Carol Deutsch

Folding zones inside the ribosomal exit tunnel

Helicity of membrane proteins can be manifested inside the ribosome tunnel, but the determinants of compact structure formation inside the tunnel are largely unexplored. Using an extended nascent peptide as a molecular tape measure of the ribosomal tunnel, we have previously demonstrated helix formation inside the tunnel. Here, we introduce a series of consecutive polyalanines into different regions of the tape measure to monitor the formation of compact structure in the nascent peptide. We find that the formation of compact structure of the polyalanine sequence depends on its location. Calculation of free energies for the equilibria between folded and unfolded nascent peptides in different regions of the tunnel shows that there are zones of secondary structure formation inside the ribosomal exit tunnel. These zones may have an active role in nascent-chain compaction.

Electrostatics in the ribosomal tunnel modulate chain elongation rates.

Electrostatic potentials along the ribosomal exit tunnel are nonuniform and negative. The significance of electrostatics in the tunnel remains relatively uninvestigated, yet they are likely to play a role in translation and secondary folding of nascent peptides. To probe the role of nascent peptide charges in ribosome function, we used a molecular tape measure that was engineered to contain different numbers of charged amino acids localized to known regions of the tunnel and measured chain elongation rates. Positively charged arginine or lysine sequences produce transient arrest (pausing) before the nascent peptide is fully elongated. The rate of conversion from transiently arrested to full-length nascent peptide is faster for peptides containing neutral or negatively charged residues than for those containing positively charged residues. We provide experimental evidence that extraribosomal mechanisms do not account for this charge-specific pausing. We conclude that pausing is due to charge-specific interactions between the tunnel and the nascent peptide.

C-type inactivation of a voltage-gated K+ channel occurs by a cooperative mechanism

The lymphocyte voltage-gated K+ channel, Kv1.3, inactivates by a C-type process. We have elucidated the molecular basis for this process using a kinetic analysis of wild-type and mutant (A413V) Kv1.3 homo- and heteromultimeric currents in a mammalian lymphoid expression system. The medians of the measured inactivation time constants for wild-type and A413V homotetrameric currents are 204 and 4 ms, respectively. Co-expression of these subunits produces heteromultimeric channels manifesting inactivation kinetics intermediate between those of wild-type and A413V homomultimers. We have considered several models in which each subunit acts either independently or cooperatively to produce the observed inactivation kinetics. The cooperative model gives excellent fits to the data for any heteromultimeric composition of subunits, clearly distinguishing it from the independent models. In the cooperative model, the difference in free energy between the open and inactivated states of the channel is invariant with subunit composition and equals approximately 1.5 kcal/mol. Each subunit contributes equally to the activation free energy for transitions between open and inactivated states, with an A413V subunit decreasing the free energy barrier for inactivation (and for recovery from inactivation) by approximately 0.6 kcal/mol. Our results are consistent with a physical model in which the outer mouth of the channel constricts during C-type inactivation (G. Yellen, D. Sodickson, T. Chen, and M.E. Jurman, 1994, Biophys. J. 66:1068-1075).

Voltage-gated potassium conductance in human T lymphocytes stimulated with phorbol ester.

The whole‐cell patch‐clamp method was used to study the voltage‐gated K+ conductance of human peripheral blood T lymphocytes. After entry into whole‐cell recording mode, there are time‐dependent changes in some properties of the conductance. Over the first 10‐30 min, the threshold for activation shifts about 10 mV more negative, and the rates of activation and inactivation increase. Inactivation is less strongly voltage dependent than activation or deactivation. Lymphocytes were stimulated to proliferate in culture with the tumour promoter 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA). No changes in K+ conductance were observed in the first few hours of TPA stimulation. At 24 h after mitogen addition, TPA‐treated cells were found to have 1.7‐fold greater average voltage‐gated K+ conductance than unstimulated control cells. At 48 h, TPA‐stimulated cells had the same average K+ conductance as at 24 h, even though the cells were now much increased in size, as measured by cell capacitance. DNA synthesis by cultures stimulated with TPA, phytohaemagglutinin or succinyl concanavalin A was depressed by the addition of 0.1 mM‐quinine at any point in the culture period. In the first 20 h after mitogen addition, DNA synthesis was more effectively inhibited by quinine than if the drug were added later. Cell proliferation was equally sensitive to quinine regardless of mitogen.

Recovery from C-type inactivation is modulated by extracellular potassium

Extracellular potassium modulates recovery from C-type inactivation of Kv1.3 in human T lymphocytes. The results of whole-cell patch clamp recordings show that there is a linear increase in recovery rate with increasing [K+]o. An increase from 5 to 150 mM K+o causes a sixfold acceleration of recovery rate at a holding potential of -90 mV. Our results suggest that 1) a low-affinity K+ binding site is involved in recovery, 2) the rate of recovery increases with hyperpolarization, 3) potassium must bind to the channel before inactivation to speed its recovery, and 4) recovery rate depends on external [K+] but not on the magnitude of the driving force through open channels. We present a model in which a bound K+ ion destabilizes the inactivated state to increase the rate of recovery of C-type inactivation, thereby providing a mechanism for autoregulation of K+ channel activity. The ability of K+ to regulate its own conductance may play a role in modulating voltage-dependent immune function.

The Birth of a Channel

An ion channel protein begins life as a nascent peptide inside a ribosome, moves to the endoplasmic reticulum where it becomes integrated into the lipid bilayer, and ultimately forms a functional unit that conducts ions in a well-regulated fashion. Here, I discuss the nascent peptide and its tasks as it wends its way through ribosomal tunnels and exit ports, through translocons, and into the bilayer. We are just beginning to explore the sequence of these events, mechanisms of ion channel structure formation, when biogenic decisions are made, and by which participants. These decisions include when to exit the endoplasmic reticulum and with whom to associate. Such issues govern the expression of ion channels at the cell surface and thus the electrical activity of a cell.

Voltage-gated K+ Channels Contain Multiple Intersubunit Association Sites

A domain in the cytoplasmic NH2 terminus of voltage-gated K+ channels supervises the proper assembly of specific tetrameric channels (Li, M., Jan, J. M., and Jan, L. Y. (1992) Science 257, 1225-1230; Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P. (1993) Neuron 11, 67-76). It is referred to as a first tetramerization domain, or T1 (Shen, N. V., Chen X., Boyer, M. M., and Pfaffinger, P. (1993) Neuron 11, 67-76). However, a deletion mutant of Kv1.3 that lacks the first 141 amino acids, Kv1.3 (T1−) forms functional channels, suggesting that additional association sites in the central core of Kv1.3 mediate oligomerization. To characterize these sites, we have tested the abilities of cRNA Kv1.3 (T1−) fragments co-injected with Kv1.3 (T1−) to suppress current in Xenopus oocytes. The fragments include portions of the six putative transmembrane segments, S1 through S6, specifically: S1, S1-S2, S1-S2-S3, S2-S3, S2-S3-S4, S3-S4, S3-S4-S5, S2 through COOH, S3 through COOH, S4 through COOH, and S5-S6-COOH. Electrophysiologic experiments show that the fragments S1-S2-S3, S3-S4-S5, S2 through COOH, and S3 through COOH strongly suppress Kv1.3 (T1−) current, while others do not. Suppression of expressed current is due to specific effects of the translated peptide Kv1.3 fragments, as validated by in vivo immunoprecipitation studies of a strong suppressor and a nonsuppressor. Pulse-chase experiments indicate that translation of truncated peptide fragments neither prevents translation of Kv1.3 (T1−) nor increases its rate of degradation. Co-immunoprecipitation experiments suggest that suppression involves direct association of a peptide fragment with Kv1.3 (T1−). Fragments that strongly suppress Kv1.3 (T1−) also suppress an analogous NH2-terminal deletion mutant of Kv2.1 (Kv2.1 (ΔN139)), an isoform belonging to a different subfamily. Our results indicate that sites in the central core of Kv1.3 facilitate intersubunit association and that there are suppression sites in the central core, which are promiscuous across voltage-gated K+ channel subfamilies.

Tertiary interactions within the ribosomal exit tunnel

Although tertiary folding of whole protein domains is prohibited by the cramped dimensions of the ribosomal tunnel, dynamic tertiary interactions may permit folding of small elementary units within the tunnel. To probe this possibility, we used a β-hairpin and an α-helical hairpin from the cytosolic N terminus of a voltage-gated potassium channel and determined a probability of folding for each at defined locations inside and outside the tunnel. Minimalist tertiary structures can form near the exit port of the tunnel, a region that provides an entropic window for initial exploration of local peptide conformations. Tertiary subdomains of the nascent peptide fold sequentially, but not independently, during translation. These studies offer an approach for diagnosing the molecular basis for folding defects that lead to protein malfunction and provide insight into the role of the ribosome during early potassium channel biogenesis.

Evidence for Dimerization of Dimers in K+ Channel Assembly

Voltage-gated K+ channels are tetrameric, but how the four subunits assemble is not known. We analyzed inactivation kinetics and peak current levels elicited for a variety of wild-type and mutant Kv1.3 subunits, expressed singly, in combination, and as tandem constructs, to show that 1) the dominant pathway involves a dimerization of dimers, and 2) dimer-dimer interaction may involve interaction sites that differ from those involved in monomer-monomer association. Moreover, using nondenaturing gel electrophoresis, we detected dimers and tetramers, but not trimers, in the translation reaction of Kv1.3 monomers.

Cross Talk between Activation and Slow Inactivation Gates of Shaker Potassium Channels

This study addresses the energetic coupling between the activation and slow inactivation gates of Shaker potassium channels. To track the status of the activation gate in inactivated channels that are nonconducting, we used two functional assays: the accessibility of a cysteine residue engineered into the protein lining the pore cavity (V474C) and the liberation by depolarization of a Cs+ ion trapped behind the closed activation gate. We determined that the rate of activation gate movement depends on the state of the inactivation gate. A closed inactivation gate favors faster opening and slower closing of the activation gate. We also show that hyperpolarization closes the activation gate long before a channel recovers from inactivation. Because activation and slow inactivation are ubiquitous gating processes in potassium channels, the cross talk between them is likely to be a fundamental factor in controlling ion flux across membranes.