Crystallographic basis for calcium regulation of sodium channels

Abstract

Voltage-gated sodium channels underlie the rapid regenerative upstroke of action potentials and are modulated by cytoplasmic calcium ions through a poorly understood mechanism. We describe the 1.35 Å crystal structure of Ca 2+ -bound calmodulin (Ca 2+ /CaM) in complex with the inactivation gate (DIII-IV linker) of the cardiac sodium channel (Na V 1.5). The complex harbors the positions of five disease mutations involved with long Q-T type 3 and Brugada syndromes. In conjunction with isothermal titration calorimetry, we identify unique inactivation-gate mutations that enhance or diminish Ca 2+ /CaM binding, which, in turn, sensitize or abolish Ca 2+ regulation of full-length channels in electrophysiological experiments. Additional biochemical experiments support a model whereby a single Ca 2+ /CaM bridges the C-terminal IQ motif to the DIII-IV linker via individual N and C lobes, respectively. The data suggest that Ca 2+ /CaM destabilizes binding of the inactivation gate to its receptor, thus biasing inactivation toward more depolarized potentials.crystallography | patch-clamp electrophysiology | structural biology | cardiac arrhythmia V oltage-gated sodium channels (Na V s) support excitability in the cardiovascular and nervous systems, where they contribute to the rhythm and rate of action potentials. These large (∼220-kDa) transmembrane protein complexes are expressed at a high density in excitable cells, where they conduct large macroscopic inward sodium currents. These channels are exquisitely sensitive to subtle changes in the transmembrane potential, and modest alterations in channel gating can fine-tune or disorder electrical signaling at the organ and systemic level. The α-subunit of the channel contains cytoplasmic amino and carboxyl termini and is composed of four homologous transmembrane domains (DI-DIV) that are connected by intracellular linkers. Each domain contains voltage-sensing (S1-S4) and pore-forming (S5 and S6) domains that form the selectivity filter and putative activation gates. A crystal structure of a bacterial Na V was recently described (1) showing a similar overall fold compared with potassium channels. However, this bacterial variant is homotetrameric, and seems to lack a conserved fast-inactivation mechanism. As such, it has no homology with several relevant domains in mammalian Na V channels, and no crystal structure of any eukaryotic Na V region has yet been reported.Calcium ions (Ca 2+ ) are universal second messengers, and in the heart they form the electrochemical link between plasma membrane depolarization and myocyte contraction. Consequently, their cytoplasmic levels oscillate between nanomolar and micromolar levels with each excitation-contraction cycle (2). Sodium channel steady-state inactivation, a process that controls channel availability at a given transmembrane potential, is modulated through interactions with Ca 2+ and calmodulin (CaM) (3-10). The mechanistic details of Ca 2+ modulation of sodium channel inactivation are sparse, but the C-terminal region of the cha...

Multiple Calmodulin-binding Sites Positively and Negatively Regulate Arabidopsis CYCLIC NUCLEOTIDE-GATED CHANNEL12

“…Our model shows CNGC12 mediating Ca 2+ influx directly; however, we cannot exclude the possibility that CNGC12 signaling triggers Ca 2+ current(s) mediated by other channel(s). Our model includes speculative features that are consistent with our results to date but which require further experimental investigation and some of which bear clear similarities to various animal channels, such as CaM-mediated bridging of inter-and intrasubunit interactions and dynamic movement of CaM between multiple CaMBDs (Fallon et al, 2009;Liu and Vogel, 2012;Sarhan et al, 2012;Morales et al, 2013;Ben Johny et al, 2013;Shao et al, 2014). Interestingly, such CaM regulatory features are widely distributed among various animal voltage-or Ca 2+ -gated cation channels for which there are no clear homologs in plants (Edel and Kudla, 2015).…”

“…association between CaM and IQ motifs is a widespread phenomenon among animal cation channels (Kovalevskaya et al, 2013) but had not previously been confirmed in plants. Our data suggest that CaM may be responsible for Ca 2+ -dependent intraand/or intersubunit interactions, another feature thought to be specific to animal cation channels (Fallon et al, 2009;Liu and Vogel, 2012;Sarhan et al, 2012). This is an interesting avenue for investigation, given that homo-or heterotetramers are hypothesized to comprise functional CNGCs in vivo (Kaplan et al, 2007;Zelman et al, 2012), though to date we have only minimal data regarding how different plant CNGC subunits associate .…”

A framework for exhaustively mapping functional missense variants

“…Secondary structures were calculated using Stride (Frishman & Argos, 1995). Solvent accessibility and interface burial were calculated using the GETAREA tool (Fraczkiewicz & Braun, 1998) on the following PDB entries: for UBE2I: 3UIP (Gareau et al , 2012); 4W5V (Boucher et al unpublished); 3KYD (Olsen et al , 2010); 2UYZ (Knipscheer et al , 2007); 4Y1L (Alontaga et al , 2015); for SUMO1: 2G4D (Xu et al , 2006); 2IO2 (Reverter & Lima, 2006); 3KYD (Olsen et al , 2010); 3UIP (Gareau et al , 2012); 2ASQ (Song et al , 2005); 4WJO (Cappadocia et al , 2015); 4WJQ (Cappadocia et al , 2015); 1WYW (Baba et al , 2005); for calmodulin: 3G43 (Fallon  et al , 2009); 4DJC (Sarhan et al , 2012); and for TPK1: 3S4Y (Baker et al , 2001). …”

Cooperativity between calmodulin-binding sites in Kv7.2 channels

“…Two helices connected by CaM have been observed or inferred in several instances (Schumacher et al, 2001;Yap et al, 2003;Houdusse et al, 2006;Wang et al, 2008;Fallon et al, 2009;Zhang et al, 2012;Sarhan et al, 2012), although the functional relevance of this engulfing arrangement has on occasion been brought into question (Kim et al, 2010). An organization of this kind is crucial for Ca 2+ -CaM mediated gating of small conductance Ca 2+ -activated K + channels (Schumacher et al, 2001).…”