Background: 

Familial hypokalemic periodic paralysis (hypoPP) is an inherited muscle disease characterized by episodic attacks of muscle paralysis. These episodes go along with an increased shift of K⁺ into muscle. Paralysis is due to muscle inexcitability, caused by fiber depolarization to membrane potentials at which voltage-gated sodium channels are inactivated. Mutations have been described in voltage-sensing domains of voltage-gated sodium (NaV1.4) or calcium channels. It has been shown, that the mutations in NaV1.4 create a cation leak, which has been suggested to be the primary cause of the disease. Periodicity seems to be based on the bistability of the resting membrane potential. Healthy muscle fibers have two stable resting membrane potentials at low extracellular potassium concentrations ([K⁺]e). It has been suggested that cation leaks shift the region of bistability to higher [K⁺]e. In this study we have tested the effect of a pharmacologically caused leak on the resting membrane potential at decreasing [K+]e. The ionophore amphothericin B (AMB) was chosen, since hypokalemic weakness is a side effect of this antifungal drug. Additionally, we have investigated the effect of acetazolamide, the most widely used medication for treating hypoPP.

Methods: 

The resting membrane potentials of rat diaphragm preparations were measured with glass microelectrodes of 10 – 15 MO filled with 3 M KCl. Muscles were incubated in a Krebs-Ringer solution with different [K⁺]e (0 – 6 mM) in which the sum of Na⁺ and K⁺ concentrations was kept constant. Drugs used were AMB (10 mM) and acetazolamide (100 mM). Control solutions contained the same amounts of solvent. Histograms of membrane potentials were smoothed by density estimation. On these density estimates a bimodal probability density function was fitted. The resulting distribution of potentials between the two populations of membrane potentials was further analyzed.Results:

We found two populations of membrane potentials in control and AMB treated muscle. The first population had a well polarized membrane potential between -70 and -100 mV, while the other was depolarized to about -60 mV. At lower [K⁺]e the fraction of depolarized fibers was increased. [K⁺]e at which 50% of fibers were depolarized was higher for AMB treated muscles compared to controls (1.5 mM vs. 2.7 mM). Acetazolamide prevented most AMB-treated fibers from depolarization (60% at [K⁺]e = 0 mM), but had no effect on controls.

Conclusions: 

A cation leak destabilizes the resting membrane potential at low [K+]e and thus increases the number of depolarized fibers. Since these fibers are less excitable, it leads to muscle weakness. This study confirms the hypothesis that a cation leak is essential for the pathogenesis of hypoPP. Additionally, AMB treated muscle can be used as a pharmacological model to study the pathogenesis of hypoPP and the mode of action of currently used medications and potential new drugs.