Small-conductance calcium-activated potassium currents in mouse hyperexcitable denervated skeletal muscle.

Denervation induces profound changes in the organization of skeletal muscle, reflecting orchestrated alterations in gene expression, including those encoding ion channels. Expression of different ion channels may be increased or decreased, including for example expression of different acetylcholine receptor isoforms as well as redistribution of the receptors, changes in Na+ channel isoforms, decreased expression of Cl− and inward rectifier K+ channels, and increased expression of a small-conductance Ca2+-activated K+ (SK) channel (Hartzell & Fambrough, 1972; Rogart & Regan, 1985; Mishina et al. 1986; Heathcote, 1989; Brenner et al. 1990; Gonoi & Hasegawa, 1991; Lupa & Caldwell, 1994; Pribnow et al. 1999). The altered ion channel expression in denervation results in a decrease in the resting membrane potential and an increase in membrane excitability (Tower, 1939; Albuquerque & Thesleff, 1968; Robbins, 1977). Skeletal muscle hyperexcitability may result from many different imbalances. In two cases, denervation and the inherited disorder myotonic muscular dystrophy, SK channel expression is implicated in the genesis of the hyperexcitability. Electromyographic recordings have shown that the selective SK channel inhibitor, apamin, suppresses the hyperexcitability in rat denervated skeletal muscle (Vergara et al. 1993) and the myotonic runs in the thenar muscle of patients with myotonic muscular dystrophy (Behrens et al. 1994). These results suggest that SK channels play an important role in the hyperexcitability of skeletal muscle. This finding presents an apparent paradox: how might expression of a hyperpolarizing K+ channel, which normally suppresses excitability, result in hyperexcitability? SK channels were first recorded from cultured skeletal muscle myotubes and are selectively blocked by the bee venom peptide toxin, apamin (Blatz & Magleby, 1986). Several reports have indicated that there is increased expression of SK channels in denervated muscle and myotonic dystrophy (Barrett et al. 1981; Schmid-Antomarchi et al. 1985; Blatz & Magleby, 1987; Kimura et al. 2000). We have previously shown that mRNA encoding SK3 is induced upon denervation in skeletal muscle (Pribnow et al. 1999). SK3 protein levels, as measured by 125I-apamin binding, were also increased after denervation but with about a 24 h delay compared with the increase in mRNA levels (Pribnow et al. 1999). SK channels are probably tetrameric assemblies of subunits that share the transmembrane topology of voltage-gated K+ channels, having six membrane-spanning domains with the N- and C-termini residing within the cell (Kohler et al. 1996). However, SK channels are not voltage activated, being gated solely by intracellular Ca2+ ions. Ca2+ gating of SK channels is accomplished through a constitutive association of calmodulin with the C-terminal portion of the channel (Ishii et al. 1997; Xia et al. 1998; Keen et al. 1999). SK channels are activated by submicromolar concentrations of Ca2+ with kinetics that are Ca2+ dependent. The time constant for activation during a jump in cytoplasmic Ca2+ from subnanomolar concentrations to 10 μm is ∼5 ms and the deactivation time constant on return to subnanomolar concentrations of Ca2+ is ∼50 ms (Hirschberg et al. 1998; Xia et al. 1998). Excitation-contraction (E-C) coupling in skeletal muscle requires physical coupling between the Ca2+ channel on the T-tubular membrane and ryanodine receptors (RyRs) on the sarcoplasmic reticulum (SR) membrane. Membrane depolarization in skeletal muscle causes a movement of the L-type Ca2+ channel voltage sensor, which in turn activates the RyRs (for reviews see Schneider, 1994; Rios & Stern, 1997). The opening of RyRs results in a rapid (∼1 ms) release of Ca2+ from the SR and a transient increase in myoplasmic [Ca2+] to about 10 μm that activates actin/myosin, resulting in filament shortening and muscle contraction. The elevated levels of myoplasmic Ca2+ are buffered by parvalbumin, limiting the duration of the contraction, and Ca2+-ATPases located in the SR subsequently return Ca2+ to the internal stores. Unlike cardiac muscle, neither extracellular Ca2+ nor Ca2+ influx through voltage-gated Ca2+ channels is required (Armstrong et al. 1972). Influx of Ca2+ through L-type Ca2+ channels in skeletal muscle is much slower (time constants, ∼30–40 ms) than the release of Ca2+ from the SR or the time delay before muscle contraction (Baylor et al. 1983; Garcia et al. 1992). In this study we characterized SK currents recorded using the whole-cell patch clamp configuration from single fast-twitch skeletal fibres acutely dissociated from innervated and denervated flexor digitorum brevis (FDB) muscle. Ca2+ released from the SR during short pulses of up to 80 ms primarily activated SK channels. In current clamp mode, the presence of SK channels decreased the action potential threshold by ∼8 mV and apamin reversed this decrease. The results are consistent with a model in which SK channel activity in the T-tubules causes a local increase in K+ concentration, resulting in an increase in the T-tubular resting membrane potential that decreases the apparent action potential threshold.