Isolation of Myocardial L-Type Calcium Channel Gating Currents with the Spider Toxin 0 -Aga-IIIA

The peptide 0~-agatoxin-IIIA (co-Aga-IIIA) blocks ionic current through L-type Ca channels in guinea pig atrial cells without affecting the associated gating currents, c0-Aga-IIIA permits the study of L-type Ca channel ionic and gating currents under nearly identical ionic conditions. Under conditions that isolate L-type Ca channel currents, 0~-Aga-IIIA blocks all ionic current during a test pulse and after repolarization. This block reveals intramembrane charge movements of equal magnitude and opposite sign at the beginning of the pulse (Qon) and after repolarization (Qo~). Qon and Qo~ are suppressed by 1 ~M felodipine, saturate with increasing test potential, and are insensitive to Cd. The decay of the transient current associated with Qon is composed of fast and slow exponential components. The slow component has a time constant similar to that for activation of L-type Ca channel ionic current, over a broad voltage range. The current associated with Qog decays monoexponentially and more slowly than ionic current. Similar charge movements are found in guinea pig tracheal myocytes, which lack Na channels and T-type Ca channels. The kinetic and pharmacological properties of Qon and Qoer indicate that they reflect gating currents associated with L-type Ca channels. o-Aga-IIIA has no effect on gating currents when ionic current is eliminated by stepping to the reversal potential for Ca or by Cd block. Gating currents constitute a significant component of total current when physiological concentrations of Ca are present and they obscure the activation and deactivation of L-type Ca channels. By using o-Aga-IIIA, we resolve the entire time course of L-type Ca channel ionic and gating currents. We also show that Land T-type Ca channel ionic currents can be accurately quantified by tail current analysis once gating currents are taken into account. I N T R O D U C T I O N The spider toxin co-Aga-IIIA blocks currents through L-type Ca channels in cardiac myocytes with no effect on T-type Ca channels (Mintz, Venema, Adams, and Bean, 1991; Cohen, Ertel, Smith, Venema, Adams, and Leibowitz, 1992a). Our earlier study Address correspondence to Dr. Eric A. Ertel, Room 80N-31C, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065. J. GEN. PHYSIOL. 9 The Rockefeller University Press 9 0022-1295/94/05/0731/23 $2.00 Volume 103 May 1994 731-753 731 on Jauary 0, 2018 jgp.rress.org D ow nladed fom 732 T H E J O U R N A L OF GENERAL PHYSIOLOGY 9 V O L U M E 103 9 1 9 9 4 with this toxin indicated a nccd to rccvaluatc the use of tail current analysis to quantify L-type Ca currents. Tail currents reflect the closing of ion channels (deactivation) and they are observed when a voltage-clamped membrane is repolarizcd after a depolarizing pulse that activates channels. L-type Ca channels deactivate more rapidly than T-type Ca channels (Cota, 1986; Matteson and Armstrong, 1986; Carbone and Lux, 1987; Cohen, McCarthy, Barrett, and Rasmussen, 1988; Hiriart and Matteson, 1988; Kostyuk and Shirokov, 1989; McCarthy and Cohen, 1989; Cohen, Spires, and Van Skiver, 1992b). Wc and others have interpreted the slowly and the rapidly decaying components of Ca channel tail current as being cntirdy duc to ionic current through T-typc and L-type Ca channels, respectively. However, in guinea pig atrial myocytcs, a substantial fraction of the rapidly decaying component is resistant to block by t0-Aga-IIIA (Cohen et al., 1992a). This result suggests that block of L-type Ca channds by co-Aga-IIIA is incomplete, and/or that intramcmbrane chargc movements can constitute a significant fraction of the tail currents in atrial cells, as shown earlier for vcntricular cells (Hadley and Lederer, 1991). Wc find that the toxin-resistant rapidly decaying tail current is entirely duc to intramembrane charge movements. Wc present evidence that these charge movements rcprcsent gating currents associated with L-type Ca channels. Gating currents arc asymmetric intramcmbranc charge movements that arise whcn charged components of voltage-gated ion channels move in response to a change in transmcmbrane voltagc (Armstrong, 1981; Bezanilla, 1985). Gating current measurements can reflect the time and voltage dependences of transitions between dosed states of ion channels and thereby complement studies of the kinetics of ionic currcnts through open channels. Gating currcnts associated with Na channels are particularly well documented. Two factors have facilitated these studies: (a) some neurons and myocytes have a high density of Na channels and few other channels that open at similar rates and voltages; and (b) several toxins, such as tctrodotoxin, block ionic current through Na channcls with little or no effect on thc voltage dependence of channel gating. Until now, such favorable conditions were not available to study gating currents associated with Ca channels. However, several recent studies have indicated that ventricular myocytcs possess a large component of dihydropyridine (DHP)-sensitive intramembranc charge movement, presumably arising from the gating of L-type Ca channels (Field, Hill, and Lamb, 1988; Bean and Rios, 1989; Hadlcy and Ledcrcr, 1991; Josephson and Sperelakis, 1992; Shirokov, Levis, Shirokova, and Rios, 1992). These studies have relied on transition metals such as Cd, Co, and/or La to suppress ionic currents because they lacked high-affinity blockers of Ca channel ionic currcnt. Unfortunately, block of L-type Ca channcls by these cations is very voltage dependent (Lansman, Hess, and Tsicn, 1986), such that high conccntrations arc requircd to isolate the gating current associated with channel deactivation. Such concentrations could affect Ca channel gating currcnts in the samc way that transition metals modify Na channel gating currents (Armstrong and Cota, 1990; Sheets and Hanck, 1992) and DHP-sensitive displacement currents in skeletal muscle (Rios and Pizarro, 1991). By using t0-Aga-IIIA, one can mcasure ionic and gating currents under nearly identical conditions bccausc the toxin blocks ionic current through L-type Ca channels and has no effect on the associated gating currents. on Jauary 0, 2018 jgp.rress.org D ow nladed fom ERTEL ET AL. Isolation of Calcium Channel Gating Currents with to-Aga-IllA 733 M A T E R I A L S AND M E T H O D S