Skeletal Muscle Calcium Channel Mutation R528G: Enhanced Channel Inactivation and Omega- Current at Hyperpolarization Contribute to Hypokalemic Periodic Paralysis

Autosomal dominant inherited hypokalemic periodic paralysis (HypoPP) is caused by S4 voltage sensor mutations in skeletal muscle CaV1.1 calcium or NaV1.4 sodium channels. In the present study, a small German family with the known CaV1.1-R528G is described. The phenotype consists of short and infrequent episodes of limb weakness with ictal respiratory and cardiac involvement. There is incomplete penetrance in women, and acetazolamide is beneficial in two patients also taking daily potassium. Expression of the mutation in the GLT mouse muscle cell line revealed accelerated kinetics of inactivation by twofold, a left-shift of the steady-state inactivation curve by 13mV and a reduced recovery from fast inactivation by up to 39%. These changes suggest a stabilization of the inactivated state. Additional significant slowing of activation may support a second open state with differing ion selectivity or decreased activation of calcium-activated potassium channels and thereby contribute to weakness similar to other CaV1.1 mutations. Also, as documented for other HypoPP mutants, we found a hyperpolarization-induced inward guanidinium current of 22nS/nF which can be interpreted as an omega current along the voltage sensor gating pore that leads to a gainoffunction at potentials near the resting membrane potential. This finding can explain the long-lasting depolarizations that are known to lead to paralysis. The omega current is large enough so that a relatively mild hypokalemic trigger of 2.4mM already produces episodes of weakness in vivo. DOI : 10.14302/issn.2470-5020.jnrt-16-993 Corresponding author: Karin Jurkat-Rott, Email: karin.jurkat-rott@uni-ulm.de, Division of Neurophysiology, Ulm University, Albert-Einstein-Allee 11, 89081 Ulm, Tel:+49 731 50


Introduction
Familial hypokalemic periodic paralysis (HypoPP) is an autosomal dominant disorder of the skeletal muscle. It is characterized by episodes of generalized paralysis caused by reduced serum potassium.
Additional permanent weakness and myopathy occurs in 68% of patients (1). Two genes of voltage-gated cation channels are genetically causative, CACNA1S coding for Ca V 1.1 (HypoPP-1) and SCN4A encoding Na V 1.4 (HypoPP Pathogenetically, a long-lasting membrane depolarization is responsible for the paralysis episodes and has been shown to be caused by an aberrant omega current that flows through the S4 mutation gating pore instead of the central ion-conducting alpha pore of the channel (originally described in 6,7). Depending on the location of the mutated residue, the voltage range in which the omega current occurs differs because S4 segments move outward during channel activation.
Briefly, the more inward the mutation the more depolarized the voltage range; therefore, the outermost mutations conduct omega currents at hyperpolarized potentials when the central alpha pore is closed (reviewed in 2, 3,4). Also, the size of the residue replacing arginine influences the size of the omega current suggesting arginine-to-glycine mutations to generate the largest omega currents and therefore the most severe phenotypes (reviewed in 8).
Even though almost all HypoPP functional studies have been done on Na V 1.4 mutations, Ca V 1.1 mutations are much more abundant and explain up to 77% of patients with hereditary HypoPP (9) with the most frequent being R528H and R1239H (10). It has not been systematically shown that the conclusions drawn for Na V 1.4 are applicable to Ca V 1.1. Until now, there is only circumstantial evidence for omega currents of R1239H in native muscle of patients (11) and measurements of omega current of R528 in native muscle of transgenic mice (12). Additionally, our group detected omega currents using a mouse cell line expressing R1242G-Ca V 1.1 which is, however, associated to normokalemic periodic paralysis with transient compartment-like syndrome (13). For several other mutations in Ca V 1.1, omega currents and alpha pore currents have not yet been studied.
In this work, we study the R528G mutation in domain II of Ca V 1.1 for which neither omega current nor alpha pore current has been examined. It has been reported in a one large HypoPP family (14) and as a de novo mutation in a single patient (15). In this study we found it in a small German family. Based on the current model of pathogenesis, we would expect i) a more severe phenotype, ii) a bad response to acetazolamide and iii) a large omega current in the range of the resting membrane potential as pathogenetic mechanism. We test these hypotheses and additionally examine alpha pore currents to discuss their possible contribution to the phenotype.

Patients :
Clinical and genetic examination was approved of by the institutional review board of Ulm and conducted according to the declaration of Helsinki. Blood was taken with informed consent from the proband, his brother, mother, and aunt. His mother sent blood but did not wish to be examined otherwise. Exons 11 and 30 of CACNA1S were studied for routine diagnosis using Sanger sequencing. Cell Culture and Transfection: Myotubes of the homozygous dysgenic cell line GLT were cultured as previously described (13,16,17). Pulse Protocols for Alpha Currents: To elicit whole-cell Ca 2+ currents, a series of 600ms pulses from -60mV to 50mV in 5mV steps from a holding potential of -90mV were performed. Steadystate activation parameters were determined by fitting the current-voltage relation with the equation I=G leak *(V inactivating 20s prepulse to 30mV was followed by a 300ms test pulse to the same potential. The peak I ca was normalized to the peak current amplitude measured during the prepulse. Test currents were recorded in both WT and R528G cells after recovery intervals ranging from 0.2ms to 247ms at -90mV, -80mV and -70mV. The recovery from fast inactivation was analyzed by fitting the data to a single exponential function, I/I max =A* is the time constant, and C is the level of noninactivating sodium current.

Results
Patients: Alpha Pore Current Activation: Figure 1 shows original traces of representative calcium alpha pore currents recorded from GLT cells expressing WT ( Figure 1A) or R528G ( Figure 1B) Figure 1C). Therefore, the voltage-dependent activation of the mutant was comparable to wildtype. In contrast, there was an indication of a change of the kinetics of activation. Figure 1D shows    Figure 1F, Table 1 Omega Currents: We found a hyperpolarization-induced inward current of the R528G mutant that was significantly higher than that of the wild-type channels and untransfected cells (Figure 2A Figure 2B).  (1,5,10,11,14,15,18,19,20 when combined with daily potassium (our family), but not when administered alone as prophylactic measure (15). The previous studies suggesting that patients with the arginine-to-glycine mutation do not experience any   (12). Because of the different expression systems used, we cannot directly conclude whether the omega current of R528G is actually larger or smaller than that of R528H, however the general magnitude range is similar.

Conclusion
Our data did not confirm the three starting hypotheses because i) the phenotype was not clearly more severe for R528G than for R528H, ii) acetazolamide ameliorated symptoms in combination with daily potassium administration and iii) the omega current for R528G was not larger than for R528H.
However, our data confirms that a large omega current is present in the resting potential range which is in agreement with current pathogenetic models of HypoPP (8).