Genetic mutations for magnesium wasting

Background:
In the recent thread that focused on the relevance of Exatest results and in response to the comment by Liz
(quote):
......There is probably no one as dedicated to eating the right diet, supplements and making sure the electrolytes were optimal than Jackie, that wasn't enough, as you probably know she has had 3 ablations, uses the exacta test, that is no cure.
....I’d like to direct Liz and other readers interested in why Exatest’s diagnostic benefit is an important clue for looking further into the role of genetic disorders related to magnesium wasting. A positive finding for wasting would be influential for a variety of ailments including arrhythmia, CV health, muscle function, inflammation, bone health, sleep, mood, anxiety and much more. Long term, it could help ward off other complications of aging from this one deficiency alone.

Magnesium is just one of critical electrolytes. They all are important to measure and ensure optimization through diet, lifestyle and supplements when needed. New afibbers should start with the knowledge that the majority of people are magnesium deficient and Afib is just one symptom of many that signals that deficiency.

It’s important to note that while we emphasize magnesium as the foundational intracellular electrolyte, potassium or lack thereof, is what initiates Afib for many because it shortens the refractory period or time between heartbeats. Therefore, the Exatest becomes a very useful diagnostic assistance.

Magnesium affects the conduction of nerve impulses, muscle contraction and normal heart rhythm through the active transport of ions such as potassium across cell membranes so Mg is a critical requirement, but potassium is the ultimate player for NSR. (Refer to the many posts about using the Cardymeter to monitor potassium levels even after an ablation to help maintain NSR.) I don’t recall reading that RBC testing reflects anything about IC potassium or calcium or the comparative ratios. It’s important to keep in mind, though, that just potassium alone doesn’t correct the problem. A substantial or optimal intracellular magnesium supply is needed before potassium can be effective; in fact, sometimes just adding potassium when magnesium is low causes more arrhythmia.

Long before I had benefit of Exatest results, I learned by trial and error that the calcium-containing bone health support supplements prescribed for post-menopausal women would put me in arrhythmia without fail. When I stopped the calcium, the Afib also stopped for a time but eventually persisted. The initial Exatest indicated I was low in magnesium and potassium and high in calcium. Classic recipe for Afib.

Younger afibbers who have otherwise healthy bodies, not compromised by other health conditions and not taking multiple drugs to manage those, are likely to respond quickly and favorably to consistent attention to electrolyte repletion and dietary improvements that eliminate electrolyte depletors (often called “muggers”). Older afibbers, perhaps ages 50+ will likely find it more of a challenge especially with long-standing, chronic interferences and influences such as poor dietary choices, environmental factors, Rx drugs and, the elusive, genetic mutations that cause various deficiencies. The more drugs prescribed for various ailments, the more mugging, and more difficulty in achieving IC optimization to any degree of consistency or reliability.

For me, and likely others with the Afib tendency, is the potential for a genetic mutation that allows for a variety of electrolyte imbalances including magnesium wasting. Although I have not had the genetic testing, my FM MD and I feel given my history of very serious kidney ailment as a child (glomerulonephritis), and now that I’m in my senior years, may indicate some kidney function impairment-- mild enough not to affect overall renal function, but significant enough to allow for some magnesium and/or potassium wasting which brings about the persistence of the Afib I’ve experienced over the past 20 years. I’ve certainly worked diligently at maintaining optimal electrolyte levels, but even with those heroics, the Exatest showed I was in the low-normal range.

I fully understand and respect the importance of Epigenetic preventive measures so emphasis on the newly-accessible genetic tests such as 23andme should also be among the first investigative tests new Afibbers should use to rule out interferences such as electrolyte wasting.

The following partial list of symptoms/ailments serves to remind that a deficiency in magnesium can produce a variety of ailments. The warning we get as afibbers…which I’ve often called “our Canary in the Coal Mine” …is that something in our body is out of balance and is facilitating arrhythmia. While it’s certainly a significant, attention-getting symptom for Afibbers, it makes sense to know by accurate testing whether or not all of our IC electrolytes are optimized so we can avoid other electrolyte deficiency consequences which may not have such clear-cut warning signals as Afib and before some other condition presents itself, or worse, just smolders behind the scenes doing irreparable damage:

Inflammation (silent)
Arrhythmia, palpitations
Ischemic Heart Disease
Blood clots
Alzheimer’s
Poor memory, confusion
Depression, anxiety
Type II Diabetes
Asthma, respiratory ailments
Hypertension
Headaches, migraines
Low bone density, osteopenia, osteoporosis
Kidney stones
Fatigue, muscle spasms, Charley horse, muscle cramps, muscle weakness, tremors
Alcoholics and those with autism are also found to be deficient in magnesium.

Here’s a report that elaborates more on symptoms of magnesium deficiency. [www.greenmedinfo.com]

Now, the final comment is the following report on one type of genetic influence that produces low magnesium which should encourage new afibbers to have Exatest as well as genetic testing.

Healthy regards,
Jackie


A missense mutation in the Kv1.1 voltage-gated potassium channel–encoding gene KCNA1 is linked to human autosomal dominant hypomagnesemia

[www.jci.org]
Published in Volume 119, Issue 4 (April 1, 2009)
J. Clin. Invest. 119(4): 936-942 (2009). doi:10.1172/JCI36948.
Copyright © 2009, American Society for Clinical Investigation
Research Article


Bob Glaudemans1, Jenny van der Wijst1, Rosana H. Scola2, Paulo J. Lorenzoni2, Angelien Heister3, AnneMiete W. van der Kemp1, Nine V. Knoers3, Joost G. Hoenderop1 and René J. Bindels1

1Department of Physiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.
2Neuromuscular Disorders Division, Clinical Hospital, Parana Federal University, Curitiba, Parana, Brazil.
3Department of Human Genetics, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands.

Address correspondence to: René J. Bindels, 286 Physiology, PO Box 9101, 6500 HB Nijmegen, The Netherlands. Phone: 31-24-3614211; Fax: 31-24-3616413; E-mail: r.bindels@ncmls.ru.nl.

First published March 23, 2009
Received for publication July 28, 2008, and accepted in revised form January 7, 2009.


Primary hypomagnesemia is a heterogeneous group of disorders characterized by renal or intestinal magnesium (Mg2+) wasting, resulting in tetany, cardiac arrhythmias, and seizures. The kidney plays an essential role in maintaining blood Mg2+ levels, with a prominent function for the Mg2+-transporting channel transient receptor potential cation channel, subfamily M, member 6 (TRPM6) in the distal convoluted tubule (DCT). In the DCT, Mg2+ reabsorption is an active transport process primarily driven by the negative potential across the luminal membrane.

Here, we studied a family with isolated autosomal dominant hypomagnesemia and used a positional cloning approach to identify an N255D mutation in KCNA1, a gene encoding the voltage-gated potassium (K+) channel Kv1.1. Kv1.1 was found to be expressed in the kidney, where it colocalized with TRPM6 along the luminal membrane of the DCT. Upon overexpression in a human kidney cell line, patch clamp analysis revealed that the KCNA1 N255D mutation resulted in a nonfunctional channel, with a dominant negative effect on wild-type Kv1.1 channel function. These data suggest that Kv1.1 is a renal K+ channel that establishes a favorable luminal membrane potential in DCT cells to control TRPM6-mediated Mg2+ reabsorption.

Introduction
Occurrence of hypomagnesemia (serum Mg2+ levels below 0.70 mmol/l) in the general population has been estimated to be around 2%, while hospitalized patients are more prone to develop hypomagnesemia (12%) (1). Recent studies of intensive care patients have even estimated frequencies as high as 60% (2). The blood Mg2+ concentration depends on the renal Mg2+ excretion in response to altered uptake by the intestine. Hence, the kidney is essential for the maintenance of the Mg2+ balance (3). The majority of filtered Mg2+ is reabsorbed along the proximal tubule and the thick ascending limb of Henle’s loop via a passive paracellular pathway (4). However, fine-tuning of Mg2+ excretion occurs in the distal convoluted tubule (DCT) in an active transcellular fashion initiated by the Mg2+-permeable transient receptor potential cation channel, subfamily M, member 6 (TRPM6) (5, 6). Since the extra- and intracellular Mg2+ concentrations are both in the millimolar range, it has been hypothesized that the membrane potential across the luminal membrane acts as the primary driving force for Mg2+ entry via TRPM6 (6, 7).

Previously, genetic studies in families with hereditary renal Mg2+ wasting syndromes revealed several new genes involved in Mg2+ homeostasis, including tight junction proteins claudin 16 and 19 (8, 9), the thiazide-sensitive sodium chloride cotransporter (NCC) (10), the γ-subunit of the Na+/K+-ATPase (FXYD2) (11), TRPM6 (12, 13), and the recently discovered magnesiotropic hormone EGF (14). Despite these discoveries, our knowledge of renal Mg2+ handling remains far from complete.

In this study, we screened a Brazilian family with isolated autosomal dominant hypomagnesemia and identified a missense mutation in KCNA1, resulting in nonfunctionality of the encoded voltage-gated potassium channel Kv1.1.

Results
A heterozygous KCNA1 A763G mutation is causative for hypomagnesemia. Here, we identified a large Brazilian family (46 family members, of which 21 are affected) with autosomal dominant hypomagnesemia (Figure 1A). Affected individuals showed low serum Mg2+ levels (<0.40 mmol/l; normal range, 0.70–0.95 mmol/l), while their urinary Mg2+ excretion was normal, suggesting impaired tubular Mg2+ reabsorption. The phenotype of the proband (IV-3, Figure 1A) starting from infancy consists of recurrent muscle cramps, tetanic episodes, tremor, and muscle weakness, especially in distal limbs. An SNP-based linkage analysis identified a 14.3-cM locus on the short arm of chromosome 12 (Figure 1, which was subsequently narrowed down by fine mapping with microsatellite markers to an 11.6-cM region containing 31 genes between the markers D12S1626 and D12S1623 (maximum multipoint lod score, 3.0) (Figure 1B and Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI36948DS1). Other genes previously associated with hypomagnesemia are located outside this critical region and therefore were excluded as candidate genes in our family. From the identified locus, we sequenced KCNA1 and identified a heterozygous mutation, A763G (Figure 1C), in the affected individual III-1 (Figure 1A) that cosegregates with the disorder and was absent in 100 control chromosomes (data not shown). The identified mutation in KCNA1, which encodes the Shaker-related voltage-gated K+ channel Kv1.1, causes substitution of the highly conserved asparagine at position 255 for an aspartic acid (N255D) (Figure 1D). The predicted amino acid topology of Kv channels shows 6 transmembrane-spanning α-helical segments (i.e., S1–S6), with the S4 segment acting as the voltage sensor and a hydrophobic pore region between S5 and S6 (Figure 1E) (15). The newly identified N255D mutation is positioned in the third transmembrane segment (S3) close to the voltage sensor (Figure 1E).

Figure 1
Heterozygous KCNA1 A763G mutation causes isolated hypomagnesemia. (A) Pedigree with 5 generations (I–V) of a Brazilian family with autosomal dominant hypomagnesemia. Affected family members are indicated in black; males and females are indicated by squares and circles, respectively. A diagonal line indicates that the individual is deceased. Numbers in red indicate individuals included in the Sequence Tagged Site (STS) mapping. ( 10K SNP array–based haplotyping analysis was performed that showed linkage to a 14.3-cM region between SNP rs717596 and rs252028 on the short arm of chromosome 12. This region was confirmed and narrowed down with STS markers to a 11.6-cM region between markers D12S1626 and D12S1623 containing 31 genes, including KCNA1. (C) KCNA1 encodes the voltage-gated potassium channel Kv1.1. Mutation analysis of KCNA1 revealed a heterozygous A763G missense mutation in affected individual III-1 that results in a N255D amino acid substitution (underlined). (D) Multiple alignment analysis shows conservation of the N255 amino acid (red bar) among species and Kv1 family members. Mutated amino acids in other families with the Kv1.1 genotype are indicated by dark blue dots (17, 19, 21, 22). Blue and black letters represent conserved and nonconserved amino acids, respectively. (E) Schematic representation of the Kv1.1 channel, which consists of a voltage sensor in transmembrane segment S4 and a pore-forming region (S5 and S6). Localization of the newly identified N255D mutation is denoted by the red dot, while other nearby mutations are indicated by dark blue dots.

Localization of Kv1.1 in the DCT of the kidney. So far, all proteins implicated in familial hypomagnesemia have been shown to be expressed in kidney, underlining the pivotal role of this organ in body Mg2+ homeostasis. To study the (sub)cellular localization of Kv1.1 in kidney, we used a rabbit polyclonal antibody raised against the Kv1.1 channel. Immunopositive staining was observed along the luminal membrane of distinct tubules present in the superficial cortex of the mouse kidney (Figure 2). Using serial kidney sections, we demonstrated that Kv1.1 colocalizes with the epithelial Mg2+ channel TRPM6 in DCT (Figure 2A). To confirm this localization, we costained kidney sections for Kv1.1 and calbindin D28K and found a partial overlap in Kv1.1 and calbindin D28K expression (Figure 2. This pattern can be explained by earlier observations that calbindin D28K is expressed not only in the DCT but also in connecting tubule (CNT) (16). Therefore, these data confirm that Kv1.1 is localized primarily in the DCT. Indeed, costaining between Kv1.1 and aquaporin-2 (AQP2), a marker for CNT and the collecting duct, was not observed (Figure 2C). These findings support the restricted localization of Kv1.1 in the Mg2+-transporting DCT segment of the kidney.

Figure 2
Immunohistochemical analysis of Kv1.1 in kidney. (A) Staining for Kv1.1 (green) and TRPM6 (red) of mouse serial kidney sections (right panels: overview of a cortical region; left panels: magnified images of immunopositive tubules). The asterisks indicate the same distal tubules on serial sections intensively stained for Kv1.1 and TRPM6. ( Mouse kidney sections were costained for Kv1.1 (green) and calbindin D28K (red) (lower panels: immunopositive tubules; upper panel: merged differential interference contrast [DIC] image). (C) Costaining of Kv1.1 (green) and AQP2 (red) in mouse kidney sections (lower panels: immunopositive tubules; upper panel: merged DIC image). G, glomerulus; 28K, calbindin D28K. Scale bars: 50 μm (A, left panels), 80 μm (A, right panels; C, bottom panels), 20 μm (B, top panel), 40 μm (B, bottom panels; C, top panel).

Kv1.1 N255D results in nonfunctional channels with dominant negative effect on wild-type channel function. To determine the effect of the Kv1.1 N255D mutation on channel activity, HEK293 cells were mock transfected or were transiently transfected with wild-type Kv1.1 and/or Kv1.1 N255D. Using the whole-cell patch clamp technique, we measured outward K+ currents by dialyzing the cells with a pipette solution containing 140 mM K+. Cells expressing wild-type Kv1.1 channels produced typical delayed rectifying currents, while Kv1.1 N255D–expressing HEK293 cells showed small currents similar to those of mock plasmid–expressing cells (Figure 3, A and . Considering the autosomal dominant inheritance in our family, we investigated a potential dominant negative effect by cotransfection of equal amounts of plasmid DNA encoding wild-type Kv1.1, Kv1.1 N255D, or mock plasmid in HEK293 cells. The K+ current amplitude in HEK293 cells coexpressing wild-type Kv1.1 and Kv1.1 N255D was significantly reduced compared with that in cells expressing wild-type Kv1.1 alone or coexpressing wild-type Kv1.1 and mock plasmid (P < 0.05) (Figure 3C). Next, the influence of Kv1.1 N255D expression on the amount of Kv1.1 channels at the plasma membrane was examined by cell surface biotinylation experiments. As shown in Figure 3D, coexpression of wild-type Kv1.1 and Kv1.1 N255D in HEK293 cells did not affect the plasma membrane localization of Kv1.1 channels. Of note, Kv1.1 was equally expressed in all conditions as analyzed in the total cell lysates (Figure 3D, bottom panel). As Kv1.1 channels are composed of 4 subunits, this result suggests that similar amounts of both homotetrameric channels of wild-type Kv1.1 or Kv1.1 N255D, and heterotetrameric channels composed of wild-type Kv1.1 with Kv1.1 N255D are located at the plasma membrane.

Continue at the website for the rest of the study and references.