Pfltigers Arch (1993) 423:189-192 EI] i "fin Journal of Physiology y Springer-Verlag 1993 Regulation of resting ionic conductances in frog skeletal muscle Domenico Triearico 1, Richard Wagner 1, Shirley H. Bryant 2, Diana Conte Camerino 1 1 Dipartimento Farmaco Biologico, Facolt~t di Farmacia, Universit~t di Bad, via Orabona 4, 1-70125 Bari, Italia 2 Department of Pharmacology and Cell Biophysics, University of Cincinnati, College of Medicine, Cincinnati, OH 45267-0575, USA Received June 25,1992/Received after revision November 9,1992/Accepted November 17,1992 Abstract. The membrane electrical properties and rest- ing ionic conductances of frog semitendinosus muscle fibres were studied in vitro at 25 ~ C with the two-micro- electrode cable technique, in the presence of an activator or inhibitor of protein kinase C (PKC) or in the presence of an activator of adenylate cyclase. The PKC activator, 4fl-phorbol 12,13-dibutyrate (4fl-PDB), reduced chloride conductance (Gc~) at concentrations greater than 1 gM and did not affect potassium conductance (GK). At 150 ~tM, the maximum concentration of 4fl-PDB tested, Gcl was reduced by 42%. The "inactive" phorbol ester 4a-phorbol 12,13-dibutyrate did not affect Gcl or GK. The inhibitory effect of 4fl-PDB on Gc~ was prevented by pretreatment of the muscle preparation with the PKC inhibitor stanrosporine. The adenylate cyclase activator forskolin (1.5-8 gM) significantly increased the GK of the fibres, without affecting Gcl. Thus, we conclude that frog skeletal muscle Gcl, unlike rat muscle Gc~, is rela- tively insensitive to activators of PKC. Moreover, in frog muscle, protein kinase A is a likely modulator of GK, but not Gc~. Key words: Frog - Skeletal muscle - Chloride con- ductance - Potassium conductance - Protein kinase C - Protein kinase A Introduction In vertebrate skeletal muscle, over two-thirds of the res- ting membrane conductance is due to C1 permeability, with the balance contributed by permeability to potassi- um [1]. Defects in the regulation of these component conductances are important in the pathophysiology of skeletal muscle. In mammalian muscle, a reduction of chloride conductance (Gcl) results in a repetitive firing abnormality known as myotonia [5]. The molecular basis for myotonia in the genetically myotonic mouse is Correspondence to: D. Conte Camerino now known to be the insertion of a transposon into the chloride channel gene [21], resulting in a disruption of the production of chloride channel mRNA. In human recessive generalized myotonia (Becker), Gc~ is de- creased by 55-100%, potassium conductance is often increased, and sodium channels exhibit "delayed re- openings" [13]. A dramatic decrease of Gc~ has also been demonstrated after denervation of mammalian muscle [3]. The molecular defect(s) responsible for these alterations in conductances are not yet known, but may involve metabolic defects in channel regulation. Recently it has been demonstrated that mammalian skeletal muscle Gc~ can be modulated by Ca 2+ and phos- pholipid-dependent protein kinase C (PKC) [2, 4, 22]. Phorbol esters, activators of PKC, produce a time and dose-dependent reduction of Gcl in rat muscle and do not affect resting potassium conductance (GK). Biochemical evidence of the presence of PKC in mammalian skeletal muscle was recently documented by Sabbadini et al. [20], who observed a localization of PKC in the junc- tional regions of triads. The channels responsible for the macroscopic chlor- ide conductance in mammalian skeletal muscle appear to be located exclusively in the T-tubule system, and not on the surface membrane [7] (personal unpublished observation). This location precludes the use of the single-channel patch-clamp technique to study the phar- macology and physiological regulation of mammalian muscle chloride channels. Frog muscle, on the other hand, has a significant population of chloride channels on the surface membrane, where they are accessible via the patch-clamp technique [24]. For technical reasons, frog skeletal muscle is also commonly used in studies of other ionic conductances and voltage-dependent charge movement [12, 18, 19, 23]. Significant similarities and differences exist between the macroscopic chloride conductances in frog and mammalian skeletal muscle. For example, although frog and mammalian chloride conductances have similar non- linear steady-state current/voltage relationships and al- most the same sensitivity to low pH [1], frog muscle
