Calcium currents and transients of native and heterologously expressed mutant skeletal muscle DHP receptor alpha1 subunits (R528H)

Rabbit cDNA of the alpha1 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 alpha1 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.

Role in fast inactivation of conserved amino acids in the IV/S4-S5 loop of the human muscle Na+ channel.

Since it has been shown that point mutations in the S4-S5 loop of the Shaker K+ channel may disrupt fast inactivation, we investigated the role of three conserved amino acids in IV/S4-S5 of the adult human muscle Na+ channel (L1471, S1478, L1482). In contrast to the K+ channel mutations, the analogous substitutions in the Na+ channel (S1478A/C, L1482A) did not substantially affect fast inactivation. Nevertheless, the mutations S1478A/C/Q shifted the voltage dependence of steady-state inactivation; L1471Q and S1478C slowed recovery from inactivation. In contrast, a novel non-conserved IV/S4-S5 mutation causing paramyotonia congenita (F1473S) slowed fast inactivation 2-fold and accelerated recovery from inactivation 5-fold. The results indicate involvement of the IV/ S4-S5 loop of the human muscle Na+ channel in fast inactivation, but different roles for conserved amino acids among Na+ and K+ channels.

Effects of the dihydropyridine receptor subunits gamma and alpha2delta on the kinetics of heterologously expressed L-type Ca2+ channels.

Ba2+ currents through L-type Ca2+ channels were measured in tsA201 cells transiently transfected with expression vectors encoding the dihydropyridine (DHP) receptor subunits alpha1C, beta1a-GFP, alpha2delta and gamma. The subunit effect on channel function was studied by omitting either alpha2delta or gamma from the transfection mixture and analyzing the voltage dependence and kinetics of activation, inactivation and recovery from inactivation. Activation could be described by a single exponential function while the time course of inactivation of the Ba2+ current followed a double exponential function. Progressively longer depolarization led to increasingly slower recovery, indicating the successive occupancy of several inactive states. Activation parameters remained largely unaffected in y-deficient cells whereas the voltage dependence of inactivation was shifted by 16 mV to more positive potentials and the larger one of the two inactivation time constants was increased by one-third. On the other hand, alpha2delta-deficient cells showed decreased current density and slowed activation and inactivation. Recovery from inactivation was significantly slowed by gamma coexpression. This and the effect of the gamma subunit on steady-state inactivation were independent of the presence of alpha2delta. We conclude that y stabilizes L-type Ca2+ channel inactivation in a way similar to certain Ca(2+)-antagonistic drugs. Alpha2delta is not needed for this effect.

Role in fast inactivation of the IV/S4-S5 loop of the human muscle Na+ channel probed by cysteine mutagenesis.

1. In order to investigate the role in fast inactivation of the cytoplasmic S4-S5 loop of the fourth domain (IV/S4-S5) within the alpha-subunit of the adult human muscle Na+ channel, every single amino acid from R1469 to G1486 was substituted by a cysteine and the mutants were studied by functional expression in human embryonic kidney cells (tsA201) using whole-cell patch clamping. Effects following intracellular application of the sulfhydryl reagents MTSET and MTSES on the mutants were investigated. 2. Sixteen of eighteen mutants resulted in the formation of functional channels. For P1480C and N1484C, no Na+ currents could be detected in transfected cells. In the absence of sulfhydryl reagents, F1473C and A1481C slowed fast Na+ channel inactivation by 2- and 1.5-fold, respectively, and L1482C induced a steady-state Na+ current (Iss) of 3% of peak current (Ipeak) (1% for wild-type). 3. Upon application of MTSET and MTSES, changes in fast inactivation gating occurred for most of the mutants. The most dramatic destabilizing effects on fast inactivation were observed for M1476C (9-fold slowing of inactivation; Iss/Ipeak, 3.6%; +15 mV shift in steady-state inactivation; 2- to 3-fold acceleration of recovery from inactivation), A1481C (3-fold; 14%; +20 mV; no change) and F1473C (2.5-fold; 2.4%; +8 mV; 1.5-fold). Less pronounced destabilizing effects were observed for M1477C and L1479C. Strongly stabilizing effects on the inactivated state, that is a 20-30 mV hyperpolarizing shift of the inactivation curve associated with a 3- to 4-fold decrease in the rate of recovery from inactivation, occurred for T1470C, L1471C and A1474C. Almost all effects were independent of the membrane potential; however, A1474C only reacted when cells were depolarized. Significant effects on activation were not observed. 4. We conclude that the IV/S4-S5 loop plays an important role in fast inactivation of the muscle Na+ channel and may contribute to the formation of a receptor for the putative inactivation particle. The effects of sulfhydryl reagents on the various mutations suggest an alpha-helical structure of IV/S4-S5 (up to P1480) with destabilizing effects on inactivation for one cluster of amino acids (1473/76/77/79) and a stabilized inactivation at the opposite side of the helix (1470/71/74).