Inward rectifier potassium currents in mammalian skeletal muscle fibres.

Inward rectifying potassium (Kir) channels play a central role in maintaining the resting membrane potential of skeletal muscle fibres. Nevertheless their role has been poorly studied in mammalian muscles. Immunohistochemical and transgenic expression were used to assess the molecular identity and subcellular localization of Kir channel isoforms. We found that Kir2.1 and Kir2.2 channels were targeted to both the surface andthe transverse tubular system membrane (TTS) compartments and that both isoforms can be overexpressed up to 3-fold 2 weeks after transfection. Inward rectifying currents (IKir) had the canonical features of quasi-instantaneous activation, strong inward rectification, depended on the external [K+], and could be blocked by Ba2+ or Rb+. In addition, IKir records show notable decays during large 100 ms hyperpolarizing pulses. Most of these properties were recapitulated by model simulations of the electrical properties of the muscle fibre as long as Kir channels were assumed to be present in the TTS. The model also simultaneously predicted the characteristics of membrane potential changes of the TTS, as reported optically by a fluorescent potentiometric dye. The activation of IKir by large hyperpolarizations resulted in significant attenuation of the optical signals with respect to the expectation for equal magnitude depolarizations; blocking IKir with Ba2+ (or Rb+) eliminated this attenuation. The experimental data, including the kinetic properties of IKir and TTS voltage records, and the voltage dependence of peak IKir, while measured at widely dissimilar bulk [K+] (96 and 24 mm), were closely predicted by assuming Kir permeability (PKir) values of ∼5.5 × 10−6 cm s−1 and equal distribution of Kir channels at the surface and TTS membranes. The decay of IKir records and the simultaneous increase in TTS voltage changes were mostly explained by K+ depletion from the TTS lumen. Most importantly, aside from allowing an accurate estimation of most of the properties of IKir in skeletal muscle fibres, the model demonstrates that a substantial proportion of IKir (>70%) arises from the TTS. Overall, our work emphasizes that measured intrinsic properties (inward rectification and external [K] dependence) and localization of Kir channels in the TTS membranes are ideally suited for re-capturing potassium ions from the TTS lumen during, and immediately after, repetitive stimulation under physiological conditions. Key points This paper provides a comprehensive electrophysiological characterization of the external [K+] dependence and inward rectifying properties of Kir channels in fast skeletal muscle fibres of adult mice. Two isoforms of inward rectifier K channels (IKir2.1 and IKir2.2) are expressed in both the surface and the transverse tubular system (TTS) membranes of these fibres. Optical measurements demonstrate that Kir currents (IKir) affect the membrane potential changes in the TTS membranes, and result in a reduction in luminal [K+]. A model of the muscle fibre assuming that functional Kir channels are equally distributed between the surface and TTS membranes accounts for both the electrophysiological and the optical data. Model simulations demonstrate that the more than 70% of IKir arises from the TTS membranes. [K+] increases in the lumen of the TTS resulting from the activation of K delayed rectifier channels (Kv) lead to drastic enhancements of IKir, and to right-shifts in their reversal potential. These changes are predicted by the model. Introduction Inward rectifier potassium (Kir) channels are known to play a crucial role in skeletal muscle physiology as, together with chloride channels (ClC-1; Bretag, 1987), they are responsible for the characteristic negative resting membrane potentials that result from potassium and chloride concentration gradients (Katz, 1948; Hodgkin & Horowicz, 1957, 1959b; Stanfield et al. 2002). The properties of Kir currents (IKir) were extensively investigated in amphibian muscle fibres (Hodgkin & Horowicz, 1959b, 1960; Standen & Stanfield, 1978b; Leech & Stanfield, 1981; Stanfield et al. 2002) where they have been shown to originate at the surface and transverse tubular system (TTS) membranes (Almers, 1972b; Standen & Stanfield, 1979; Ashcroft et al. 1985). In contrast, IKir measurements in mammalian skeletal muscle fibres are scarce (Duval & Leoty, 1980; Beam & Donaldson, 1983; Barrett-Jolley et al. 1999) and have yielded an incomplete characterization of the functional properties of Kir channels in their natural environment. It has been reported, by immunohistochemistry (IH) and molecular biological methods, that mammalian skeletal muscles express several types of Kir channels, including Kir1.1, Kir2.1, Kir2.2, Kir2.6 and Kir6.2 (KATP) channels (Kubo et al. 1993; Raab-Graham et al. 1994; Takahashi et al. 1994; Doupnik et al. 1995; Inagaki et al. 1995; Kondo et al. 1996; Kristensen et al. 2006; Dassau et al. 2011). Nevertheless, it is generally accepted that Kir channels in skeletal muscle belong to the Kir2.x family, and that Kir2.1 and Kir2.2 may be the most prevalent isoforms (Doupnik et al. 1995; Stanfield et al. 2002; Hibino et al. 2010; Dassau et al. 2011); yet, the evidence about their location in the surface and TTS membrane is less conclusive (Kristensen et al. 2006; Dassau et al. 2011). Also, heterologously expressed Kir2.1 and Kir2.2 channels display strong inward rectification, are constitutively active and are able to form heterotetramers (Doupnik et al. 1995; Stanfield et al. 2002; Hibino et al. 2010). However, their functional characterization in the naive environment of skeletal muscle fibres, together with a quantitative balance of the respective IKir contributions from the surface and TTS membranes, as estimated for amphibian muscle, is missing. This paper reports results from experiments that have been carefully designed to characterize IKir in murine fast muscle fibres and to ultimately define the distribution of Kir channels between the surface and TTS membrane compartments. To this end, we have combined electrophysiological and optical methods and model simulations to quantitatively assess not only the physiological properties of ion channels in the context of adult mammalian skeletal muscle fibres, but also their relative distribution between surface and TTS membranes (S/TTS ratio). This approach has been used previously to perform similar studies of the ClC-1 currents (DiFranco et al. 2011a), sodium (NaV1.4; DiFranco & Vergara, 2011) and potassium delayed rectifier channels (KV1.4 and KV3.4; DiFranco et al. 2012) of adult murine fibres. We also performed IH and transient expression experiments to study the targeting of native and transgenic IKir2.1 and 2.2 channels. The present work extends previous studies from our laboratory characterizing other ion conductances present in the sarcolemma and TTS membranes that are responsible for the electrical properties of mammalian skeletal muscle fibres. The knowledge gained here is vital to understand the homeostasis of potassium ions during sustained activity, and how it may affect the fibres’ excitability.