Cibulsky SM , Sather WA .

MH11717 NIMH NIH HHS, NS35245 NINDS NIH HHS , F31 MH011717 NIMH NIH HHS, R01 NS035245 NINDS NIH HHS

	Figure 1. Determination of side-chain orientation of pore-lining amino acids in the AAAA channel. (A) Superimposed two-electrode voltage clamp records illustrating the effects of MTSEA upon whole-cell Ca2+ channel currents. Currents (100 mM Li+) were elicited by step depolarization (150 ms) from the holding potential of −80 to −20 mV every 5 s. In each recording, after a stable baseline was established, MTSEA·Br (final concentration of MTSEA: 2 mM) was dissolved in the 100 mM Li+ solution and applied immediately to the oocyte via bath perfusion. (B) Time course of MTSEA action on peak inward Li+ currents.
	Figure 2. Summary of MTSEA block of substituted-cysteine mutants. (A) Percent block (±SEM) for the quadruple alanine mutant (AAAA) and cysteine substitutions in the AAAA channel. Block was calculated as the average for n = 4–9 oocytes, except for F1144C/AAAA, for which n = 3. Cysteine-substituted mutants differed from the AAAA channel in that block of the mutants was not reversible with wash, and their block was larger (P < 0.00005). Current was carried by Li+, and data were collected as described for Fig. 1. (B) Percent block (±SEM) for the EEEE channel and cysteine substitutions in the EEEE channel. Block was calculated as the average for n = 4–6 oocytes. Block of the cysteine-substituted mutant versions of the EEEE channel was not reversible with wash, unlike the rapidly reversible block of the EEEE channel, and block of the mutants was much larger than for the EEEE channel (P < 0.00002). Current was carried by Ba2+ (40 mM Ba2+ solution), holding potential was −80 mV, test potential was +20 mV, and 150-ms step depolarizations were applied every 15 s. Open bars indicate reversible block (AAAA and EEEE channels only), filled bars indicate −1 position cysteine substitution mutants, and hatched bars indicate 0-position cysteine substitutions. For reference, EEEE locus glutamates are located at positions 393, 736, 1145, and 1446 in motifs I–IV, respectively.
	Figure 3. Absence of divalent cation block or flux in the AAAA mutant Ca2+ channel. (A) High-affinity block of single-channel inward Li+ currents by external Ca2+ was abolished by replacing all four EEEE locus residues with alanine. Inward Li+ currents were studied in cell-attached patches on oocytes expressing either WT or AAAA channels. The patch pipet contained 100 mM Li+ plus various [Ca2+]. Control solution contained 3 nM Ca2+. Holding potential was −100 mV, channels were activated by 25–50-ms prepulses to +40 mV, and the illustrated single-channel currents were recorded at −40 mV. (B) Li+, but not Ba2+, permeates AAAA channels. Example current–voltage relationships and current records from two-electrode voltage-clamp recordings from individual oocytes expressing either WT or AAAA are illustrated. The bath solution contained either 100 mM Li+ (•) or 40 mM Ba2+ (□). Currents were recorded during step depolarizations from a holding potential of −100 mV (Li+) or −80 mV (Ba2+) every 15 s. Reversal potentials in Li+ were 3.7 ± 1.2 mV (n = 6) for WT and −8.0 ± 1.7 (n = 6) for AAAA. Smooth curves drawn through the data were best-fits calculated according to: I = (1/{1 + exp[(V0.5 − VM)/b]}) · ([A1 · exp(zFδVM/RT)] − {A2 · exp[−2zF(1 − δ)VM/RT]}), where I = membrane current, VM = membrane potential, V0.5 = half-activation voltage, b = Boltzmann slope factor, A1 and A2 are amplitude factors related to the concentrations and permeabilities of the internal and external ions, z = ion valence, δ = electrical distance, and F, R, and T are Faraday's constant, ideal gas law constant, and temperature (K).
	Figure 4. Whole-cell current densities in HEK293 cells. (A) Examples of current–voltage relationships from individual cells expressing WT (○), AAAA (▴), or GFP (control, □). The bath solution contained 100 mM Li+ and the pipet contained 135 mM Cs+. Currents were elicited every 5 s by step depolarizations from a holding potential of −100 mV. (B) Average current densities in WT-, AAAA-, and GFP-transfected (control) HEK293 cells. Inward current density was measured at the inward peak of the I-V, and outward current density was measured at +80 mV. Data plotted as mean ± SEM (n = 10–13).
	Figure 5. Concentration and voltage dependence of Ca2+ block of outward Li+ current carried through WT α1C channels in inside-out patches excised from HEK293 cells. (A) Internal Ca2+ blocked outward Li+ currents through single WT channels with high affinity. The bath solution contained 300 mM Li+ plus various [Ca2+], and the patch pipet contained 55 mM Li+. Control Li+ solution contained 3 nM Ca2+. Single-channel currents were recorded during a test depolarization to +20 mV from a holding potential of −100 mV. In some cases, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates are plotted as mean ± SEM versus [Ca2+] for n = 3–5. The linear regression fit through the on-rate data points has a slope of 2.3 × 108 M−1 s−1. The horizontal line fit to the off-rate data indicates the average off rate was 2,965 s−1. (B) Voltage dependence of internal Ca2+ block of outward Li+ currents through single WT channels. The bath solution contained 300 mM Li+ and 3 μM Ca2+, while the pipet solution contained 55 mM Li+. Single-channel currents were recorded during a step depolarization from the holding potential of −100 mV. In some experiments, a 25- or 50-ms prepulse to +100 mV was given to facilitate channel activation. Ca2+ on (○) and off (▪) rates plotted as mean ± SEM versus test potential (n = 4–5). The horizontal line fit to the on-rate data indicates the average on rate was 846 s−1. The exponential curve fit to the off-rate data indicates that off rate increased e-fold per 19 mV.
	Figure 6. Internal Ca2+ does not block outward single-channel Li+ currents through AAAA channels. Currents were studied in inside-out patches excised from HEK293 cells expressing AAAA channels (WT records shown for comparison). The bath solution contained 300 mM Li+ plus various [Ca2+], and the patch pipet contained 55 mM Li+. [Ca2+] was 3 nM in the control solution. Single-channel currents were recorded during a test depolarization to +20 mV, following a 25- or 50-ms prepulse to +100 mV. Holding potential was −100 mV.
	Figure 7. Outward Li+ currents through QQQQ channels are not blocked by internal Ca2+. Currents were studied in inside-out patches excised from HEK293 cells expressing QQQQ channels. The bath contained 300 mM Li+ plus either 3 nM Ca2+ (control) or 1 mM Ca2+, and the patch pipet contained 55 mM Li+. Single-channel currents were recorded during a test depolarization to +20 mV, following a 25- or 50-ms prepulse to +100 mV. Holding potential was −100 mV.

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed, Xenbase

Akabas, Acetylcholine receptor channel structure probed in cysteine-substitution mutants. 1992, Pubmed , Xenbase
Almers, Non-selective conductance in calcium channels of frog muscle: calcium selectivity in a single-file pore. 1984, Pubmed
Armstrong, Voltage-activated calcium channels that must be phosphorylated to respond to membrane depolarization. 1987, Pubmed
Armstrong, Ion permeation through calcium channels. A one-site model. 1991, Pubmed
Berjukow, Endogenous calcium channels in human embryonic kidney (HEK293) cells. 1996, Pubmed
Chen, Molecular basis of proton block of L-type Ca2+ channels. 1996, Pubmed , Xenbase
Chen, Aspartate substitutions establish the concerted action of P-region glutamates in repeats I and III in forming the protonation site of L-type Ca2+ channels. 1997, Pubmed , Xenbase
Cloues, Permeant ion binding affinity in subconductance states of an L-type Ca2+ channel expressed in Xenopus laevis oocytes. 2000, Pubmed , Xenbase
Dang, Ion channel selectivity through stepwise changes in binding affinity. 1998, Pubmed
Doyle, The structure of the potassium channel: molecular basis of K+ conduction and selectivity. 1998, Pubmed
Ellinor, Ca2+ channel selectivity at a single locus for high-affinity Ca2+ interactions. 1995, Pubmed , Xenbase
Friel, Voltage-gated calcium channels: direct observation of the anomalous mole fraction effect at the single-channel level. 1989, Pubmed
Fukushima, Currents carried by monovalent cations through calcium channels in mouse neoplastic B lymphocytes. 1985, Pubmed
Furukawa, Selectivities of dihydropyridine derivatives in blocking Ca(2+) channel subtypes expressed in Xenopus oocytes. 1999, Pubmed , Xenbase
Gomez, Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle. 1995, Pubmed
HODGKIN, The potassium permeability of a giant nerve fibre. 1955, Pubmed
Hao, Run-down of L-type Ca2+ channels occurs on the alpha 1 subunit. 1998, Pubmed
Heinemann, Calcium channel characteristics conferred on the sodium channel by single mutations. 1992, Pubmed , Xenbase
Hess, Mechanism of ion permeation through calcium channels. , Pubmed
Hess, Calcium channel selectivity for divalent and monovalent cations. Voltage and concentration dependence of single channel current in ventricular heart cells. 1986, Pubmed
Hille, Potassium channels as multi-ion single-file pores. 1978, Pubmed
Hullin, Calcium channel beta subunit heterogeneity: functional expression of cloned cDNA from heart, aorta and brain. 1992, Pubmed , Xenbase
Kim, Structural determinants of ion selectivity in brain calcium channel. 1993, Pubmed , Xenbase
Klöckner, Molecular studies of the asymmetric pore structure of the human cardiac voltage- dependent Ca2+ channel. Conserved residue, Glu-1086, regulates proton-dependent ion permeation. 1996, Pubmed , Xenbase
Kostyuk, Some predictions concerning the calcium channel model with different conformational states. 1986, Pubmed
Kuo, Ion permeation through the L-type Ca2+ channel in rat phaeochromocytoma cells: two sets of ion binding sites in the pore. 1993, Pubmed
Kuo, Characterization of the high-affinity Ca2+ binding sites in the L-type Ca2+ channel pore in rat phaeochromocytoma cells. 1993, Pubmed
Kuo, Block of the L-type Ca2+ channel pore by external and internal Mg2+ in rat phaeochromocytoma cells. 1993, Pubmed
Lambert, Maintained L-type Ca2+ channel activity in excised patches of PTX-treated granule cells of the cerebellum. 1995, Pubmed
Lansman, Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore. 1986, Pubmed
Liman, Subunit stoichiometry of a mammalian K+ channel determined by construction of multimeric cDNAs. 1992, Pubmed , Xenbase
Lu, Architecture of a K+ channel inner pore revealed by stoichiometric covalent modification. 1999, Pubmed , Xenbase
Lux, Na+ currents through low-voltage-activated Ca2+ channels of chick sensory neurones: block by external Ca2+ and Mg2+. 1990, Pubmed
Matsuda, Sodium conductance in calcium channels of guinea-pig ventricular cells induced by removal of external calcium ions. 1986, Pubmed
McCleskey, Calcium channel permeation: A field in flux. 1999, Pubmed
Mikala, Differential contribution by conserved glutamate residues to an ion-selectivity site in the L-type Ca2+ channel pore. 1993, Pubmed
Mikami, Primary structure and functional expression of the cardiac dihydropyridine-sensitive calcium channel. 1989, Pubmed , Xenbase
Miller, Ionic hopping defended. 1999, Pubmed
Neyton, Potassium blocks barium permeation through a calcium-activated potassium channel. 1988, Pubmed
Neyton, Discrete Ba2+ block as a probe of ion occupancy and pore structure in the high-conductance Ca2+ -activated K+ channel. 1988, Pubmed
Nonner, Ion permeation and glutamate residues linked by Poisson-Nernst-Planck theory in L-type calcium channels. 1998, Pubmed
Parent, Glutamate substitution in repeat IV alters divalent and monovalent cation permeation in the heart Ca2+ channel. 1995, Pubmed , Xenbase
Polo-Parada, Block of N-type calcium channels in chick sensory neurons by external sodium. 1997, Pubmed
Rosenberg, Characterization and localization of two ion-binding sites within the pore of cardiac L-type calcium channels. 1991, Pubmed
Sather, Distinctive biophysical and pharmacological properties of class A (BI) calcium channel alpha 1 subunits. 1993, Pubmed , Xenbase
Sjöholm, Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity. 1993, Pubmed
Tang, Molecular localization of ion selectivity sites within the pore of a human L-type cardiac calcium channel. 1993, Pubmed
Yang, Molecular determinants of Ca2+ selectivity and ion permeation in L-type Ca2+ channels. 1993, Pubmed , Xenbase
Yatani, Single amino acid substitutions within the ion permeation pathway alter single-channel conductance of the human L-type cardiac Ca2+ channel. 1994, Pubmed , Xenbase
Yu, Endogenous voltage-gated potassium channels in human embryonic kidney (HEK293) cells. 1998, Pubmed
Yue, Permeation in the dihydropyridine-sensitive calcium channel. Multi-ion occupancy but no anomalous mole-fraction effect between Ba2+ and Ca2+. 1990, Pubmed
Zhu, Identification of endogenous outward currents in the human embryonic kidney (HEK 293) cell line. 1998, Pubmed