The homeostasis of animals is regulated not only by the growth and differentiation of cells, but also by cell death through a process known as apoptosis. Apoptosis is mediated by members of the caspase family of proteases, and eventually causes the degradation of chromosomal DNA. A caspase-activated deoxyribonuclease (CAD) and its inhibitor (ICAD) have now been identified in the cytoplasmic fraction of mouse lymphoma cells. CAD is a protein of 343 amino acids which carries a nuclear-localization signal; ICAD exists in a long and a short form. Recombinant ICAD specifically inhibits CAD-induced degradation of nuclear DNA and its DNase activity. When CAD is expressed with ICAD in COS cells or in a cell-free system, CAD is produced as a complex with ICAD: treatment with caspase 3 releases the DNase activity which causes DNA fragmentation in nuclei. ICAD therefore seems to function as a chaperone for CAD during its synthesis, remaining complexed with CAD to inhibit its DNase activity; caspases activated by apoptotic stimuli then cleave ICAD, allowing CAD to enter the nucleus and degrade chromosomal DNA.

A caspase-activated DNase that degrades DNA during apoptosis, and its inhibitor ICAD

Voltage-dependent potassium ion (K+) channels (Kv channels) conduct K+ ions across the cell membrane in response to changes in the membrane voltage, thereby regulating neuronal excitability by modulating the shape and frequency of action potentials. Here we report the crystal structure, at a resolution of 2.9 angstroms, of a mammalian Kv channel, Kv1.2, which is a member of the Shaker K+ channel family. This structure is in complex with an oxido-reductase beta subunit of the kind that can regulate mammalian Kv channels in their native cell environment. The activation gate of the pore is open. Large side portals communicate between the pore and the cytoplasm. Electrostatic properties of the side portals and positions of the T1 domain and beta subunit are consistent with electrophysiological studies of inactivation gating and with the possibility of K+ channel regulation by the beta subunit.

http://science.sciencemag.org/content/sci/309/5736/897.full.pdf

Crystal Structure of a Mammalian Voltage-Dependent Shaker Family K + Channel

K+ channel principal subunits are by far the largest and most diverse of the ion channels. This diversity originates partly from the large number of genes coding for K+ channel principal subunits, but also from other processes such as alternative splicing, generating multiple mRNA transcripts from a single gene, heteromeric assembly of different principal subunits, as well as possible RNA editing and posttranslational modifications. In this chapter, we attempt to give an overview (mostly in tabular format) of the different genes coding for K+ channel principal and accessory subunits and their genealogical relationships. We discuss the possible correlation of different principal subunits with native K+ channels, the biophysical and pharmacological properties of channels formed when principal subunits are expressed in heterologous expression systems, and their patterns of tissue expression. In addition, we devote a section to describing how diversity of K+ channels can be conferred by heteromultimer formation, accessory subunits, alternative splicing, RNA editing and posttranslational modifications. We trust that this collection of facts will be of use to those attempting to compare the properties of new subunits to the properties of others already known or to those interested in a comparison between native channels and cloned candidates.

Molecular Diversity of K+ Channels

https://www.cell.com/neuron/pdf/S0896-6273(00)80708-4.pdf

A new ER trafficking signal regulates the subunit stoichiometry of plasma membrane K(ATP) channels.

The heart is a rhythmic electromechanical pump, the functioning of which depends on action potential generation and propagation, followed by relaxation and a period of refractoriness until the next...

Molecular Physiology of Cardiac Repolarization

https://www.cell.com/neuron/pdf/S0896-6273(00)80104-X.pdf

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

The voltage-gated delayed-rectifier-type K+ channel Kv2.1 is expressed in high-density clusters on the soma and proximal dendrites of mammalian central neurons; thus, dynamic regulation of Kv2.1 would be predicted to have an impact on dendritic excitability. Rat brain Kv2.1 polypeptides are phosphorylated extensively, leading to a dramatically increased molecular mass on sodium dodecyl sulfate gels. Phosphoamino acid analysis of Kv2.1 expressed in transfected cells and labeled in vivo with 32P shows that phosphorylation was restricted to serine residues and that a truncation mutant, DeltaC318, which lacks the last 318 amino acids in the cytoplasmic carboxyl terminus, was phosphorylated to a much lesser degree than was wild-type Kv2.1. Whole-cell patch-clamp studies showed that the voltage-dependence of activation of DeltaC318 was shifted to more negative membrane potentials than Kv2.1 without differences in macroscopic kinetics; however, the differences in the voltage-dependence of activation between Kv2.1 and DeltaC318 were eliminated by in vivo intracellular application of alkaline phosphatase, suggesting that these differences were due to differential phosphorylation. Similar analyses of other truncation and point mutants indicated that the phosphorylation sites responsible for the observed differences in voltage-dependent activation lie between amino acids 667 and 853 near the distal end of the Kv2.1 carboxyl terminus. Together, these parallel biochemical and electrophysiological results provide direct evidence that the voltage-dependent activation of the delayed-rectifier K+ channel Kv2. 1 can be modulated by direct phosphorylation of the channel protein; such modulation of Kv2.1 could dynamically regulate dendritic excitability.

Phosphorylation of the Kv2.1 K+ Channel Alters Voltage-Dependent Activation

Selective interaction of voltage-gated K+ channel β-subunits with α-subunits

Properties of Kv2.1 K+ channels expressed in transfected mammalian cells.

Glycosylation of ion channel proteins dramatically impacts channel function. Here we characterize the asparagine (N)-linked glycosylation of voltage-gated K+ channel α subunits in rat brain and transfected cells. We find that in brain Kv1.1, Kv1.2 and Kv1.4, which have a single consensus glycosylation site in the first extracellular interhelical domain, are N-glycosylated with sialic acid-rich oligosaccharide chains. Kv2.1, which has a consensus site in the second extracellular interhelical domain, is not N-glycosylated. This pattern of glycosylation is consistent between brain and transfected cells, providing compelling support for recent models relating oligosaccharide addition to the location of sites on polytopic membrane proteins. The extent of processing of N-linked chains on Kv1.1 and Kv1.2 but not Kv1.4 channels expressed in transfected cells differs from that seen for native brain channels, reflecting the different efficiencies of transport of K+ channel polypeptides from the endoplasmic reticulum to the Golgi apparatus. These data show that addition of sialic acid-rich N-linked oligosaccharide chains differs among highly related K+ channel α subunits, and given the established role of sialic acid in modulating channel function, provide evidence for differential glycosylation contributing to diversity of K+ channel function in mammalian brain.

Gongyi Shi

Papers

βSubunits Promote K+ Channel Surface Expression through Effects Early in Biosynthesis

Phosphorylation of the Kv2.1 K+ Channel Alters Voltage-Dependent Activation

Selective interaction of voltage-gated K+ channel β-subunits with α-subunits

Properties of Kv2.1 K+ channels expressed in transfected mammalian cells.

Differential Asparagine-Linked Glycosylation of Voltage-Gated K+ Channels in Mammalian Brain and in Transfected Cells