Werner Melzer

A mutation in the GABAA receptor α1-subunit is associated with absence epilepsy

To detect mutations in GABRA1 in idiopathic generalized epilepsy.

Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor α1 subunits (R528H)

Rabbit cDNA of the α1 subunit of the skeletal muscle dihydropyridine (DHP) receptor was functionally expressed in a muscular dysgenesis mouse (mdg) cell line, GLT. L‐type calcium currents and transients were recorded for the wild type and a mutant α1 subunit carrying an R528H substitution in the supposed voltage sensor of the second channel domain that is linked to a human disease, hypokalemic periodic paralysis. L‐type channels expressed in GLT myotubes exhibited currents similar to those described for primary cultured mdg cells injected with rabbit wild type cDNA, indicating this system to be useful for functional studies of heterologous DHP receptors. Voltage dependence and kinetics of activation and inactivation of L‐type calcium currents from mutant and wild type channels did not differ significantly. Intracellular calcium release activation measured by fura‐2 microfluorimetry was not grossly altered by the mutation either. Analogous measurements on myotubes of three human R528H carriers revealed calcium transients comparable to controls while the voltage dependence of both activation and inactivation of the L‐type current showed a shift to more negative potentials of approximately 6 mV. Similar effects on the voltage dependence of the fast T‐type current and changes in the expression level of the third‐type calcium current point to factors not primarily associated with the mutation perhaps participating in disease pathogenesis.

Skeletal muscle DHP receptor mutations alter calcium currents in human hypokalaemic periodic paralysis myotubes.

1. Mutations in the gene encoding the alpha 1‐subunit of the skeletal muscle dihydropyridine (DHP) receptor are responsible for familial hypokalaemic periodic paralysis (HypoPP), an autosomal dominant muscle disease. We investigated myotubes cultured from muscle of patients with arginine‐to‐histidine substitutions in putative voltage sensors, IIS4 (R528H) and IVS4 (R1239H), of the DHP receptor alpha 1‐subunit. 2. Analysis of the messenger ribonucleic acid (mRNA) in the myotubes from such patients indicated transcription from both the normal and mutant genes. 3. In control myotubes, the existence of the slow L‐type current and of two rapidly activating and inactivating calcium current components (T‐type with a maximum at about ‐20 mV and ‘third type’ with a maximum at +10 to +20 mV) was confirmed. In the myotubes from patients with either mutation, the third‐type current component was seen more frequently and, on average, with larger amplitude. 4. In myotubes with the IVS4 mutation (R1239H) the maximum L‐type current density was smaller than control (‐0.53 +/‐ 0.31 vs. ‐1.41 +/‐ 0.71 pA pF‐1). The voltage dependence of activation was normal, and hyperpolarizing prepulses to ‐120 mV for 20 s did not increase the reduced current amplitude during test pulses. 5. In myotubes with the IIS4 mutation (R528H) the L‐type current‐voltage relation, determined at a holding potential of ‐90 mV, was normal. However, the voltage dependence of inactivation was shifted by about 40 mV to more negative potentials (voltage at half‐maximum inactivation, V1/2 = ‐41.5 +/‐ 8.2 vs. ‐4.9 +/‐ 4.3 mV in normal controls).(ABSTRACT TRUNCATED AT 250 WORDS)

Malignant hyperthermia mutation Arg615Cys in the porcine ryanodine receptor alters voltage dependence of Ca2+ release

1 Ca2+ inward current and fura‐2 Ca2+ transients were simultaneously recorded in porcine myotubes. Myotubes from normal pigs and cells from specimens homozygous for the Arg615Cys (malignant hyperthermia) mutation of the skeletal muscle ryanodine receptor RyR1 were investigated. We addressed the question whether this mutation alters the voltage dependence of Ca2+ release from the sarcoplasmic reticulum. 2 The time course of the total flux of Ca2+ into the myoplasm was estimated. Analysis showed that the largest input Ca2+ flux occurred immediately after depolarization. Amplitude and time course of the Ca2+ flux at large depolarizations were not significantly different in the Arg615Cys myotubes. 3 Ca2+ release from the sarcoplasmic reticulum was activated at more negative potentials than the L‐type Ca2+ conductance. In the controls, the potentials for half‐maximal activation (V½) were ‐9.0 mV and 16.5 mV, respectively. 4 In myotubes expressing the Arg615Cys mutation, Ca2+ release was activated at significantly lower depolarizing potentials (V½= ‐23.5 mV) than in control myotubes. In contrast, V½ of conductance activation (13.5 mV) was not significantly different from controls. 5 The specific shift in the voltage dependence of Ca2+ release caused by this mutation can be well described by altering a voltage‐independent reaction of the ryanodine receptor that is coupled to the voltage‐dependent transitions of the L‐type Ca2+ channel.

A retrograde signal from RyR1 alters DHP receptor inactivation and limits window Ca2+ release in muscle fibers of Y522S RyR1 knock-in mice

Malignant hyperthermia (MH) is a life-threatening hypermetabolic condition caused by dysfunctional Ca2+ homeostasis in skeletal muscle, which primarily originates from genetic alterations in the Ca2+ release channel (ryanodine receptor, RyR1) of the sarcoplasmic reticulum (SR). Owing to its physical interaction with the dihydropyridine receptor (DHPR), RyR1 is controlled by the electrical potential across the transverse tubular (TT) membrane. The DHPR exhibits both voltage-dependent activation and inactivation. Here we determined the impact of an MH mutation in RyR1 (Y522S) on these processes in adult muscle fibers isolated from heterozygous RyR1Y522S-knock-in mice. The voltage dependence of DHPR-triggered Ca2+ release flux was left-shifted by ≈8 mV. As a consequence, the voltage window for steady-state Ca2+ release extended to more negative holding potentials in muscle fibers of the RyR1Y522S-mice. A rise in temperature from 20° to 30 °C caused a further shift to more negative potentials of this window (by ≈20 mV). The activation of the DHPR-mediated Ca2+ current was minimally changed by the mutation. However, surprisingly, the voltage dependence of steady-state inactivation of DHPR-mediated calcium conductance and release were also shifted by ≈10 mV to more negative potentials, indicating a retrograde action of the RyR1 mutation on DHPR inactivation that limits window Ca2+ release. This effect serves as a compensatory response to the lowered voltage threshold for Ca2+ release caused by the Y522S mutation and represents a novel mechanism to counteract excessive Ca2+ leak and store depletion in MH-susceptible muscle.

Expression and functional characterization of the cardiac L-type calcium channel carrying a skeletal muscle DHP-receptor mutation causing hypokalaemic periodic paralysis

A histidine substitution for the outermost arginine in II/S4 of the α1 subunit of the human skeletal muscle dihydropyridine (DHP) receptor has been reported to cause hypokalaemic periodic paralysis (HypoPP). This mutation shifts the voltage dependence of L-type Ca current inactivation in myotubes from HypoPP patients by −40 mV without affecting activation. Based on the strong homology of II/S4 in cardiac and skeletal muscle α1, we introduced the corresponding mutation into the rabbit cardiac α1 subunit (R650H). Wild type (WT) and mutant constructs were transiently transfected in HEK cells together with β and α2δ subunits and Ca and Ba currents were studied using the whole-cell patch-clamp technique. In contrast to the results obtained from human myotubes, R650H produced a small (−5 mV) but significant shift of both the steady-state activation and inactivation curves. When external pH was increased from 7.4 to 8.4 in order to favour deprotonization of H650, the only difference between WT and mutant channels was a slightly reduced steepness of the inactivation curve. Additional cotransfection of the γ subunit which is only found in skeletal but not in heart muscle, shifted the inactivation curves of both WT and R650H by −20 mV. We conclude that R650 plays a different role in voltage-dependent gating of the cardiac L-type Ca channel than the corresponding residue in the human skeletal muscle L-type channel, since a distinct and selective effect on the midpoint voltage of steady-state inactivation could not be found for R650H.

Muscle weakness in Ryr1I4895T/WT knock-in mice as a result of reduced ryanodine receptor Ca2+ ion permeation and release from the sarcoplasmic reticulum

The type 1 isoform of the ryanodine receptor (RYR1) is the Ca2+ release channel of the sarcoplasmic reticulum (SR) that is activated during skeletal muscle excitation–contraction (EC) coupling. Mutations in the RYR1 gene cause several rare inherited skeletal muscle disorders, including malignant hyperthermia and central core disease (CCD). The human RYR1I4898T mutation is one of the most common CCD mutations. To elucidate the mechanism by which RYR1 function is altered by this mutation, we characterized in vivo muscle strength, EC coupling, SR Ca2+ content, and RYR1 Ca2+ release channel function using adult heterozygous Ryr1I4895T/+ knock-in mice (IT/+). Compared with age-matched wild-type (WT) mice, IT/+ mice exhibited significantly reduced upper body and grip strength. In spite of normal total SR Ca2+ content, both electrically evoked and 4-chloro-m-cresol–induced Ca2+ release were significantly reduced and slowed in single intact flexor digitorum brevis fibers isolated from 4–6-mo-old IT/+ mice. The sensitivity of the SR Ca2+ release mechanism to activation was not enhanced in fibers of IT/+ mice. Single-channel measurements of purified recombinant channels incorporated in planar lipid bilayers revealed that Ca2+ permeation was abolished for homotetrameric IT channels and significantly reduced for heterotetrameric WT:IT channels. Collectively, these findings indicate that in vivo muscle weakness observed in IT/+ knock-in mice arises from a reduction in the magnitude and rate of RYR1 Ca2+ release during EC coupling that results from the mutation producing a dominant-negative suppression of RYR1 channel Ca2+ ion permeation.

Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin-mediated glucose uptake in adult skeletal muscle cells

The involvement of Ca2+ in the insulin‐mediated signaling cascade, resulting in glucose uptake in skeletal muscle, is uncertain. Here, we test the hypothesis that Ca2+ influx through canonical transient receptor potential 3 (TRPC3) channels modulates insulin‐mediated glucose uptake in adult skeletal muscle. Experiments were performed on adult skeletal muscle cells of wild‐type (WT) and obese, insulin‐resistant ob/ob mice. Application of the diacylglycerol analog 1‐oleyl‐2‐acetyl‐sn‐glycerol (OAG) induced a nonselective cation current, which was inhibited by the addition of anti‐TRPC3 antibody in the patch pipette and smaller in ob/ob than in WT cells. Knockdown of TRPC3, using a novel technique based on small interfering RNA (siRNA) coupled to functionalized carbon nanotubes, resulted in pronounced (~70%) decreases in OAG‐induced Ca2+ influx and insulin‐mediated glucose uptake. TRPC3 and the insulin‐sensitive glucose transporter 4 (GLUT4) coimmunoprecipitated, and immunofluorescence staining showed that they were colocalized in the proximity of the transverse tubular system, which is the predominant site of insulin‐mediated glucose transport in skeletal muscle. In conclusion, our results indicate that TRPC3 interacts functionally and physically with GLUT4, and Ca2+ influx through TRPC3 modulates insulin‐mediated glucose uptake. Thus, TRPC3 is a potential target for treatment of insulin‐resistant conditions.—Lanner, J.T., Bruton, J.D., Assefaw‐Redda, Y.,Andronache, Z., Zhang, S.‐J., Severa, D., Zhang, Z.‐B., Melzer, W., Zhang, S.‐L., Katz, A., Westerblad, H. Knockdown of TRPC3 with siRNA coupled to carbon nanotubes results in decreased insulin‐mediated glucose uptake in adult skeletal muscle cells. FASEB J. 23, 1728–1738 (2009)

Calcium transients in developing mouse skeletal muscle fibres

Ca2+ transients elicited by action potentials were measured using MagFluo‐4, at 20–22°C, in intact muscle fibres enzymatically dissociated from mice of different ages (7, 10, 15 and 42 days). The rise time of the transient (time from 10 to 90% of the peak) was 2.4 and 1.1 ms in fibres of 7‐ and 42‐day‐old mice, respectively. The decay of the transient was described by a double exponential function, with time constants of 1.8 and 16.4 ms in adult, and of 4.6 and 105 ms in 7‐day‐old animals. The fractional recovery of the transient peak amplitude after 10 ms, F2(10)/F1, determined using twin pulses, was 0.53 for adult fibres and ranged between 0.03 and 0.60 in fibres of 7‐day‐old animals This large variance may indicate differences in the extent of inactivation of Ca2+ release, possibly related to the difference in ryanodine receptor composition between young and old fibres. At the 7 and 10 day stages, fibres responded to Ca2+‐free solutions with a larger decrease in the transient peak amplitude (25%versus 11% in adult fibres), possibly indicating a contribution of Ca2+ influx to the Ca2+ transient in younger animals. Cyclopiazonic acid (1 μm), an inhibitor of the sarcoplasmic reticulum (SR) Ca2+‐ATPase, abolished the Ca2+ transient decay in fibres of 7‐ and 10‐day‐old animals and significantly reduced its rate in older animals. Analysis of the transients with a Ca2+ removal model showed that the results are consistent with a larger relative contribution of the SR Ca2+ pump and a lower expression of myoplasmic Ca2+ buffers in fibres of young versus old animals.

Voltage‐controlled Ca2+ release and entry flux in isolated adult muscle fibres of the mouse

The voltage‐activated fluxes of Ca2+ from the sarcoplasmic reticulum (SR) and from the extracellular space were studied in skeletal muscle fibres of adult mice. Single fibres of the interosseus muscle were enzymatically isolated and voltage clamped using a two‐electrode technique. The fibres were perfused from the current‐passing micropipette with a solution containing 15 mm EGTA and 0.2 mm of either fura‐2 or the faster, lower affinity indicator fura‐FF. Electrical recordings in parallel with the fluorescence measurements allowed the estimation of intramembrane gating charge movements and transmembrane Ca2+ inward current exhibiting half‐maximal activation at −7.60 ± 1.29 and 3.0 ± 1.44 mV, respectively. The rate of Ca2+ release from the SR was calculated after fitting the relaxation phases of fluorescence ratio signals with a kinetic model to quantify overall Ca2+ removal. Results obtained with the two indicators were similar. Ca2+ release was 2–3 orders of magnitude larger than the flux carried by the L‐type Ca2+ current. At maximal depolarization (+50 mV), release flux peaked at about 3 ms after the onset of the voltage pulse and then decayed in two distinct phases. The slower phase, most likely resulting from SR depletion, indicated a decrease in lumenal Ca2+ content by about 80% within 100 ms. Unlike in frog fibres, the kinetics of the rapid phase of decay showed no dependence on the filling state of the SR and the results provide little evidence for a substantial increase of SR permeability on depletion. The approach described here promises insight into excitation–contraction coupling in future studies of genetically altered mice.