Iberiotoxin-Induced Block of Ca -Activated K Channels Induces Dihydropyridine Sensitivity of ACh Release from Mammalian Motor Nerve Terminals

The role which Ca -activated K (KCa) channels play in regulating acetylcholine (ACh) release was examined at mouse motor nerve terminals. In particular, the ability of the antagonist iberiotoxin to recruit normally silent L-type Ca channels to participate in nerve-evoked release was examined using conventional intracellular electrophysiological techniques. Incubation of cut hemidiaphragm preparations with 10 M nimodipine, a dihydropyridine L-type Ca channel antagonist, had no significant effect on quantal content of end-plate potentials. Nevertheless, 1 M S-( )-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2[trifluoromethyl]phenyl)-3-pyridine carboxylic acid methyl ester (Bay K 8644) enhanced quantal content to 134.7 3.5% of control. Iberiotoxin (150 nM) increased quantal content to 177.5 9.9% of control, whereas iberiotoxin plus nimodipine increased quantal content to only 145.7 10.4% of control. Coapplication of 1 M Bay K 8644 with iberiotoxin did not significantly increase quantal content further than did treatment with iberiotoxin alone. The effects of iberiotoxin and nimodipine alone or in combination on the miniature end-plate potential (MEPP) frequency following KCl-induced depolarization were examined using uncut hemidiaphragm preparations. Nimodipine alone had no effect on MEPP frequency from preparations incubated in physiological saline containing 5 to 20 mM KCl. Moreover, iberiotoxin alone or combined with nimodipine also had no effect on MEPP frequency in physiological salines containing 5 to 15 mM KCl. At 20 mM KCl, however, iberiotoxin significantly increased MEPP frequency to 125.6% of iberiotoxin-free values; combined treatment with nimodipine and iberiotoxin prevented this increase in MEPP frequency. Thus, loss of functional KCa channels unmasks normally silent L-type Ca channels to participate in ACh release from motor nerve terminals, particularly under conditions of intense nerve terminal depolarization. Release of acetylcholine (ACh) from motor nerves is a highly controlled process that requires precisely controlled entry of Ca through voltage-dependent Ca channels into the nerve terminal (Augustine et al., 1987). Multiple Ca channel subtypes have been demonstrated to exist based on differential pharmacological, biophysical, and molecular characteristics; thus far L-, T-, N-, P-, Q-, and R-type (now designated Cav1.1–1.4, Cav2.1–2.3, Cav 3.1–3.3; Ertel et al., 2000) channels have been described (for review, see Catterall, 1998). Often more than one subtype of Ca channel coexists at the same nerve terminal to control transmitter release (Lemos and Nowycky, 1989; Turner et al., 1993; Elhamdani et al., 1998). Nevetheless, the specific Ca channel phenotype primarily involved in ACh release is both speciesdependent (Sano et al., 1987; De Luca et al., 1991; Protti et al., 1996) and age-dependent (Sugiura and Ko, 1997). Release of ACh from mature mammalian motor nerves relies primarily on entry of Ca through P/Q-type (Uchitel et al., 1992; Protti et al., 1996) and not N-type Ca channels, which control release of ACh from motor nerves of amphibians (Sano et al., 1987) and birds (De Luca et al., 1991). Developing mammalian motor nerves, on the other hand, possess multiple subtypes of Ca channels that are involved in the release of ACh, some of which become less important during maturation (Sugiura and Ko, 1997). L-Type Ca channels, which colocalize with other Ca This work was submitted by M.T.F. in partial fulfillment of the requirements for the Ph.D. degree in Pharmacology and Toxicology as part of the combined degree Medical Scientist Training Program in the College of Osteopathic Medicine at Michigan State University. A portion of these results was presented at the 2000 Annual Meeting of the Society for Neuroscience (New Orleans, LA) on November 4 to 9, 2000 and published in abstract form in Soc Neurosci Abstr 26:88. Supported by National Institutes of Health Grant ES05822 (Bethesda, MD) and a Viets Fellowship from the Myasthenia Gravis Foundation to Michael T. Flink. Article, publication date, and citation information can be found at http://jpet.aspetjournals.org. DOI: 10.1124/jpet.102.046102. ABBREVIATIONS: ACh, acetylcholine; KCa, calcium-activated potassium; LEMS, Lambert-Eaton myasthenic syndrome; MEPP, miniature endplate potential; DAP, 3,4-diaminopyridine; EPP, end-plate potential; BSA, bovine serum albumin; Cav, voltage-activated calcium channel; DHP, dihydropyridine. 0022-3565/03/3052-646–652$7.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 305, No. 2 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 46102/1056804 JPET 305:646–652, 2003 Printed in U.S.A. 646 at A PE T Jornals on A ril 5, 2017 jpet.asjournals.org D ow nladed from channel phenotypes, participate in release of noradrenaline from chromaffin cells of the adrenal medulla (Owen et al., 1989) and in the release of oxytocin and vasopressin (Lemos and Nowycky, 1989). Pharmacological evidence has implicated a potential role of normally silent L-type Ca channels in release of ACh from mature mammalian motor nerves (Atchison and O’Leary, 1987; Atchison, 1989). Moreover, during certain pathological conditions, L-type Ca channels can participate in release of ACh as well (Katz et al., 1996; Santafe et al., 2000; Flink and Atchison, 2002; Giovannini et