Structure and Function of Voltage-Dependent Ion Channel Regulatory β Subunits

The Saccharomyces cerevisiae MID1 gene product, Mid1, is composed of 548 amino acid residues, has four relatively hydrophobic segments named H1-H4, and functions as a Ca 2؉ -permeable, stretch-activated channel when expressed in mammalian cells. In some conditions Mid1 cooperates with Cch1, a yeast homolog of the ␣1 subunit of mammalian voltage-gated channels. To identify the important regions or amino acid residues necessary for Mid1 function, we employed in vitro sitedirected mutagenesis on H3 and H4 of Mid1 and expressed the resulting mutant genes in a mid1 null mutant to examine whether the mutant gene products are functional or not in vivo. Mutant Mid1 proteins lacking the whole H3 or H4 segment, H3De or H4De, did not complement the lethality and low Ca 2؉ accumulation activity of the mid1 mutant, although their localization and contents appeared to be normal, indicating that H3 and H4 are required for Mid1 function itself. Single amino acid exchange experiments on individual amino acid residues of H3 and H4 showed that 10 of 20 residues in H3 and 14 of 23 residues in H4 were important for the normal function of Mid1. In particular, we found four severe loss-of-function mutations, D341E, F356S, C373D, and C373R, and two interesting mutations leading to a high level of Ca 2؉ accumulation with a slightly low complementing activity, G342A and Y355A. The importance of these amino acid residues will be discussed.

Section: H3 and H4 May Not Be Required For The Localization Of The MImentioning

confidence: 94%

mentioning

confidence: 99%

Molecular Dissection of the Hydrophobic Segments H3 and H4 of the Yeast Ca2+ Channel Component Mid1

Tada¹,

Ohmori²,

Iida³

2003

“…2 In non-injected X. laevis oocytes, the level of expression of the endogenous NCS-1 was barely detectable in Western-blots and much lower than in human embryonic kidney 293 and COS-7 cells (see Fig. 1A).…”

Section: Expression Of Ncs-1 In Xenopusmentioning

confidence: 95%

“…Several types of Ca 2ϩ channels have been characterized (T, L, N, P/Q, and R) that appear to play a specific role in each of these functions. These channels share a common architecture composed of a major ␣ 1 subunit (for which ten genes are known) tightly associated with regulatory subunits ␣ 2 -␦ (four different genes), ␤ (four genes), and possibly ␥ (eight genes) in a functional multimeric complex (1)(2)(3)(4). This molecular diversity, further expanded by the existence of several splice variants for each of these genes, produces a large number of possible Ca 2ϩ channel subunit combinations with different pharmacological and biophysical properties and specific cellular and subcellular localization (5).…”

mentioning

confidence: 99%

Down-regulation of Voltage-gated Ca2+ Channels by Neuronal Calcium Sensor-1 Is β Subunit-specific

Rousset¹,

Cens²,

Gavarini³

et al. 2003

Neuronal Ca 2؉ sensor protein-1 (NCS-1) is a member of the Ca 2؉ binding protein family, with three functional Ca 2؉ binding EF-hands and an N-terminal myristoylation site. NCS-1 is expressed in brain and heart during embryonic and postnatal development. In neurons, NCS-1 facilitates neurotransmitter release, but both inhibition and facilitation of the Ca 2؉ current amplitude have been reported. In heart, NCS-1 co-immunoprecipitates with K ؉ channels and modulates their activity, but the potential effects of NCS-1 on cardiac Ca 2؉ channels have not been investigated. To directly assess the effect of NCS-1 on the various types of Ca 2؉ channels we have co-expressed NCS-1 in Xenopus oocytes, with Ca V 1.2, Ca V 2.1, and Ca V 2.2 Ca 2؉ channels, using various subunit combinations. The major effect of NCS-1 was to decrease Ca 2؉ current amplitude, recorded with the three different types of ␣ 1 subunit. When expressed with Ca V 2.1, the depression of Ca 2؉ current amplitude induced by NCS-1 was dependent upon the identity of the ␤ subunit expressed, with no block recorded without ␤ subunit or with the ␤ 3 subunit. Current-voltage and inactivation curves were also slightly modified and displayed a different specificity toward the ␤ subunits. Taken together, these data suggest that NCS-1 is able to modulate cardiac and neuronal voltage-gated Ca 2؉ channels in a ␤ subunit specific manner.

“…The correct functioning of various ion channels depends upon the interaction with auxiliary subunits (15) and with scaffolding proteins, which co-localize channels and regulatory components in subcellular compartments for improved efficiency of channel modulation (16 -18). The aim of this work was to investigate whether HCN1 channels interact with partner proteins in the brain and to check whether existing associations with any such proteins have a role in the specific cellular localization of the channels and modulation of channel function.…”

mentioning

confidence: 99%

Interaction of the Pacemaker Channel HCN1 with Filamin A

Gravante

Barbuti

Milanesi

et al. 2004

109

Pacemaker channels are encoded by the HCN gene family and are responsible for a variety of cellular functions including control of spontaneous activity in cardiac myocytes and control of excitability in different types of neurons. Some of these functions require specific membrane localization. Although several voltagegated channels are known to interact with intracellular proteins exerting auxiliary functions, no cytoplasmic proteins have been found so far to modulate HCN channels. Through the use of a yeast two-hybrid technique, here we showed that filamin A interacts with HCN1, an HCN isoform widely expressed in the brain, but not with HCN2 or HCN4. Filamin A is a cytoplasmic scaffold protein with actin-binding domains whose main function is to link transmembrane proteins to the actin cytoskeleton. Using several HCN1 C-terminal constructs, we identified a filamin A-interacting region of 22 amino acids located downstream from the cyclic nucleotide-binding domain; this region is not conserved in HCN2, HCN3, or HCN4. We also verified by immunoprecipitation from bovine brain that the filamin A-HCN1 interaction is functional in vivo. In filamin A-expressing cells (filamin ؉ ), HCN1 (but not HCN4) channels were expressed in hot spots, whereas they were evenly distributed on the membrane of cells lacking filamin A (filamin ؊ ) indicating that interaction with filamin A affects membrane localization. Also, in filamin ؊ cells the gating kinetics of HCN1 were strongly accelerated relative to filamin ؉ cells. The interaction with filamin A may contribute to localizing HCN1 channels to specific neuronal areas and to modulating channel activity.