Jose Mercado

Ca2+-Dependent Desensitization of TRPV2 Channels Is Mediated by Hydrolysis of Phosphatidylinositol 4,5-Bisphosphate

TRPV2 is a member of the transient receptor potential family of ion channels involved in chemical and thermal pain transduction. Unlike the related TRPV1 channel, TRPV2 does not appear to bind either calmodulin or ATP in its N-terminal ankyrin repeat domain. In addition, it does not contain a calmodulin-binding site in the distal C-terminal region, as has been proposed for TRPV1. We have found that TRPV2 channels transiently expressed in F-11 cells undergo Ca2+-dependent desensitization, similar to the other TRPVs, suggesting that the mechanism of desensitization may be conserved in the subfamily of TRPV channels. TRPV2 desensitization was not altered in whole-cell recordings in the presence of calmodulin inhibitors or on coexpression of mutant calmodulin but was sensitive to changes in membrane phosphatidylinositol 4,5-bisphosphate (PIP2), suggesting a role of membrane PIP2 in TRPV2 desensitization. Simultaneous confocal imaging and electrophysiological recording of cells expressing TRPV2 and a fluorescent PIP2-binding probe demonstrated that TRPV2 desensitization was concomitant with depletion of PIP2. We conclude that the decrease in PIP2 levels on channel activation underlies a major component of Ca2+-dependent desensitization of TRPV2 and may play a similar role in other TRP channels.

Charged Residues in the α1 and β2 Pre-M1 Regions Involved in GABAA Receptor Activation

For Cys-loop ligand-gated ion channels (LGIC), the protein movements that couple neurotransmitter binding to channel gating are not well known. The pre-M1 region, which links the extracellular agonist-binding domain to the channel-containing transmembrane domain, is in an ideal position to transduce binding site movements to gating movements. A cluster of cationic residues in this region is observed in all LGIC subunits, and in particular, an arginine residue is absolutely conserved. We mutated charged pre-M1 residues in the GABAA receptor α1 (K219, R220, K221) and β2 (K213, K215, R216) subunits to cysteine and expressed the mutant subunits with wild-type β2 or α1 in Xenopus oocytes. Cysteine substitution of β2R216 abolished channel gating by GABA without altering the binding of the GABA agonist [3H]muscimol, indicating that this residue plays a key role in coupling GABA binding to gating. Tethering thiol-reactive methanethiosulfonate (MTS) reagents onto α1K219C, β2K213C, and β2K215C increased maximal GABA-activated currents, suggesting that structural perturbations of the pre-M1 regions affect channel gating. GABA altered the rates of sulfhydryl modification of α1K219C, β2K213C, and β2K215C, indicating that the pre-M1 regions move in response to channel activation. A positively charged MTS reagent modified β2K213C and β2K215C significantly faster than a negatively charged reagent, and GABA activation eliminated modification of β2K215C by the negatively charged reagent. Overall, the data indicate that the pre-M1 region is part of the structural machinery coupling GABA binding to gating and that the transduction of binding site movements to channel movements is mediated, in part, by electrostatic interactions.

Local control of TRPV4 channels by akap150-targeted PKC in arterial smooth muscle

Angiotensin signaling promotes interactions between AKAP150, PKC, and TRPV4 channels to form signaling domains that control Ca2+ influx into arterial myocytes.

L-type Ca2+ channel function during Timothy Syndrome

Voltage-gated, dihydropyridine-sensitive L-type Ca(2+) channels are multimeric proteins composed of a pore-forming transmembrane α(1) subunit (Ca(v)1.2) and accessory β, α(2)δ, and γ subunits. Ca(2+) entry via Ca(v)1.2 channels shapes the action potential (AP) of cardiac myocytes and is required for excitation-contraction coupling. Two de novo point mutations of Ca(v)1.2 glycine residues, G406R and G402S, cause a rare multisystem disorder called Timothy syndrome (TS). Here, we discuss recent work on the mechanisms by which Ca(v)1.2 channels bearing TS mutations display slowed inactivation that leads to increased Ca(2+) influx, prolonging the cardiac AP and promoting lethal arrhythmias. Based on these studies, we propose a model in which the scaffolding protein AKAP79/150 stabilizes the open conformation of Ca(v)1.2-TS channels and facilitates physical interactions among adjacent channels via their C-tails, increasing the activity of adjoining channels and amplifying Ca(2+) influx.

γ-Aminobutyric Acid (GABA) and Pentobarbital Induce Different Conformational Rearrangements in the GABAA Receptor α1 and β2 Pre-M1 Regions

γ-Aminobutyric acid (GABA) binding to GABAA receptors (GABAARs) triggers conformational movements in the α1 and β2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABAAR channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABAAR pre-M1 regions. α1β2 GABAARs containing cysteine substitutions in the pre-M1 α1 (K219C, K221C) and β2 (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto α1K219C and α1K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For α1K221Cβ2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For α1β2K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.

Gamma-aminobutyric acid (GABA) and pentobarbital induce different conformational rearrangements in the GABA A receptor alpha1 and beta2 pre-M1 regions.

γ-Aminobutyric acid (GABA) binding to GABAA receptors (GABAARs) triggers conformational movements in the α1 and β2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABAAR channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABAAR pre-M1 regions. α1β2 GABAARs containing cysteine substitutions in the pre-M1 α1 (K219C, K221C) and β2 (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto α1K219C and α1K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For α1K221Cβ2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For α1β2K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.

Regulation of L-type calcium channel sparklet activity by c-Src and PKC-α

The activity of persistent Ca²⁺ sparklets, which are characterized by longer and more frequent channel open events than low-activity sparklets, contributes substantially to steady-state Ca²⁺ entry under physiological conditions. Here, we addressed two questions related to the regulation of Ca²⁺ sparklets by PKC-α and c-Src, both of which increase whole cell Cav1.2 current: 1) Does c-Src activation enhance persistent Ca²⁺ sparklet activity? 2) Does PKC-α activate c-Src to produce persistent Ca²⁺ sparklets? With the use of total internal reflection fluorescence microscopy, Ca²⁺ sparklets were recorded from voltage-clamped tsA-201 cells coexpressing wild-type (WT) or mutant Cav1.2c (the neuronal isoform of Cav1.2) constructs ± active or inactive PKC-α/c-Src. Cells expressing Cav1.2c exhibited both low-activity and persistent Ca²⁺ sparklets. Persistent Ca²⁺ sparklet activity was significantly reduced by acute application of the c-Src inhibitor PP2 or coexpression of kinase-dead c-Src. Cav1.2c constructs mutated at one of two COOH-terminal residues (Y²¹²²F and Y²¹³⁹F) were used to test the effect of blocking putative phosphorylation sites for c-Src. Expression of Y²¹²²F but not Y²¹³⁹F Cav1.2c abrogated the potentiating effect of c-Src on Ca²⁺ sparklet activity. We could not detect a significant change in persistent Ca²⁺ sparklet activity or density in cells coexpressing Cav1.2c + PKC-α, regardless of whether WT or Y²¹²²F Cav1.2c was used, or after PP2 application, suggesting that PKC-α does not act upstream of c-Src to produce persistent Ca²⁺ sparklets. However, our results indicate that persistent Ca²⁺ sparklet activity is promoted by the action of c-Src on residue Y²¹²² of the Cav1.2c COOH terminus.

γ-Aminobutyric Acid (GABA) and Pentobarbital Induce Different Conformational Rearrangements in the GABAAReceptor α1and β2Pre-M1 Regions

γ-Aminobutyric acid (GABA) binding to GABAA receptors (GABAARs) triggers conformational movements in the α1 and β2 pre-M1 regions that are associated with channel gating. At high concentrations, the barbiturate pentobarbital opens GABAAR channels with similar conductances as GABA, suggesting that their open state structures are alike. Little, however, is known about the structural rearrangements induced by barbiturates. Here, we examined whether pentobarbital activation triggers movements in the GABAAR pre-M1 regions. α1β2 GABAARs containing cysteine substitutions in the pre-M1 α1 (K219C, K221C) and β2 (K213C, K215C) subunits were expressed in Xenopus oocytes and analyzed using two-electrode voltage clamp. The cysteine substitutions had little to no effect on GABA and pentobarbital EC50 values. Tethering chemically diverse thiol-reactive methanethiosulfonate reagents onto α1K219C and α1K221C affected GABA- and pentobarbital-activated currents differently, suggesting that the pre-M1 structural elements important for GABA and pentobarbital current activation are distinct. Moreover, pentobarbital altered the rates of cysteine modification by methanethiosulfonate reagents differently than GABA. For α1K221Cβ2 receptors, pentobarbital decreased the rate of cysteine modification whereas GABA had no effect. For α1β2K215C receptors, pentobarbital had no effect whereas GABA increased the modification rate. The competitive GABA antagonist SR-95531 and a low, non-activating concentration of pentobarbital did not alter their modification rates, suggesting that the GABA- and pentobarbital-mediated changes in rates reflect gating movements. Overall, the data indicate that the pre-M1 region is involved in both GABA- and pentobarbital-mediated gating transitions. Pentobarbital, however, triggers different movements in this region than GABA, suggesting their activation mechanisms differ.

Local control of TRPV4 channels by AKAP150-targeted PKC in arterial smooth muscle

Arterial myocytes have the intrinsic ability to contract in response to increases in intravascular pressure (Bayliss, 1902). It has been proposed that this myogenic response is initiated by the stretch-induced activation of the nonselective cation channels TRPP2, TRPC6, and TRPM4, which depolarize arterial myocytes (Welsh et al., 2002; Earley et al., 2004; Spassova et al., 2006; Narayanan et al., 2013). Membrane depolarization activates voltage-gated L-type CaV1.2 channels (Harder et al., 1987; Fleischmann et al., 1994; Rubart et al., 1996; Jaggar et al., 1998). The influx of Ca via a single CaV1.2 channel can be optically detected in the form of a “CaV1.2 sparklet” (Navedo et al., 2005; Amberg et al., 2007). Simultaneous activation of multiple CaV1.2 sparklets induces a cell-wide increase in intracellular Ca ([Ca]i) that activates myosin light chain kinase and thus triggers contraction. This myogenic

Adding Accessories for Hypertension: α2δ-1 Subunits Upregulate CaV1.2 Channels in Arterial Myocytes in a Model of Genetic Hypertension

See related article, pp 1006–1015 Hypertension is a major risk factor for the development of stroke, coronary artery disease, heart failure, and renal disease.1 Although the principal cause of hypertension is likely renal, vascular dysfunction is critical,2 and the increased arterial tone associated with hypertension contributes to the development of the pathology. This is highlighted by recent studies indicating that the endogenous vasoconstrictor angiotensin II is a likely contributor to vascular dysfunction in human3 and hypertension models.4 In the current issue of Hypertension , a study from Bannister et al5 addressed an important and yet unresolved question in vascular physiology: what are the molecular mechanisms underlying changes in the function of dihydropyridine-sensitive, voltage-gated L-type Ca2+ channels in arterial myocytes during hypertension? Through a series of elegant experiments they provide an interesting and unexpected answer to this difficult conundrum. Below, we describe the context and implications of their findings. In arterial smooth muscle, L-type Ca2+ currents are produced by channels composed of pore-forming CaV1.2 α1 subunits and accessory β and α2δ-1 subunits. …