Robert O. Blaustein

Parasympathetic response in chick myocytes and mouse heart is controlled by SREBP.

Parasympathetic stimulation of the heart, which provides protection from arrhythmias and sudden death, involves activation of the G protein-coupled inward rectifying K+ channel GIRK1/4 and results in an acetylcholine-sensitive K+ current, I KACh. We describe a unique relationship between lipid homeostasis, the lipid-sensitive transcription factor SREBP-1, regulation of the cardiac parasympathetic response, and the development of ventricular arrhythmia. In embryonic chick atrial myocytes, lipid lowering by culture in lipoprotein-depleted serum increased SREBP-1 levels, GIRK1 expression, and I KACh activation. Regulation of the GIRK1 promoter by SREBP-1 and lipid lowering was dependent on interaction with 2 tandem sterol response elements and an upstream E-box motif. Expression of dominant negative SREBP-1 (DN-SREBP-1) reversed the effect of lipid lowering on I KACh and GIRK1. In SREBP-1 knockout mice, both the response of the heart to parasympathetic stimulation and the expression of GIRK1 were reduced compared with WT. I KACh, attenuated in atrial myocytes from SREBP-1 knockout mice, was stimulated by SREBP-1 expression. Following myocardial infarction, SREBP-1 knockout mice were twice as likely as WT mice to develop ventricular tachycardia in response to programmed ventricular stimulation. These results demonstrate a relationship between lipid metabolism and parasympathetic response that may play a role in arrhythmogenesis.

The N-terminal half of the heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayers

The heavy chain of botulinum type A neurotoxin forms channels in planar phospholipid bilayer membranes. Channel activity is confined to the N‐terminal half of this chain; the C‐terminal half is inactive. Channel activity is stimulated by low pH (4.5–5.5) on the cis side (the side to which protein is added), neutral pH on the opposite (trans) side, and cis positive voltages. These findings are strikingly similar to those previously reported for analogous fragments of diphtheria and tetanus toxins.

Tethered blockers as molecular 'tape measures' for a voltage-gated K+ channel

The propagation of electrical signals in excitable cells is orchestrated by a molecular family of voltage-dependent ion channel proteins. These K+, Na+, and Ca++ channels are all composed of four identical or similar units, each containing six transmembrane segments (S1–S6) in a roughly four-fold symmetric structure. The S5–S6 sequences fold into a central pore unit, which is surrounded by a voltage-gating module composed of S1–S4. The recent structure of KcsA, a two-transmembrane bacterial K+ channel, illuminates the physical character of the pore unit, but little is known about the arrangement of the surrounding S1–S4 sequences. To locate regions of this gating module in space, we synthesized a series of compounds of varying length that function as molecular ‘tape measures’: quaternary ammonium (QA) pore blockers that can be tethered to specific test residues. We show that in a Shaker K+ channel, the extracellular ends of S1 and S3 are ∼30 Å from the tetraethylammonium (TEA) blocking site at the external opening of the pore. A portion of the S3-S4 loop is, at 17–18 Å, considerably closer.

Ion channels: Shake, rattle or roll?

Nerve transmission depends on voltage-gated ion-channel proteins, which in turn depend on the behaviour of a membrane domain called the voltage sensor. Therein lies the latest episode in a continuing story.

Role of SREBP-1 in the Development of Parasympathetic Dysfunction in the Hearts of Type 1 Diabetic Akita Mice

Rationale: Diabetic autonomic neuropathy (DAN), a major complication of diabetes mellitus, is characterized, in part, by impaired cardiac parasympathetic responsiveness. Parasympathetic stimulation of the heart involves activation of an acetylcholine-gated K+ current, IKAch, via a (GIRK1)2/(GIRK4)2 K+ channel. Sterol regulatory element binding protein-1 (SREBP-1) is a lipid-sensitive transcription factor. Objective: We describe a unique SREBP-1–dependent mechanism for insulin regulation of cardiac parasympathetic response in a mouse model for DAN. Methods and Results: Using implantable EKG transmitters, we demonstrated that compared with wild-type, Ins2Akita type I diabetic mice demonstrated a decrease in the negative chronotropic response to carbamylcholine characterized by a 2.4-fold decrease in the duration of bradycardia, a 52±8% decrease in atrial expression of GIRK1 (P<0.01), and a 31.3±2.1% decrease in SREBP-1 (P<0.05). Whole-cell patch-clamp studies of atrial myocytes from Akita mice exhibited a markedly decreased carbamylcholine stimulation of IKAch with a peak value of −181±31 pA/pF compared with −451±62 pA/pF (P<0.01) in cells from wild-type mice. Western blot analysis of extracts of Akita mice demonstrated that insulin treatment increased the expression of GIRK1, SREBP-1, and IKAch activity in atrial myocytes from these mice to levels in wild-type mice. Insulin treatment of cultured atrial myocytes stimulated GIRK1 expression 2.68±0.12-fold (P<0.01), which was reversed by overexpression of dominant negative SREBP-1. Finally, adenoviral expression of SREBP-1 in Akita atrial myocytes reversed the impaired IKAch to levels in cells from wild-type mice. Conclusions: These results support a unique molecular mechanism for insulin regulation of GIRK1 expression and parasympathetic response via SREBP-1, which might play a role in the pathogenesis of DAN in response to insulin deficiency in the diabetic heart.

Sizing the Protein Translocation Pathway of Colicin Ia Channels

The bacterial toxin colicin Ia forms voltage-gated channels in planar lipid bilayers. The toxin consists of three domains, with the carboxy-terminal domain (C-domain) responsible for channel formation. The C-domain contributes four membrane-spanning segments and a 68-residue translocated segment to the open channel, whereas the upstream domains and the amino-terminal end of the C-domain stay on the cis side of the membrane. The isolated C-domain, lacking the two upstream domains, also forms channels; however, the amino terminus and one of the normally membrane-spanning segments can move across the membrane. (This can be observed as a drop in single-channel conductance.) In longer carboxy-terminal fragments of colicin Ia that include ≤169 residues upstream from the C-domain, the entire upstream region is translocated. Presumably, a portion of the C-domain creates a pathway for the polar upstream region to move through the membrane. To determine the size of this translocation pathway, we have attached “molecular stoppers,” small disulfide-bonded polypeptides, to the amino terminus of the C-domain, and determined whether they could be translocated. We have found that the translocation rate is strongly voltage dependent, and that at voltages ≥90 mV, even a 26-Å stopper is translocated. Upon reduction of their disulfide bonds, all of the stoppers are easily translocated, indicating that it is the folded structure, rather than some aspect of the primary sequence, that slows translocation of the stoppers. Thus, the pathway for translocation is ≥26 Å in diameter, or can stretch to this value. This is large enough for an α-helical hairpin to fit through.

Kinetics of Tethering Quaternary Ammonium Compounds to K+ Channels

Polymeric maleimido–quaternary ammonium (QA) compounds have been shown to function as molecular tape measures when covalently tethered to external cysteine residues of a Shaker K+ channel (Blaustein R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309–311). For sufficiently long compounds, the cysteine–maleimide tethering reaction creates a high concentration, at the channels pore, of a TEA-like moiety that irreversibly blocks current. This paper investigates a striking feature of the maleimide–cysteine tethering kinetics. Strong blockers—those that induce substantial levels (>80%) of irreversible inhibition of current—react with channel cysteines much more rapidly than weak blockers and, when delivered to channels with four cysteine targets, react with multiexponential kinetics. This behavior is shown to arise from the ability of a strong blocker to concentrate its maleimide end near a channels cysteine target by exploiting the reversible pore-blocking affinity of its QA headgroup.

Constraints on voltage sensor movement in the shaker K+ channel.

In nerve and muscle cells, the voltage-gated opening and closing of cation-selective ion channels is accompanied by the translocation of 12–14 elementary charges across the membranes electric field. Although most of these charges are carried by residues in the S4 helix of the gating module of these channels, the precise nature of their physical movement is currently the topic of spirited debate. Broadly speaking, two classes of models have emerged: those that suggest that small-scale motions can account for the extensive charge displacement, and those that invoke a much larger physical movement. In the most recent incarnation of the latter type of model, which is based on structural and functional data from the archaebacterial K+ channel KvAP, a “voltage-sensor paddle” comprising a helix-turn-helix of S3–S4 translocates ∼20 Å through the bilayer during the gating cycle (Jiang, Y., A. Lee, J. Chen, V. Ruta, M. Cadene, B.T. Chait, and R. MacKinnon. 2003. Nature. 423:33–41; Jiang, Y., V. Ruta, J. Chen, A. Lee, and R. MacKinnon. 2003. Nature. 423:42–48.; Ruta, V., J. Chen, and R. MacKinnon. 2005. Cell. 123:463–475). We used two methods to test for analogous motions in the Shaker K+ channel, each examining the aqueous exposure of residues near S3. In the first, we employed a pore-blocking maleimide reagent (Blaustein, R.O., P.A. Cole, C. Williams, and C. Miller. 2000. Nat. Struct. Biol. 7:309–311) to probe for state-dependent changes in the chemical reactivity of substituted cysteines; in the second, we tested the state-dependent accessibility of a tethered biotin to external streptavidin (Qiu, X.Q., K.S. Jakes, A. Finkelstein, and S.L. Slatin. 1994. J. Biol. Chem. 269:7483–7488; Slatin, S.L., X.Q. Qiu, K.S. Jakes, and A. Finkelstein. 1994. Nature. 371:158–161). In both types of experiments, residues predicted to lie near the top of S3 did not exhibit any change in aqueous exposure during the gating cycle. This lack of state dependence argues against large-scale movements, either axially or radially, of Shakers S3–S4 voltage-sensor paddle.

Structural insights into neuronal K+ channel-calmodulin complexes.

Calmodulin (CaM) is a ubiquitous intracellular calcium sensor that directly binds to and modulates a wide variety of ion channels. Despite the large repository of high-resolution structures of CaM bound to peptide fragments derived from ion channels, there is no structural information about CaM bound to a fully folded ion channel at the plasma membrane. To determine the location of CaM docked to a functioning KCNQ K+ channel, we developed an intracellular tethered blocker approach to measure distances between CaM residues and the ion-conducting pathway. Combining these distance restraints with structural bioinformatics, we generated an archetypal quaternary structural model of an ion channel–CaM complex in the open state. These models place CaM close to the cytoplasmic gate, where it is well positioned to modulate channel function.

Preventing Voltage-dependent Gating of Anthrax Toxin Channels Using Engineered Disulfides

The channel-forming component of anthrax toxin, (PA63)7, is a heptameric water-soluble protein at neutral pH, but under acidic conditions it spontaneously inserts into lipid bilayers to form a 14-stranded β-barrel ion-conducting channel. This channel plays a vital role in anthrax pathogenesis because it serves as a conduit for the membrane translocation of the two enzymatic components of anthrax toxin, lethal factor and edema factor. Anthrax channels open and close in response to changes in transmembrane voltage, a property shared by several other pore-forming toxins. We have discovered an unexpected phenomenon in cysteine-substituted channels that provides a window into this gating process: their normal voltage-dependent gating can be abolished by reaction with methanethiosulfonate (MTS) reagents or exposure to oxidizing conditions. Remarkably, this perturbation is seen with cysteines substituted at sites all along the ∼100 Å length of the channels β-barrel. In contrast, reaction with N-ethylmaleimide, a thiol-reactive compound that does not form a mixed disulfide, does not affect gating at any of the sites tested. These findings, coupled with our biochemical detection of dimers, have led us to conclude that MTS reagents are catalyzing the formation of intersubunit disulfide bonds that lock channels in a conducting state, and that voltage gating requires a conformational change that involves the entire β-barrel.