The electrostatic interactions governing binding and electron transfer from cytochrome c(2) (cyt c(2)) to the reaction center (RC) from the photosynthetic bacteria Rhodobacter sphaeroides were studied by using site-directed mutagenesis to change the charges of residues on the RC surface. Charge-reversing mutations (acid --> Lys) decreased the binding affinity for cyt c(2). Dissociation constants, K(D) (0.3--250 microM), were largest for mutations of Asp M184 and nearby acid residues, identifying the main region for electrostatic interaction with cyt c(2). The second-order rate constants, k(2) (1--17 x 10(8) M(-1) s(-1)), increased with increasing binding affinity (log k(2) vs log 1/K(D) had a slope of approximately 0.4), indicating a transition state structurally related to the final complex. In contrast, first-order electron transfer rates, k(e), for the bound cyt did not change significantly (<3-fold), indicating that electron tunneling pathways were unchanged by mutation. Charge-neutralizing mutations (acid --> amide) showed changes in binding free energies of approximately 1/2 the free energy changes due to the corresponding charge-reversing mutations, suggesting that the charges in the docked complex remain well solvated. Charge-enhancing mutations (amide --> acid) produced free energy changes of the same magnitude (but opposite sign) as changes due to the charge-neutralizing mutations in the same region, indicating a diffuse electrostatic potential due to cyt c(2). A two-domain model is proposed, consisting of an electrostatic docking domain with charged surfaces separated by a water layer and a hydrophobic tunneling domain with atomic contacts that provide an efficient pathway for electron transfer.

Background: Missense mutations in Ca V ␣2␦1, an auxiliary subunit of cardiac L-type Ca V 1.2 channels, are associated with arrhythmias. Results:The reduction in the cell surface density of Ca V ␣2␦1 D550Y/Q917H was sufficient to impair Ca V 1.2 currents. Conclusion: Defects in the cell surface trafficking of Ca V ␣2␦1 mutants down-regulate L-type currents. Significance: CACNA2D1 genetic variants may trigger arrhythmias by reducing L-type Ca 2ϩ currents.

Cytochrome c2 (cyt) is the mobile electron donor to the reaction center (RC) in photosynthetic bacteria. The electrostatic interactions involved in the dynamics of docking of cyt onto the RC were examined by double mutant studies of the rates of electron transfer between six modified Rhodobacter sphaeroides RCs in which negatively charged acid residues were replaced with Lys and five modified Rhodobacter capsulatus Cyt c2 molecules in which positively charged Lys residues were replaced with Glu. We measured the second-order rate constant, k2, for electron transfer from the reduced cyt to the oxidized primary donor on the RC, which reflects the energy of the transition state for the formation of the active electron transfer complex. Strong interactions were found between Lys C99 and Asp M184/Glu M95, and between Lys C54 and Asp L261/Asp L257. The interacting residues were found to be located close to each other in the recently determined crystal structure of the cyt-RC complex [Axelrod, H., et al. (2002) J. Mol. Biol. (in press)]. The interaction energies were approximately inversely proportional to the distances between charges. These results support earlier suggestions [Tetreault, M., et al. (2001) Biochemistry 40, 8452-8462] that the structure of the transition state in solution resembles the structure of the cyt-RC complex in the cocrystal and indicate that specific electrostatic interactions facilitate docking of the cyt onto the RC in a configuration optimized for both binding and electron transfer. The specific interaction between Asp M184 and Lys C99 may help to nucleate short-range hydrophobic contacts.

Alteration in the L-type current density is one aspect of the electrical remodeling observed in patients suffering from cardiac arrhythmias. Changes in channel function could result from variations in the protein biogenesis, stability, post-translational modification, and/or trafficking in any of the regulatory subunits forming cardiac L-type Ca 2؉ channel complexes. Ca V ␣2␦1 is potentially the most heavily N-glycosylated subunit in the cardiac L-type Ca V 1.2 channel complex. Here, we show that enzymatic removal of N-glycans produced a 50-kDa shift in the mobility of cardiac and recombinant Ca V ␣2␦1 proteins. This change was also observed upon simultaneous mutation of the 16 Asn sites. Nonetheless, the mutation of only 6/16 sites was sufficient to significantly 1) reduce the steady-state cell surface fluorescence of Ca V ␣2␦1 as characterized by two-color flow cytometry assays and confocal imaging; 2) decrease protein stability estimated from cycloheximide chase assays; and 3) prevent the Ca V ␣2␦1-mediated increase in the peak current density and voltage-dependent gating of Ca V 1.2. Reversing the N348Q and N812Q mutations in the non-operational sextuplet Asn mutant protein partially restored Ca V ␣2␦1 function. Single mutation N663Q and double mutations N348Q/N468Q, N348Q/N812Q, and N468Q/N812Q decreased protein stability/synthesis and nearly abolished steady-state cell surface density of Ca V ␣2␦1 as well as the Ca V ␣2␦1-induced up-regulation of L-type currents. These results demonstrate that Asn-663 and to a lesser extent Asn-348, Asn-468, and Asn-812 contribute to protein stability/synthesis of Ca V ␣2␦1, and furthermore that N-glycosylation of Ca V ␣2␦1 is essential to produce functional L-type Ca 2؉ channels.The regulation of Ca 2ϩ influx in cardiac cells is critical to the generation of the force necessary for the myocardium to meet the physiological needs of the body (1). In resting cells, intracellular free ionized Ca 2ϩ is maintained at a low concentration (high nanomolar range) by the concerted action of mechanisms that prevent Ca 2ϩ entry, promote its extrusion (mostly via the Na ϩ /Ca 2ϩ exchanger), and ensure its storage in the sarcoplasmic reticulum (2). Ca 2ϩ entry is mediated mainly by the cardiac L-type Ca 2ϩ channel, which is central to the initiation of excitation-contraction coupling via Ca 2ϩ -induced Ca 2ϩ release from the sarcoplasmic reticulum. Regulation of the L-type Ca 2ϩ current has profound physiological significance. Indeed, alterations in density or the activation/inactivation gating of L-type Ca 2ϩ channels have been implicated in a variety of cardiovascular diseases (3, 4), including cardiac arrhythmias such as atrial fibrillation (5-8), heart failure (9, 10), and ischemic heart disease (10). The molecular mechanisms underlying changes in the activity of the L-type Ca 2ϩ channel remain under study for most pathologies.The L-type Ca V 1.2 channel belongs to the molecular family of high voltage-activated Ca V channels. High voltage-activated Ca V 1.2 channels are hetero-oligo...