Papers by Richard Aldrich
KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+.... more KCa2.1-3 Ca2+-activated K+-channels (SK) require calmodulin to gate in response to cellular Ca2+. A model for SK gating proposes that the N-terminal domain (N-lobe) of calmodulin is required for activation, but an immobile C-terminal domain (C-lobe) has constitutive, Ca2+-independent binding. Although structures support a domain-driven hypothesis of SK gate activation by calmodulin, only a partial understanding is possible without measuring both channel activity and protein binding. We measured SK2 (KCa2.2) activity using inside-out patch recordings. Currents from calmodulin-disrupted SK2 channels can be restored with exogenously applied calmodulin. We find that SK2 activity only approaches full activation with full-length calmodulin with both an N- and C-lobe. We measured calmodulin binding to a C-terminal SK peptide (SKp) using both composition-gradient multi-angle light scattering and tryptophan emission spectra. Isolated lobes bind to SKp with high affinity, but isolated lobes d...
The Journal of Neuroscience, Oct 1, 1990
Forskolin (FSK) directly blocks a distinct class of voltagedependent K+ channels in pheochromocyt... more Forskolin (FSK) directly blocks a distinct class of voltagedependent K+ channels in pheochromocytoma cells. We have studied the biophysical mechanism of FSK action on these channels. The mean open duration decreased linearly with [FSK], indicating that a single molecule of FSK interacts with a single open K+ channel. FSK did not alter the voltage dependence of activation or the latency to first opening. Wholecell currents in the presence of FSK did not show a rising phase in tail currents, suggesting that FSK-bound channels can close. We used a kinetic scheme in which FSK binds preferentially to the open state of the channel to describe its interaction with the K+ channel. This scheme is analogous to the modulated receptor hypothesis used to describe the interaction of local anesthetics with voltage-dependent Na+ channels. Forskolin (FSK), a diterpene isolated from the roots of Coleus forskohlii,
The Journal of Neuroscience, Nov 1, 1989
Growth factors and hormones induce differentiation of clonal pheochromocytoma (PC1 2) cells, whic... more Growth factors and hormones induce differentiation of clonal pheochromocytoma (PC1 2) cells, which are derived from rat adrenal medulla chromaffin cells. On application of nerve growth factor (NGF), PC1 2 cells extend neurites and express properties characteristic of autonomic ganglion cells. In contrast, incubation of PC12 cells with a corticosteroid, dexamethasone (DEX), does not induce neurite formation but causes an increase in tyrosine hydroxylase activity, suggesting that the cells become chromaffin cell-like. The ability of NGF and DEX to regulate ionic currents has been less well studied. Therefore, we examined how long-term NGF and DEX treatments affected voltage-dependent Na, Ca, and K currents in PC12 cells. Voltage-dependent Na currents were observed only in a small fraction of the PC12 cells in the absence of NGF or DEX. Virtually all NGF-treated cells expressed Na currents within 7 d. DEX increased the number of cells expressing voltage-dependent Na current slowly over 3 weeks, but, unlike NGF, DEX did not change Na current density. Both NGF and DEX also affected the expression of voltage-dependent Ca currents. Most of the untreated cells had only sustained, high-threshold voltage-dependent Ca currents. Chronic application of NGF or DEX increased the fractions of the cells that showed transient, low-threshold T-type Ca currents in addition to the high-threshold currents. The T-type Ca current density, however, increased significantly only in NGF-treated cells. Neither DEX nor NGF affected the voltage-dependent K currents. These results suggest that the expression of voltage-dependent Na and Ca currents are differentially regulated by NGF and DEX. The distinction between treated and untreated cells decreased after 3 weeks in culture as older untreated cells showed increases in the fraction of cells expressing both Na and low-threshold Ca currents. A PC1 2 subline selected for adherence to uncoated plastic also showed increased fraction of cells expressing these currents, suggesting that interactions with substrate may also influence ionic current expression. Peptide growth factors and steroid hormones play important roles in the differentiation of excitable cells such as neurons and endocrine cells. Differentiation of excitable cells is often accom
Neuron, Dec 1, 1989
We have used P element-mediated germline transformation to express ShB channels in Shaker mutant ... more We have used P element-mediated germline transformation to express ShB channels in Shaker mutant Drosophila and have examined their properties by patchclamp of embryonic myotubes. The transformed ShB cDNA was placed under the transcriptional control of a heat shock promoter (hsp70). Northern blots revealed that transformed DNA is efficiently transcribed in response to heat shock. Cultured myotubes from the transformants produced large A-type potassium currents in response to heat shock. Although qualitatively similar to native Shaker A-currrents in wild-type myotubes, transformant A-currrent inactivates more rapidly and recovers from inactivation more rapidly, similar to ShB channels expressed in Xenopus oocytes. Unlike the channels in oocytes, however, the transformant A-current is insensitive to 50 nM charybdotoxin.
ABSTRACTNew proteomics methods make it possible to determine protein interaction maps at the prot... more ABSTRACTNew proteomics methods make it possible to determine protein interaction maps at the proteome scale without the need for genetically encoded tags, opening up new organisms and tissue types to investigation. Current molecular and computational methods are oriented towards protein complexes that are soluble, stable, and discrete. However, the mammalian brain, among the most complicated and most heavily studied tissue types, derives many of its unique functions from protein interactions that are neither discrete nor soluble. Proteomics investigations into the global protein interaction landscape of the brain have therefore leveraged non-proteomics datasets to supplement their experiments. Here, we develop a novel, integrative proteomics pipeline and apply it to infer a global map of functional protein neighborhoods in the mouse brain without the aid of external datasets. By leveraging synaptosome enrichment and interactomics methods that target both soluble and insoluble protei...
IUPHAR/BPS Guide to Pharmacology CITE, 2019
Calcium- and sodium- activated potassium channels are members of the 6TM family of K channels whi... more Calcium- and sodium- activated potassium channels are members of the 6TM family of K channels which comprises the voltage-gated KV subfamilies, including the KCNQ subfamily, the EAG subfamily (which includes herg channels), the Ca2+-activated Slo subfamily (actually with 6 or 7TM) and the Ca2+- and Na+-activated SK subfamily (nomenclature as agreed by the NC-IUPHAR Subcommittee on Calcium- and sodium-activated potassium channels [124]). As for the 2TM family, the pore-forming a subunits form tetramers and heteromeric channels may be formed within subfamilies (e.g. KV1.1 with KV1.2; KCNQ2 with KCNQ3).
Biophysical Journal, 2019
The Journal of general physiology, 1994
Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB d... more Voltage-dependent gating behavior of Shaker potassium channels without N-type inactivation (ShB delta 6-46) expressed in Xenopus oocytes was studied. The voltage dependence of the steady-state open probability indicated that the activation process involves the movement of the equivalent of 12-16 electronic charges across the membrane. The sigmoidal kinetics of the activation process, which is maintained at depolarized voltages up to at least +100 mV indicate the presence of at least five sequential conformational changes before opening. The voltage dependence of the gating charge movement suggested that each elementary transition involves 3.5 electronic charges. The voltage dependence of the forward opening rate, as estimated by the single-channel first latency distribution, the final phase of the macroscopic ionic current activation, the ionic current reactivation and the ON gating current time course, showed movement of the equivalent of 0.3 to 0.5 electronic charges were associat...
Journal of General Physiology, 1990
The voltage-dependent gating of transient A2-type potassium channels from primary cultures of lar... more The voltage-dependent gating of transient A2-type potassium channels from primary cultures of larval Drosophila central nervous system neurons was studied using whole-cell and single-channel voltage clamp. A2 channels are genetically distinct from the Shaker A1 channels observed in Drosophila muscle, and differ in single-channel conductance, voltage dependence, and gating kinetics. Single A2 channels were recorded and analyzed at -30, -10, +10, and +30 mV. The channels opened in bursts in response to depolarizing steps, with three to four openings per burst and two to three bursts per 480-ms pulse (2.8-ms burst criterion). Mean open durations were in a range of 2-4 ms and mean burst durations in a range of 9-17 ms. With the exception of the first latency distributions, none of the means of the distributions measured showed a consistent trend with voltage. Macroscopic inactivation of both whole-cell A currents and ensemble average currents of single A2 channels was well fitted by a s...
The molecular and cellular basis of novelty is a major open question in evolutionary biology. Unt... more The molecular and cellular basis of novelty is a major open question in evolutionary biology. Until very recently, the vast majority of cellular phenomena were so difficult to sample that cross-species studies of biochemistry were rare and comparative analysis at the level of biochemical systems was almost impossible. Recent advances in systems biology are changing what is possible, however, and comparative phylogenetic methods that can handle this new data are wanted. Here, we introduce the term “phylogenetic latent variable models” (PLVMs, pronounced “plums”) for a class of models that has recently been used to infer the evolution of cellular states from systems-level molecular data, and develop a new parameterization and fitting strategy that is useful for comparative inference of biochemical networks. We deploy this new framework to infer the ancestral states and evolutionary dynamics of protein-interaction networks by analyzing >16,000 predominantly metazoan co-fractionation...
The Journal of Neuroscience, 1990
Forskolin (FSK) directly blocks a distinct class of voltagedependent K+ channels in pheochromocyt... more Forskolin (FSK) directly blocks a distinct class of voltagedependent K+ channels in pheochromocytoma cells. We have studied the biophysical mechanism of FSK action on these channels. The mean open duration decreased linearly with [FSK], indicating that a single molecule of FSK interacts with a single open K+ channel. FSK did not alter the voltage dependence of activation or the latency to first opening. Wholecell currents in the presence of FSK did not show a rising phase in tail currents, suggesting that FSK-bound channels can close. We used a kinetic scheme in which FSK binds preferentially to the open state of the channel to describe its interaction with the K+ channel. This scheme is analogous to the modulated receptor hypothesis used to describe the interaction of local anesthetics with voltage-dependent Na+ channels. Forskolin (FSK), a diterpene isolated from the roots of Coleus forskohlii,
The Journal of Neuroscience, 1989
Growth factors and hormones induce differentiation of clonal pheochromocytoma (PC1 2) cells, whic... more Growth factors and hormones induce differentiation of clonal pheochromocytoma (PC1 2) cells, which are derived from rat adrenal medulla chromaffin cells. On application of nerve growth factor (NGF), PC1 2 cells extend neurites and express properties characteristic of autonomic ganglion cells. In contrast, incubation of PC12 cells with a corticosteroid, dexamethasone (DEX), does not induce neurite formation but causes an increase in tyrosine hydroxylase activity, suggesting that the cells become chromaffin cell-like. The ability of NGF and DEX to regulate ionic currents has been less well studied. Therefore, we examined how long-term NGF and DEX treatments affected voltage-dependent Na, Ca, and K currents in PC12 cells. Voltage-dependent Na currents were observed only in a small fraction of the PC12 cells in the absence of NGF or DEX. Virtually all NGF-treated cells expressed Na currents within 7 d. DEX increased the number of cells expressing voltage-dependent Na current slowly over 3 weeks, but, unlike NGF, DEX did not change Na current density. Both NGF and DEX also affected the expression of voltage-dependent Ca currents. Most of the untreated cells had only sustained, high-threshold voltage-dependent Ca currents. Chronic application of NGF or DEX increased the fractions of the cells that showed transient, low-threshold T-type Ca currents in addition to the high-threshold currents. The T-type Ca current density, however, increased significantly only in NGF-treated cells. Neither DEX nor NGF affected the voltage-dependent K currents. These results suggest that the expression of voltage-dependent Na and Ca currents are differentially regulated by NGF and DEX. The distinction between treated and untreated cells decreased after 3 weeks in culture as older untreated cells showed increases in the fraction of cells expressing both Na and low-threshold Ca currents. A PC1 2 subline selected for adherence to uncoated plastic also showed increased fraction of cells expressing these currents, suggesting that interactions with substrate may also influence ionic current expression. Peptide growth factors and steroid hormones play important roles in the differentiation of excitable cells such as neurons and endocrine cells. Differentiation of excitable cells is often accom
Biochemistry, May 4, 2018
Drugs do not act solely by canonical ligand-receptor binding interactions. Amphiphilic drugs part... more Drugs do not act solely by canonical ligand-receptor binding interactions. Amphiphilic drugs partition into membranes thereby perturbing bulk lipid bilayer properties and possibly altering the function of membrane proteins. Distinguishing membrane perturbation from more direct protein-ligand interactions is an ongoing challenge in chemical biology. Herein, we present one strategy for doing so using dimeric 6-bromo-2-mercaptotryptamine (BrMT) and synthetic analogs. BrMT is a chemically unstable marine snail toxin that has unique effects on voltage-gated K+ channel proteins, making it an attractive medicinal chemistry lead. BrMT is amphiphilic and perturbs lipid bilayers, raising the question of whether its action against K+ channels is merely a manifestation of membrane perturbation. To determine whether medicinal chemistry approaches to improve BrMT might be viable, we synthesized BrMT and 11 analogs and determined their activities in parallel assays measuring K+ channel activity an...
Proceedings of the National Academy of Sciences of the United States of America, Apr 15, 2018
The Ca-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is ... more The Ca-sensing protein calmodulin (CaM) is a popular model of biological ion binding since it is both experimentally tractable and essential to survival in all eukaryotic cells. CaM modulates hundreds of target proteins and is sensitive to complex patterns of Caexposure, indicating that it functions as a sophisticated dynamic transducer rather than a simple on/off switch. Many details of this transduction function are not well understood. Fourier transform infrared (FTIR) spectroscopy, ultrafast 2D infrared (2D IR) spectroscopy, and electronic structure calculations were used to probe interactions between bound metal ions (Caand several trivalent lanthanide ions) and the carboxylate groups in CaM's EF-hand ion-coordinating sites. Since Tbis commonly used as a luminescent Caanalog in studies of protein-ion binding, it is important to characterize distinctions between the coordination of Caand the lanthanides in CaM. Although functional assays indicate that Tbfully activates many ...
The Journal of general physiology, Jan 5, 2018
Journal of General Physiology, 2016
A critical but often overlooked question in the study of ligands binding to proteins is whether t... more A critical but often overlooked question in the study of ligands binding to proteins is whether the parameters obtained from analyzing binding data are practically identifiable (PI), i.e., whether the estimates obtained from fitting models to noisy data are accurate and unique. Here we report a general approach to assess and understand binding parameter identifiability, which provides a toolkit to assist experimentalists in the design of binding studies and in the analysis of binding data. The partial fraction (PF) expansion technique is used to decompose binding curves for proteins with n ligand-binding sites exactly and uniquely into n components, each of which has the form of a one-site binding curve. The association constants of the PF component curves, being the roots of an n-th order polynomial, may be real or complex. We demonstrate a fundamental connection between binding parameter identifiability and the nature of these one-site association constants: all binding parameters...
Journal of General Physiology, 2016
Understanding the interactions of proteins with their ligands requires knowledge of molecular pro... more Understanding the interactions of proteins with their ligands requires knowledge of molecular properties, such as binding site affinities and the effects that binding at one site exerts on binding at other sites (cooperativity). These properties cannot be measured directly and are usually estimated by fitting binding data with models that contain these quantities as parameters. In this study, we present a general method for answering the critical question of whether these parameters are identifiable (i.e., whether their estimates are accurate and unique). In cases in which parameter estimates are not unique, our analysis provides insight into the fundamental causes of nonidentifiability. This approach can thus serve as a guide for the proper design and analysis of protein–ligand binding experiments. We show that the equilibrium total binding relation can be reduced to a conserved mathematical form for all models composed solely of bimolecular association reactions and to a related, ...
Proceedings of the National Academy of Sciences of the United States of America, Jan 16, 2016
Calmodulin (CaM) is a Ca(2+)-sensing protein that is highly conserved and ubiquitous in eukaryote... more Calmodulin (CaM) is a Ca(2+)-sensing protein that is highly conserved and ubiquitous in eukaryotes. In humans it is a locus of life-threatening cardiomyopathies. The primary function of CaM is to transduce Ca(2+) concentration into cellular signals by binding to a wide range of target proteins in a Ca(2+)-dependent manner. We do not fully understand how CaM performs its role as a high-fidelity signal transducer for more than 300 target proteins, but diversity among its four Ca(2+)-binding sites, called EF-hands, may contribute to CaM's functional versatility. We therefore looked at the conservation of CaM sequences over deep evolutionary time, focusing primarily on the four EF-hand motifs. Expanding on previous work, we found that CaM evolves slowly but that its evolutionary rate is substantially faster in fungi. We also found that the four EF-hands have distinguishing biophysical and structural properties that span eukaryotes. These results suggest that all eukaryotes require C...
The Journal of general physiology, 2015
Uploads
Papers by Richard Aldrich