Differential contribution by conserved glutamate residues to an ion‐selectivity site in the L‐type Ca²⁺ channel pore

Abstract

In voltage-gated cation channels, it is thought that residues responsible for ion-selectivity are located within the pore-lining SSl-SSZ segments.In this study, we compared the ion permeation properties of mutant calcium channels in which highly conserved glutamate residues, located at analogous positions in the SS2 regions of all four motifs, were individually replaced. All of the mutants exhibited a loss of selectivity for divalent over monovalent cations. However, the permeation properties of the individual mutants varied in a position dependent manner. The results provide strong evidence that these glutamate residues, positioned at equivalent locations in the aligned sequences, play significantly different roles in forming the selectivity barrier of the calcium channel, and are probably arranged in an asymmetrical manner inside the ion-conducting pore.

Molecular Pharmacology of Voltage-Dependent Calcium Channels

“…Further mutation studies have located amino acids that are the most critical for highaffinity w-CgTx-GVIA inhibition, within the putative extracellular loop between S5 and P region in repeat III. Considering the previous reports locating the glutamate residue with the highest Ca 2+ affinity in the repeat III P loop (149)(150)(151)(152)(153)(154) as well as the critical importance of motif III in the proton block of the channel (246,247), the results obtained are consistent with a direct pore-blocking mechanism (Fig. 2).…”

“…2). The glutamic acid residues in the P ("pore") region (or SS1-SS2 segment), which is critical for ion selectivity of Ca 21 channels (94,(150)(151)(152)(153)(154), are conserved among the a1-subunits (see below in the section V.C.3.). At the initial stage of the molecular approach, identification of Ca 2+ channels was more directed towards the L-type isoforms.…”

“…Structural determinants of E-C coupling (232) and channel activation kinetics (233) have been located in the primary structure of the DHP-sensitive L-type Ca 2+ channel al using chimeras between skeletal muscle als and cardiac a,c expressed in mdg myotubes. Furthermore, it has been demonstrated that the glutamic acid residues in the pore-forming "P" region of the four repeats, equivalently positioned in the aligned sequences, play different roles in the selective permeability of Ca 2+ channels (151)(152)(153)(154), in agreement with the data and model demonstrated for Na+ channels and K+ channels (150,(234)(235)(236)(237)(238)(239). The advantage of the approach is that structural evaluation is always carried out relying upon the functional channel proteins free from any conformational "destruction" caused during biochemical procedures.…”

Control of ion flux and selectivity by negatively charged residues in the outer mouth of rat sodium channels.

“…The permeation segments of Na+ and Ca2+ channels each contain two sets of charged residues, separated by three to four uncharged amino acids, in the so-called SS2 region (Noda, Suzuki, Numa & Stiihmer, 1989;Mikala, Bahinski, Yatani, Tang & Schwartz, 1993). The more N-terminal set includes acidic residues that are well recognized as critical determinants of selectivity (Heinemann et al 1992;Yang et al 1993).…”

Architecture of Ca2+ Channel Pore-lining Segments Revealed by Covalent Modification of Substituted Cysteines

“…Despite the large pore size of the calcium channel, the narrowest point being 6 Å (10 Ϫ10 m) in diameter (1), the channel is specifically permeable to calcium at concentrations as low as 1 M (2). For all calcium channels, the selectivity filter consists of four glutamate residues (3)(4)(5)(6)(7)(8)(9), one residing in each of the four motifs, with the exception of the newly cloned T-type channel family (10 -12), which contains two glutamates (motifs I and II) and two aspartates (motifs III and IV).…”