰ between the two positively charged side chains of Arg⁷⁰⁴ and Lys⁷²⁴. B, close up of interaction network around Arg⁵⁹³ and Met⁷³¹. C, close up of hydrophobic/van der Waals interaction network around Val⁶²⁸ (Pro⁵⁹² and Val⁵⁹⁶ also indicated in A).

FIGURE 11.

Side view of the membrane interface of the P-domain. The structure used is the same as in Fig. 1 with the same color codes for P-, A-, N-, and M-domain, and the phosphate analog MgF₄²⁻ (MgF) shown as one green and four cyan spheres. Side chains corresponding to mutated residues and interaction partners (numbered according to the human α2-isoform) are highlighted as sticks, as are key residues in the catalytic site. Atoms are colored according to the elements (carbon, gray; oxygen, red; nitrogen, blue; sulfur, yellow). P-domain helices P1–P3 and P5–P7 as well as the interposed β-strands (yellow numbers) and transmembrane helices M3–M5 are indicated. Note the location of the P1 helix with Val³⁶² interacting with Thr³⁴⁵, and Glu³⁶³ and Ser³⁶⁷ interacting with Arg⁸³⁴. Glu⁷⁶¹ (M5) may also interact with Thr³⁴⁵, depending on the actual rotational state of Thr³⁴⁵. In addition, Glu²⁸⁵ (M3) interacts with Arg⁸³⁴.

The valine replaced in mutant V628M is positioned at the end of the P3 helix in the Rossmann fold. It is part of a hydrophobic/van der Waals interaction network (Fig. 10, A and C), and the interaction with Pro⁵⁹² and Val⁵⁹⁶ at the N-terminal end of the P2 helix may contribute to stabilize the positions of helices P2 and P3. The V628M mutation might lead to a shift of the position of Pro⁵⁹², thereby disturbing the interactions of the adjacent Arg⁵⁹³ with Gly³⁷⁷ and Thr³⁷⁸ mentioned above.

The glutamate replaced in E700K is located in the P5 helix of the Rossmann fold. In the crystal structure of the Na⁺,K⁺-ATPase E₂[K₂] state with bound MgF₄²⁻, Glu⁷⁰⁰ is positioned between two positively charged residues, Arg⁷⁰⁴ (at the end of the P5 helix) and Lys⁷²⁴ (P6 helix), with a distance of 4.6 Å to each (Fig. 10A). Moreover, Lys⁷²⁴ is within hydrogen bonding distance to Gln⁷⁰³ in the P5 helix (2.7 Å). It can be imagined that the extra positive charge introduced between Arg⁷⁰⁴ and Lys⁷²⁴ by E700K leads to a repulsion between the three positive charges disturbing the positioning of P5 and P6. Such a disturbance could lead to a shift of β5 and the loop between β5 and P6 containing Asp⁷¹⁴ and Asp⁷¹⁸ critical to Mg²⁺ binding (Fig. 10A). We wondered whether Glu⁷⁰⁰ and either Lys⁷²⁴ or Arg⁷⁰⁴ might actually be closer to each other in the vanadate-bound E₂ state than the 4.6 Å in the E₂[K₂] crystal structure. The Na⁺,K⁺-ATPase residues Glu⁷⁰⁰, Lys⁷²⁴, and Gln⁷⁰³ are conserved as Glu⁶⁸⁹, Lys⁷¹³, and Gln⁶⁹² in the Ca²⁺-ATPase, which has been crystallized not only in the MgF₄²⁻-bound E₂ form similar to the Na⁺,K⁺-ATPase crystal form but also in several other E₂ states, including the AlF₄⁻-bound form. The latter is considered the state closest to the E₂ form with vanadate bound, as AlF₄⁻ and vanadate are both believed to mimic the trigonal bipyramidal structure of the penta-coordinated phosphate in the transition state of E₂P dephosphorylation (39, 40). Interestingly, among the AlF₄⁻-bound E₂ crystal structures of the Ca²⁺-ATPase, 4 out of 7 have a distance between Glu⁶⁸⁹ and Lys⁷¹³ <4 Å, i.e. short enough to indicate the presence of a salt link between these residues in the E₂P transition state. In contrast, in the MgF₄²⁻-bound Ca²⁺-ATPase structures, the corresponding distance is 4–5 Å, consistent with the distance in the MgF₄²⁻-bound form of Na⁺,K⁺-ATPase (cf. supplemental Table S1). If this scenario for the E₂P transition state were extrapolated to the Na⁺,K⁺-ATPase, it would explain the very strong destabilization of the vanadate-bound state of E700K (Fig. 8). If in addition Glu⁷⁰⁰ and Lys⁷²⁴ were further apart in the E₁[Na₃] state, the lack of effect of the mutation on the phosphorylation from ATP would be understandable. The Ca²⁺-ATPase E₁ structures do not provide a clear answer to this question, but it is noteworthy that 4 out of 8 E₁ structures show a distance larger than 4 Å (cf. supplemental Table S1).

T345A in the cytoplasmic extension of M4 and R834Q in the L6–7 loop are both located at the boundary between the P-domain and the transmembrane region, and the reason that these mutations also reduce the phosphorylation rate significantly may be the participation of Thr³⁴⁵ and Arg⁸³⁴ in interaction networks involving helices connected with the Rossmann fold (Fig. 11). Thr³⁴⁵ might participate in van der Waals interactions with Val³⁶² at the N-terminal end of the P-domain helix P1, from which the central β-strand of the Rossmann fold (β-strand “1” in Fig. 11) leads to the phosphorylation site with Asp³⁷⁴. Interestingly, mutation V362E has also been found in patients with FHM2 (41). Depending on the actual rotational state of the Thr³⁴⁵ side chain, the hydroxyl group might form a hydrogen bond with Glu⁷⁶¹ of M5. Mutation R834Q seems to disrupt bonds from P1-helix residues Glu³⁶³ and Ser³⁶⁷ (C-terminal end of the P1-helix) to the L6–7 loop (19). R834Q furthermore disrupts the bond between Arg⁸³⁴ and Glu²⁸⁵ of M3 (Fig. 11), which may explain the reduction of Na⁺ affinity caused by R834Q, since mutation of Glu²⁸⁵ has been shown to reduce Na⁺ affinity, possibly a consequence of the involvement of Glu²⁸⁵ in control of the cytoplasmic entrance pathway for Na⁺ (22).

Surprisingly, the mutation V138A also had significant impact on the phosphorylation rate, even though Val¹³⁸ is positioned in the transmembrane segment M2 far from the phosphorylation site, thus indicating a long range effect, which might be exerted through interference with the hydrophobic/van der Waals interactions between M1 and M2 (cf. Fig. 12A). These interactions seem to allow M1 to close the Na⁺ binding pocket through contact with the ion binding Glu³³² of M4 (42), a conformational change believed to be propagated to the phosphorylation site in a yet undefined way and result in phosphoryl transfer (35). Indeed, a reduced rate of phosphorylation was previously observed following replacement of M1 residues Leu⁹⁴ and Gly⁹⁷ (42, 43).

FIGURE 12.

Structural relationships of residues altered in mutants V138A, R202Q, and T263M. The structure used in A–C is the same as in Fig. 1 with the same color codes for P-, A-, N-, and M-domain. Side chains corresponding to mutated residues and interaction partners (numbered according to the human α2-isoform) are highlighted as sticks. Atoms are colored according to the elements (carbon, gray; oxygen, red; nitrogen, blue). A, Val¹³⁸ in M2 interacts with M1 residues Leu⁹⁴ and Trp¹⁰³. B, interaction between Arg²⁰² and Pro²²⁷ may stabilize the loop containing the conserved TGES motif with Glu²¹⁹. C, Thr²⁶³ is located in the AM3 linker segment connecting the A-domain to the M3 transmembrane segment (cf. Fig. 1). In the E₂ and E₂P conformations, part of the AM3 linker is coiled up, forming an α-helical arrangement, which is partially unwound at Thr²⁶³, with the two parts of the α-helix being almost orthogonal to each other. The side chain hydroxyl group of the threonine is within hydrogen bonding distance to the backbone amide nitrogen of Gly²⁶⁶. D, homology model of the Na⁺,K⁺-ATPase in E₁ based on the structure of the closely related Ca²⁺-ATPase in E₁ (Protein Data Bank code 1T5T). The helical structure of the AM3 linker is much more unwound than in E₂ conformations of Na⁺,K⁺-ATPase and Ca²⁺-ATPase, with less strict requirement for the bond between Thr²⁶³ and Gly²⁶⁶.

For R202Q and T263M, the phosphorylation rate was wild type-like, and the reason for the reduced catalytic turnover rate seems to be a slow conversion of E₁P to E₂P, in particular T263M showed a conspicuous accumulation of E₁P. Furthermore, both R202Q and T263M appeared to shift the E₁-E₂ distribution of the dephosphoenzyme in favor of E₁. These mutations are both associated with the A-domain, which undergoes drastic structural rearrangements during the E₁-E₂ and E₁P-E₂P transitions (35, 44). Arg²⁰² is positioned in a β-strand of the A-domain, where it appears to form a hydrogen bond with the backbone carbonyl oxygen of Pro²²⁷ (2.7 Å distance, Fig. 12B). This bond could be important for stabilization of the loop containing the conserved TGES motif, and hence for interaction of TGES with the P-domain in E₂ and E₂P. Thr²⁶³ is located in the AM3 linker segment connecting the A-domain to M3 and appears to be involved in stabilization of the kinked α-helix present in the AM3 linker in E₂, but not in E₁ (see Fig. 12, C and D). Hence, in E₂ the side chain hydroxyl group of the threonine seems to form a hydrogen bond with the backbone amide nitrogen of Gly²⁶⁶ (2.8 Å distance). This structural arrangement is likely destabilized by the T263M substitution due to the bulkiness of the methionine side chain. The ability of the E₁ conformation to better accommodate the methionine, due to a looser structure compared with the helical arrangement in E₂ (Fig. 12D), explains that the conformational equilibrium is shifted toward E₁ in the mutant. In line with this interpretation, mutation of Gly²⁶⁶ to alanine, disturbing the kinked helix arrangement, was previously shown to cause accumulation of E₁ (45).

To summarize, if disturbance of K⁺ clearance by glial cells is the reason for development of FHM2, this disturbance must be attributed to a low maximum turnover rate of the Na⁺,K⁺-ATPase and not to a reduced affinity for external K⁺. In several of the FHM2 mutants studied here, the function of the catalytic site in phosphorylation (V138A, T345A, R593W, V628M, M731T, and R834Q) or dephosphorylation (E700K) is affected, involving local effects on the catalytic assembly as well as helices connected with the Rossmann fold, and long range effects transmitted from as far away as the membrane domain. The last two mutations (R202Q and T263M) affect the maximum turnover rate by destabilizing the A-domain in the E₂/E₂P conformations.