Identification and functional characterization of Kir2.6 mutations associated with non-familial hypokalemic periodic paralysis

Abstract

Hypokalemic periodic paralysis (hypoKPP) is characterized by episodic flaccid paralysis of muscle and acute hypokalemia during attacks. Familial forms of hypoKPP are predominantly caused by mutations of either voltage-gated Ca(2+) or Na(+) channels. The pathogenic gene mutation in non-familial hypoKPP, consisting mainly of thyrotoxic periodic paralysis (TPP) and sporadic periodic paralysis (SPP), is largely unknown. Recently, mutations in KCNJ18, which encodes a skeletal muscle-specific inwardly rectifying K(+) channel Kir2.6, were reported in some TPP patients. Whether mutations of Kir2.6 occur in other patients with non-familial hypoKPP and how mutations of the channel predispose patients to paralysis are unknown. Here, we report one conserved heterozygous mutation in KCNJ18 in two TPP patients and two separate heterozygous mutations in two SPP patients. These mutations result in V168M, R43C, and A200P amino acid substitution of Kir2.6, respectively. Compared with the wild type channel, whole-cell currents of R43C and V168M mutants were reduced by ∼78 and 43%, respectively. No current was detected for the A200P mutant. Single channel conductance and open probability were reduced for R43C and V168M, respectively. Biotinylation assays showed reduced cell surface abundance for R43C and A200P. All three mutants exerted dominant negative inhibition on wild type Kir2.6 as well as wild type Kir2.1, another Kir channel expressed in the skeletal muscle. Thus, mutations of Kir2.6 are associated with SPP as well as TPP. We suggest that decreased outward K(+) current from hypofunction of Kir2.6 predisposes the sarcolemma to hypokalemia-induced paradoxical depolarization during attacks, which in turn leads to Na(+) channel inactivation and inexcitability of muscles.

FIGURE 1.

KCNJ18 mutations in TPP and SPP patients. A, chromatograms of partial sequences of KCNJ18 showing one heterozygous point mutation with guanine 501 mutated to adenosine (GTG → ATG), leading to a single amino acid substitution from valine to methionine (V168M), was found in two TPP patient (26- and 32-year-old males), one heterozygous point mutation with cytosine 127 mutated to thymidine (CGC → TGC), leading to the substitution of arginine 43 by cysteine (R43C), was found in a SPP patient (24-year-old male), and one heterozygous point mutation with guanine 598 mutated to cytosine (GCC → CCC), leading to alanine 200 to proline (A200P) mutation, was found in another SPP patient (52-year-old male). Bold letters above the nucleotide sequence represent corresponding coding amino acids. Mutated nucleotides are pointed by arrows, and mutated codons are surrounded by a red box. WT and Mu designate wild type allele and mutant allele respectively. B, membrane topology of Kir2.6 shows the relative locations of mutations. C, amino acid sequence alignment of three mutation sites from each of the four members of the inward rectifying K⁺ channels is shown. Mutations are indicated by red rectangles.

FIGURE 2.

Functional characteristics of R43C, V168M, and A200P KCNJ18 mutations. A, configuration of ruptured whole-cell recording, voltage clamp protocol (from −60 to 60 mV with 10-mV increment), and representative currents from Kir2.6- and mock-transfected cells are shown. The bath and pipette solutions contained 30 mm KCl and 20 mm KCl plus 110 mm potassium gluconate, respectively. The activity of K⁺ in potassium gluconate is estimated to be ∼46% that in KCl (based on the result that currents reverse at 0 mV when bath and pipette solutions contain 70 mm KCl and 20 mm KCl plus 110 mm potassium gluconate, respectively; not shown). Thus, under 30 mm KCl in the bath and 20 mm KCl plus 110 mm potassium gluconate in the pipette, the reversal potential for K⁺ (E_k) is estimated at −22 mV. B, protein expression of WT and three mutant (R43C, V168M, A200) GFP-tagged Kir2.6 channels (GFP-Kir2.6) was detected by Western blot. β-Actin was used as a loading control. C, the current-voltage (I-V) relationship curve of WT and mutant Kir2.6 channels are shown. Currents shown are after subtraction of residual currents in the presence of 30 μm Ba²⁺. The portion of outward hump current in this I-V curve was magnified (dotted circle, in the range of holding potential from −30 to 20 mV) to the right. D and E, shown are representative bar graphs of wild type and three mutant Kir2.6 current densities (pA/pF; normalized to the cell surface area) at holding potential −60 mV (D, inward current) or −20 mV (E, outward current) (mean ± S.E., n ≥ 6 for each). The single and double asterisks denote p < 0.05 and p < 0.01, respectively, between wild type and each mutation by unpaired two-tailed Student's t test.

FIGURE 3.

Cell surface abundance of mutant Kir2.6 channels. A, shown is the effect of H118K mutation on wild type and three mutant Kir2.6 channels. Upper, protein expression of wild type and mutant Kir2.6 channels was detected by Western blotting analysis. β-Actin was used as a loading control. Lower, Kir2.6 inward current density at a holding potential −60 mV was measured and is presented as a bar graph (mean ± S.E., n ≥ 6 for each). The asterisk denotes p < 0.01 between H118-Kir2.6 and each double mutant channel. NS denotes not statistically significant. B, shown is the effect of three human Kir2.6 mutations on membrane abundance of channel. HEK cells were transfected with wild type and/or mutant Kir2.6 cDNAs (in μg of cDNA as indicated). 2WT (lane 2) and WT (lane 6) reflect the ratio of protein expression from two or single allele of KCNJ18, respectively. Homozygous (lane 3–5) and heterozygous groups (lane 7–9) (marked by a box) stand for homozygous mutation with two mutant alleles and heterozygous mutation with one mutant allele and one wild type allele, respectively. Lane 1 is a non-biotinylated group for negative control. Lane 10 shows low efficiency of the biotinylation reaction in wild type Kir2.6. Lysate-Kir2.6 and Biotin-Kir2.6 represent Kir2.6 channels in HEK cell lysates and in the eluate from a mixture of HEK cell lysates and streptavidin-agarose beads, respectively. The bar graph at the bottom is the relative surface density of Kir2.6 channel (normalized to lane 6). The mean ± S.E. from four separate experiments is shown on the top of each bar. Asterisks denote p < 0.01 each group versus lane 2. # denotes p < 0.01 in lane 9 versus lane 6. The gel shown is representative of four experiments with similar results. The abundance of each band in the gel was measured by densitometry by the Image J program available at the NIH website.

FIGURE 4.

Single channel properties of wild type and mutant Kir2.6 channels. A, configuration of cell-attached single channel recording of Kir2.6 is shown. B, left, representative tracings are shown of WT (upper), R43C (middle), and V168M (lower) Kir2.6 single channel recording at membrane potential −80 mV. The dotted line indicates channel closed. O and C indicate open and closed state, respectively. Right, shown is an all-point histogram of the representative tracings. Bin width = 0.1 pA. Curve lines indicate fits by the sum of two Gaussian distributions. C, upper, single channel current-voltage (I-V) relationship is shown. Single channel currents were recorded at membrane holding potentials (HP) ranging from −40 to −120 mV. Slope conductances (dotted line) were calculated by linear regression. Lower, shown is a bar graph of single channel conductance. D, upper, open channel probability (P_o) of WT and mutant single channels were analyzed by an amplitude histogram at holding potentials ranging from −40 to −120 mV. Dotted lines were linear regressions of P_o. Lower, shown is a bar graph of open channel probability of WT and mutants at membrane potential −80 mV. Single and double asterisks denote p < 0.05 and p < 0.01, respectively, between wild type and each mutation. NS denotes statistically not significant.

FIGURE 5.

Dominant negative effects of mutant Kir2.6 on wild type Kir2.6. A, B, and C, shown is the dominant negative effect of three individual mutations on wild type Kir2.6 current. WT and mutant pEGFP-Kir2.6 cDNAs (in μg as indicated) were cotransfected into HEK cells to test the dominant negative effect of R43C (A), V168M (B), and A200P (C) on WT Kir2.6 current. Upper, the bar graph shows whole-cell outward (at membrane potential −10 mV) and inward current density (at membrane potential −60 mV) for wild type Kir2.6 and/or mutant Kir2.6 channels (mean ± S.E., n ≥ 6 for each). Lower figures display current-voltage (I-V) relationship curves for each group (mean ± S.E., n ≥ 6 for each). WT/R, WT/V, and WT/A denote the expressed protein ratio of wild type versus R43C, V168M, or A200P mutants, respectively (for example, WT/A = 1/3 indicates the ratio of wild type Kir2.6 channel versus A200P Kir2.6 channel is 1:3). Single and double asterisks denote p < 0.05 and p < 0.01 respectively between indicated groups.

FIGURE 6.

Formation of functional heteromultimers between Kir2.1 and Kir2.6 and disease mutant Kir2.6 exerts dominant negative inhibition on Kir2.1. A and B, currents and I-V relationships of homomeric and heteromeric Kir2.1 and Kir2.6 channels are shown. C, dose-dependent dominant negative effect of A200P Kir2.6 mutant on Kir2.1 is shown. E, the dominant negative effect of disease mutant Kir2.6 on Kir2.1 is shown. Kir2.1 and either wild type Kir 2.6 (A) or A200P Kir2.6 (C) (in μg of cDNA as indicated) were cotransfected in HEK cells. In E, the cDNA ratio of Kir2.1 versus either WT or disease mutant (R43C, V168M, A200P) Kir2.6 are all 1/3 (Kir2.1, 0.15 μg; Kir2.6, 0.45 μg). Upper and lower bar figures represent outward current densities at a pipette holding potential of −10 mV and inward current densities at a pipette holding potential −60 mV, respectively (mean ± S.E., n ≥ 6 for each group). Numbers above or below each bar represent the mean current for each group. Numbers in parentheses in A indicate the absolute value of the ratio between inward current at −60 mV and outward current at −10 mV for each group. The inset in A shows the overlap of normalized current voltage (I-V) relationships curves of Kir 2.1 and wild type Kir2.6. The dotted box in C represents a separate experiment with Kir2.1 and G145A (GA) Kir2.6 cotransfected in HEK cells. B, D, and F, shown are current voltage (I-V) relationships curves of each group in A, C, and E, respectively. 2.1/2.6WT and 2.1/A200P, 2.1/R43C, or 2.1/V168M denote the cDNA ratio (for example, 2.1/2.6 = 1/1 indicates that the same amounts of Kir2.1 and wild type Kir2.6 cDNA were cotransfected), respectively.

FIGURE 7.

Schematic models illustrating reduced outward current of Kir could induce paradoxical depolarization in patients with Kir2.6 mutation. The steady-state current-voltage (I-V) relationship curve for K⁺ current in mammalian skeletal muscle is a combination of I-V curves of inward rectifying K⁺ channel (Kir, red line) and delayed rectifying K⁺ channel (KDR, green line). Leakage current (yellow dotted line) has a reversal potential of 0 mV. Because gating pore currents from mutations of the voltage sensor only occur in hyperpolarized potentials, the inward leak current in familial hypoKPP patients is not linear (not illustrated here). The model is intended for conceptual understanding. The numerical value may be slightly different from the true in vivo value. See “Discussion” and Struyk and Cannon (19) for details. E_k, equilibrium potential of Kir channel; E_r, resting membrane potential; I_o, outward cation current; I_i, inward cation current; I_Kir, current of inward rectifying potassium channel; I_KDR, current of delayed rectifying potassium channel; I_Leak, inward cation leak current; [K]_o, extracellular potassium concentration.