title: A single amino acid mutation attenuates rundown of voltage-gated calcium channels.
abstract: The activity of voltage-gated calcium channels (VGCCs) decreases with time in whole-cell and inside-out patch-clamp recordings. In this study we found that substituting a single amino acid (I1520) at the intracellular end of IIIS6 in the alpha(1) subunit of P/Q-type Ca(2+) channels with histidine or aspartate greatly attenuated channel rundown in inside-out patch-clamp recordings. The homologous mutations also slowed rundown of N- and L-type Ca(2+) channels, albeit to a lesser degree. In P/Q-type channels, the attenuation of rundown is accompanied by an increased apparent affinity for phosphatidylinositol-4,5-bisphosphate, which has been shown to be critical for maintaining Ca(2+) channel activity [L. Wu, C.S. Bauer, X.-G. Zhen, C. Xie, J. Yang, Dual regulation of voltage-gated calcium channels by PtdIns(4,5)P2. Nature 419 (2002) 947-952]. Furthermore, the histidine mutation significantly stabilized the open state, making the channels easier to open, slower to close, harder to inactivate and faster to recover from inactivation. Our finding that mutation of a single amino acid can greatly attenuate rundown provides an easy and efficient way to slow the rundown of VGCCs, facilitating functional studies that require direct access to the cytoplasmic side of the channel.
authors.affiliation: Department of Biological Sciences, 917 Fairchild Center, MC2462, Columbia University, New York, NY 10027, USA.
authors.forename: Xiao-Guang
authors.initials: XG
authors.lastname: Zhen
authors.forename: Cheng
authors.initials: C
authors.lastname: Xie
authors.forename: Yoichi
authors.initials: Y
authors.lastname: Yamada
authors.forename: Yun
authors.initials: Y
authors.lastname: Zhang
authors.forename: Christina
authors.initials: C
authors.lastname: Doyle
authors.forename: Jian
authors.initials: J
authors.lastname: Yang
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004594:Electrophysiology; D015640:Ion Channel Gating; D007532:Isoleucine / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009865:Oocytes; D018408:Patch-Clamp Techniques; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D016415:Sequence Alignment; D014982:Xenopus laevis
chemical_list: D015220:Calcium Channels; D019269:Phosphatidylinositol 4,5-Diphosphate; D021122:Protein Subunits; D007532:Isoleucine