ABSTRACT Calcium-dependent potassium (BK-type) Ca^sup 2+^ and voltage-dependent K+ channels in chromaffin cells exhibit an inactivation that probably arises from coassembly of Slo 1 a subunits with auxiliary beta subunits. One goal of this work was to determine whether the Ca^sup 2+^ dependence of inactivation arises from any mechanism other than coupling of inactivation to the Ca^sup 2+^ dependence of activation. Steady-state inactivation and the onset of inactivation were studied in inside-out patches and whole-cell recordings from rat adrenal chromaffin cells with parallel experiments on inactivating BK channels resulting from cloned alpha + beta 2 subunits. In both cases, steady-state inactivation was shifted to more negative potentials by increases in submembrane [Ca^sup 2+^] from 1 to 60 AM. At 10 and 60 (mu) M Ca^sup 2+^, the maximal channel availability at negative potentials was similar despite a shift in the voltage of half availability, suggesting there is no strictly Ca^sup 2+^-dependent inactivation. In contrast, in the absence of Ca^sup 2+^, depolarization to potentials positive to +20 mV induces channel inactivation. Thus, voltagedependent, but not solely Ca-dependent, kinetic steps are required for inactivation to occur. Finally, under some conditions, BK channels are shown to inactivate as readily from closed states as from open states, indicative that a key conformational change required for inactivation precedes channel opening.
We have now proposed two general sorts of physical models that might account for the properties of inactivation we have observed. In one case, inactivation is proposed to result from a conformational change specifically associated with voltage-sensor movement. In the other, a conformational change within the closed channel, allosterically regulated by Ca^sup 2+^ and voltage, is the essential step required for inactivation. Future work will be required to evaluate whether either sort of physical mechanism provides a better account of BK channel inactivation.
This work was supported by DK-46564 and NS-37671 from the National Institutues of Health
Adelman, J. P., K. Z. Shen, M. P. Kavanaugh, R. A. Warren, Y. N. Wu, A. Lagrutta, C. T. Bond, and R. A. North. 1992. Calcium-activated potassium channels expressed from cloned complementary DNAs. Neuron. 9:209-216.
Aldrich, R. W., D. P. Corey, and C. F. Stevens. 1983. A reinterpretation of mammalian sodium channel gating based on single channel recording. Nature. 306:436-441.
Aldrich, R. W., and C. F. Stevens. 1987. Voltage-dependent gating of single sodium channels from mammalian neuroblastoma cells. J. Neurosci. 7:418-431.
Brenner, R., T. J. Jegla, A. Wickenden, Y. Liu, and R. W. Aldrich. 2000. Cloning and functional characterization of novel large conductance calcium-activated potassium channel beta subunits, hKCNMB3 and hKCNMB4. J. Biol. Chem. 275:6453-6461.
Butler, A., S. Tsunoda, D. P. McCobb, A. Wei, and L. Salkoff. 1993. mSlo, A complex mouse gene encoding "maxi" calcium-activated potassium channels. Science. 261:221-224.
Cannell, M. B., and C. G. Nichols. 1991. Effects of pipette geometry on the time course of solution change in patch clamp experiments. Biophys. J. 60:1156-1163.
Cox, D., and R. Aldrich. 2000. Role of the 31 subunit in large-conductance Ca2+-activated K+ channel gating energetics. Mechanisms of enhanced Ca 21 sensitivity. J. Gen. Physiol. 116:411-432.
Cox, D. H., J. Cui, and R. W. Aldrich. 1997. Allosteric gating of a large conductance Ca-activated K+ channel. J. Gen. Physiol. 110:257-281. Cui, J., and R. W. Aldrich. 2000. Allosteric linkage between voltage and
Ca2+-dependent activation of BK-type mslol K+ channels. Biochemistry. 39:15612-15619.
Demo, S. D., and G. Yellen. 1992. Ion effects on gating of the Ca 2+activated K+ channel correlate with occupancy of the pore. Biophys. J. 61:639-648.
Ding, J. P., Z. W. Li, and C. J. Lingle. 1998. Inactivating BK channels in rat chromaffin cells may arise from heteromultimeric assembly of dis
tinct inactivation-competent and noninactivating subunits. Biophys. J. 74:268-289.
Fenwick, E. M., A. Marty, and E. Neher. 1982. Sodium and calcium channels in bovine chromaffin cells. J. Physiol. (Land.). 331:599-635. Hamill, 0. P., A. Marty, E. Neher, B. Sakmann, and F. J. Sigworth. 1981.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches. Pflugers Arch. 391:85-100. Herrington, J., C. R. Solaro, A. Neely, and C. J. Lingle. 1995. The
suppression of Ca"- and voltage-dependent outward K+ current during mAChR activation in rat adrenal chromaffin cells. J. Physiol. (Load.). 485:297-318.
Horrigan, F. T., and R. W. Aldrich. 1999. Allosteric voltage gating of potassium channels II. Mslo channel gating charge movement in the absence of Ca2+. J. Gen. Physiol. 114:305-336.
Horrigan, F. T., J. Cui, and R. W. Aldrich. 1999. Allosteric voltage gating of potassium channels I. Mslo ionic currents in the absence of Ca 21. J. Gen. Physiol. 114:277-304.
Jerng, H. H., M. Shahidullah, and M. Covarrubias. 1999. Inactivation gating of Kv4 potassium channels: molecular interactions involving the inner vestibule of the pore. J. Gen. Physiol. 113:641-660.
Kilpatrick, D. L., R. V. Lewis, S. Stein, and S. Udenfriend. 1980. Release of enkephalins and enkephalin-containing polypeptides from perfused beef adrenal glands. Proc. Natl. Acad. Sci. U.S.A. 77:7473-7475.
Kuo, C. C. 1997. Deactivation retards recovery from inactivation in Shaker K+ channels. J. Neurosci. 17:3436-3444.
Li, Z. W., J. P. Ding, V. Kalyanaraman, and C. J. Lingle. 1999. RINm5f cells express inactivating BK channels whereas HIT cells express noninactivating BK channels. J. Neurophysiol. 81:611-624.
Lingle, C., X.-H. Zeng, J.-P. Ding, and X.-M. Xia. 2001. Inactivation of BK channels mediated by the N-terminus of the /3b auxiliary subunit involves a two-step mechanism: possible separation of binding and blockade. J. Gen. Physiol. 117:583-605.
Lingle, C. J., C. R. Solaro, M. Prakriya, and J. P. Ding. 1996. Calciumactivated potassium channels in adrenal chromaffin cells. Ion Channels. 4:261-301.
Livett, B. G. 1984. Adrenal medullary chromaffin cells in vitro. Physiol. Rev. 64:1103-1161.
Markwardt, F., and G. Isenberg. 1992. Gating of maxi K+ channels studied by Ca 21 concentration jumps in excised inside-out multi-channel patches (myocytes from guinea pig urinary bladder). J. Gen. Physiol. 99:841-862.
Neely, A., and C. J. Lingle. 1992. Two components of calcium-activated potassium current in rat adrenal chromaffin cells. J. Physiol. 453: 97-131.
Piskorowski, R., and R. Aldrich. 2001. Altering the gating of largeconductance Ca"-activated K+ channels with different permeant ions. Biophys. J. 80:221a. (Abstract)
Prakriya, M., C. R. Solaro, and C. J. Lingle. 1996. i elevations detected by BK channels during Ca2' influx and muscarine-mediated release of Ca 21 from intracellular stores in rat chromaffin cells. J. Neurosci. 16:4344-4359.
Role, L., and R. Perlman. 1980. Purification of adrenal medullary chromaffin cells by density gradient centrifugation. J. Neurosci. Methods. 2:253-265.
Rothberg, B. S., and K. L. Magleby. 1999. Gating kinetics of single large-conductance Ca 2'-activated K+ channels in high Caz+ suggest a two-tiered allosteric gating mechanism. J. Gen. Physiol. 114:93-124.
Rothberg, B. S., and K. L. Magleby. 2000. Voltage and Ca2+ activation of single large-conductance Ca"-activated K+ channels described by a two-tiered allosteric gating mechanism. J. Gen. Physiol. 116:75-99.
Ruppersberg, J. P., R. Frank, 0. Pongs, and M. Stocker. 1991. Cloned neuronal IK(A) channels reopen during recovery from inactivation. Nature. 353:657-660.
Solaro, C. R., J. P. Ding, Z. W. Li, and C. J. Lingle. 1997. The cytosolic inactivation domains of BK; channels in rat chromaffin cells do not behave like simple, open-channel blockers. Biophys. J. 73:819-830.
Solaro, C. R., and C. J. Lingle. 1992. Trypsin-sensitive, rapid inactivation of a calcium-activated potassium channel. Science. 257:1694-1698.
Solaro, C. R., M. Prakriya, J. P. Ding, and C. J. Lingle. 1995. Inactivating and noninactivating Ca 2'- and voltage-dependent K+ current in rat adrenal chromaffin cells. J. Neurosci. 15:6110-6123.
Uebele, V. N., A. Lagrutta, T. Wade, D. J. Figueroa, Y. Liu, E. McKenna, C. P. Austin, P. B. Bennett, and R. Swanson. 2000. Cloning and functional expression of two families of beta-subunits of the large conductance calcium-activated K+ channel. J. Biol. Chem. 275: 23211-23218.
Wallner, M., P. Meera, and L. Toro. 1999. Molecular basis of fast inactivation in voltage and Ca 2+-activated K+ channels: a transmembrane beta-subunit homolog. Proc. Natl. Acad. Sci. U.S.A. 96:4137-4142.
Wei, A., C. Solaro, C. Lingle, and L. Salkoff. 1994. Calcium sensitivity of BK-type KCa channels determined by a separable domain. Neuron. 13:671-681.
Xia, X. M., J. P. Ding, and C. J. Lingle. 1999. Molecular basis for the inactivation of Ca"- and voltage-dependent BK channels in adrenal chromaffin cells and rat insulinoma tumor cells. J. Neurosci. 19: 5255-5264.
Xia, X.-M., J. Ding, X.-H. Zeng, K.-L. Duan, and C. Lingle. 2000. Rectification and rapid activation at low Ca2+ of Ca2+-activated, voltage-dependent BK currents: consequences of rapid inactivation by a novel/3 subunit. J. Neurosci. 20:4890-4903.
Zeng, X.-H., X.-M. Xia, and C. J. Lingle. 2001. Gating properties conferred on BK channels by the/33b auxiliary subunit in the absence of its N- and C-termini. J. Gen. Physiol. 117:607-627.
Zhou, M., J. H. Morais-Cabral, S. Mann, and R. MacKinnon. 2001. Potassium channel receptor site for the inactivation gate and quaternary amine inhibitors. Nature. 411:657-661.
Jiu Ping Ding and Christopher J. Lingle
Washington University School of Medicine, Department of Anesthesiology, St. Louis, Missouri 63110 USA
Submitted November 2, 2001, and accpeted for publication December 12, 2001
Address reprint requests to Chris Lingle, 660 S. Euclid Ave., St. Louis, MO 63110. Tel : 314-362-8558; Fax: 314-362-8571; E-mail: clingle@ morpheus.wustl.edu
Address reprint requests to Chris Lingle, 660 S. Euclid Ave., St. Louis, MO 63110. Tel.: 314-362-8558; Fax: 314-362-8571; E-mail: clingle@ morpheus.wustl.edu.
2002 by the Biophysical Society 0006-3495/02/05/2448/18 $2.00
Copyright Biophysical Society May 2002
Provided by ProQuest Information and Learning Company. All rights Reserved