Biomedical subjects

William A Catterall

Publications and source records attributed to William A Catterall.

At least 19 recordsLinked to original sources

PubMed2007

Bidirectional modulation of transmitter release by calcium channel/syntaxin interactions in vivo.

Protein interactions within the active zone of the nerve terminal are critical for regulation of transmitter release. The SNARE protein syntaxin 1A, primarily known for important interactions that control vesicle fusion, also interacts with presynaptic voltage-gated calcium channels. Based on recordings of calcium channel function in vitro, it has been hypothesized that syntaxin 1A-calcium channel interactions could alter calcium channel function at synapses. However, results at synapses in vitro suggest two potentially opposing roles: enhancement of neurotransmitter release by positioning docked vesicles near calcium channels and inhibition of calcium channel function by interaction with SNARE proteins. We have examined the possibility that these two effects of syntaxin can occur at synapses by studying the effects on transmitter release of manipulating syntaxin 1A-calcium channel interactions at Xenopus tadpole tail neuromuscular synapses in vivo. Introduction of synprint peptides, which competitively perturb syntaxin 1A-calcium channel interactions, decreased quantal content at these synapses and increased paired-pulse and tetanic facilitation. In contrast, injecting mRNA for mutant (A240V, V244A) syntaxin 1A, which reduces calcium channel modulation but not binding in vitro, increased quantal content and decreased paired-pulse and tetanic facilitation. Injection of wild-type syntaxin 1A mRNA had no effect. The opposing effects of synprint peptides and mutant syntaxin 1A provide in vivo support for the hypothesis that these interactions serve both to colocalize calcium channels with the release machinery and to modulate the functional state of the calcium channel. As such, these two effects of syntaxin on calcium channels modulate transmitter release in a bidirectional manner.

Animals↗

PubMed2006

Painful channels.

Paroxysmal extreme pain disorder (PEPD), previously known as familial rectal pain (FRP, OMIM 167400), is an inherited disease causing intense burning rectal, ocular, and submandibular pain and flushing. Fertleman et al. (this issue of Neuron) show that mutations in SCN9A, the gene encoding the sodium channel Na(V)1.7 channels, are responsible for this syndrome. Together with earlier work implicating a distinct class of functional mutations in SCN9A in a distinct inherited pain syndrome, these results point to Na(V)1.7 channels as key players in signaling nociceptive information and as a potential target for drug therapy of chronic pain.

Humans↗

PubMed2006

Phosphorylation of serine 1928 in the distal C-terminal domain of cardiac CaV1.2 channels during beta1-adrenergic regulation.

During the fight-or-flight response, epinephrine and norepinephrine released by the sympathetic nervous system increase L-type calcium currents conducted by Ca(V)1.2a channels in the heart, which contributes to enhanced cardiac performance. Activation of beta-adrenergic receptors increases channel activity via phosphorylation by cAMP-dependent protein kinase (PKA) tethered to the distal C-terminal domain of the alpha(1) subunit via an A-kinase anchoring protein (AKAP15). Here we measure phosphorylation of S1928 in dissociated rat ventricular myocytes in response to beta-adrenergic receptor stimulation by using a phosphospecific antibody. Isoproterenol treatment increased phosphorylation of S1928 in the distal C-terminal domain, and a similar increase was observed with a direct activator of adenylyl cyclase, forskolin, confirming that the cAMP and PKA are responsible. Pretreatment with selective beta1- and beta2-adrenergic antagonists reduced the increase in phosphorylation by 79% and 42%, respectively, and pretreatment with both agents completely blocked it. In contrast, treatment with these agents in the presence of 1,2-bis(2-aminophenoxy)ethane-N',N'-tetraacetic acid (BAPTA)-acetoxymethyl ester to buffer intracellular calcium results in only beta1-stimulated phosphorylation of S1928. Whole-cell patch clamp studies with intracellular BAPTA demonstrated that 98% of the increase in calcium current was attributable to beta1-adrenergic receptors. Thus, beta-adrenergic stimulation results in phosphorylation of S1928 on the Ca(V)1.2 alpha1 subunit in intact ventricular myocytes via both beta1- and beta2-adrenergic receptor pathways, but the beta2-dependent increase in phosphorylation depends on elevated intracellular calcium and does not contribute to regulation of whole-cell calcium currents at basal calcium levels. Our results correlate phosphorylation of S1928 with beta1-adrenergic functional up-regulation of cardiac calcium channels in the presence of BAPTA in intact ventricular myocytes.

Animals↗

PubMed2006

Reduced sodium current in GABAergic interneurons in a mouse model of severe myoclonic epilepsy in infancy.

Voltage-gated sodium channels (Na(V)) are critical for initiation of action potentials. Heterozygous loss-of-function mutations in Na(V)1.1 channels cause severe myoclonic epilepsy in infancy (SMEI). Homozygous null Scn1a-/- mice developed ataxia and died on postnatal day (P) 15 but could be sustained to P17.5 with manual feeding. Heterozygous Scn1a+/- mice had spontaneous seizures and sporadic deaths beginning after P21, with a notable dependence on genetic background. Loss of Na(V)1.1 did not change voltage-dependent activation or inactivation of sodium channels in hippocampal neurons. The sodium current density was, however, substantially reduced in inhibitory interneurons of Scn1a+/- and Scn1a-/- mice but not in their excitatory pyramidal neurons. An immunocytochemical survey also showed a specific upregulation of Na(V)1.3 channels in a subset of hippocampal interneurons. Our results indicate that reduced sodium currents in GABAergic inhibitory interneurons in Scn1a+/- heterozygotes may cause the hyperexcitability that leads to epilepsy in patients with SMEI.

Action Potentials↗

PubMed2006

Autoinhibitory control of the CaV1.2 channel by its proteolytically processed distal C-terminal domain.

Voltage-gated Ca(2+) channels of the Ca(V)1 family initiate excitation-contraction coupling in cardiac, smooth, and skeletal muscle and are primary targets for regulation by the sympathetic nervous system in the 'fight-or-flight' response. In the heart, activation of beta-adrenergic receptors greatly increases the L-type Ca(2+) current through Ca(V)1.2 channels, which requires phosphorylation by cyclic AMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15). Surprisingly, the site of interaction of PKA and AKAP15 lies in the distal C-terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the proteolytically cleaved distal C-terminal domain forms a specific molecular complex with the truncated alpha(1) subunit and serves as a potent autoinhibitory domain. Formation of the autoinhibitory complex greatly reduces the coupling efficiency of voltage sensing to channel opening and shifts the voltage dependence of activation to more positive membrane potentials. Ab initio structural modelling and site-directed mutagenesis revealed a binding interaction between a pair of arginine residues in a predicted alpha-helix in the proximal C-terminal domain and a set of three negatively charged amino acid residues in a predicted helix-loop-helix bundle in the distal C-terminal domain. Disruption of this interaction by mutation abolished the inhibitory effects of the distal C-terminus on Ca(V)1.2 channel function. These results provide the first functional characterization of this autoinhibitory complex, which may be a major form of the Ca(V)1 family Ca(2+) channels in cardiac and skeletal muscle cells, and reveal a unique ion channel regulatory mechanism in which proteolytic processing produces a more effective autoinhibitor of Ca(V)1.2 channel function.

Animals↗

PubMed2006

Structure and function of the voltage sensor of sodium channels probed by a beta-scorpion toxin.

Voltage sensing by voltage-gated sodium channels determines the electrical excitability of cells, but the molecular mechanism is unknown. beta-Scorpion toxins bind specifically to neurotoxin receptor site 4 and induce a negative shift in the voltage dependence of activation through a voltage sensor-trapping mechanism. Kinetic analysis showed that beta-scorpion toxin binds to the resting state, and subsequently the bound toxin traps the voltage sensor in the activated state in a voltage-dependent but concentration-independent manner. The rate of voltage sensor trapping can be fit by a two-step model, in which the first step is voltage-dependent and correlates with the outward gating movement of the IIS4 segment, whereas the second step is voltage-independent and results in shifted voltage dependence of activation of the channel. Mutations of Glu(779) in extracellular loop IIS1-S2 and both Glu(837) and Leu(840) in extracellular loop IIS3-S4 reduce the binding affinity of beta-scorpion toxin. Mutations of positively charged and hydrophobic amino acid residues in the IIS4 segment do not affect beta-scorpion toxin binding but alter voltage dependence of activation and enhance beta-scorpion toxin action. Structural modeling with the Rosetta algorithm yielded a three-dimensional model of the toxin-receptor complex with the IIS4 voltage sensor at the extracellular surface. Our results provide mechanistic and structural insight into the voltage sensor-trapping mode of scorpion toxin action, define the position of the voltage sensor in the resting state of the sodium channel, and favor voltage-sensing models in which the S4 segment spans the membrane in both resting and activated states.

Amino Acid Sequence↗

PubMed2006

Allosteric interactions required for high-affinity binding of dihydropyridine antagonists to Ca(V)1.1 Channels are modulated by calcium in the pore.

Dihydropyridines (DHPs) are an important class of drugs, used extensively in the treatment of angina pectoris, hypertension, and arrhythmia. The molecular mechanism by which DHPs modulate Ca(2+) channel function is not known in detail. We have found that DHP binding is allosterically coupled to Ca(2+) binding to the selectivity filter of the skeletal muscle Ca(2+) channel Ca(V)1.1, which initiates excitation-contraction coupling and conducts L-type Ca(2+) currents. Increasing Ca(2+) concentrations from approximately 10 nM to 1 mM causes the DHP receptor site to shift from a low-affinity state to a high-affinity state with an EC(50) for Ca(2+) of 300 nM. Substituting each of the four negatively charged glutamate residues that form the ion selectivity filter with neutral glutamine or positively charged lysine residues results in mutant channels whose DHP binding affinities are decreased up to 10-fold and are up to 150-fold less sensitive to Ca(2+) than wild-type channels. Analysis of mutations of amino acid residues adjacent to the selectivity filter led to identification of Phe-1013 and Tyr-1021, whose mutation causes substantial changes in DHP binding. Thermo-dynamic mutant cycle analysis of these mutants demonstrates that Phe-1013 and Tyr-1021 are energetically coupled when a single Ca(2+) ion is bound to the channel pore. We propose that DHP binding stabilizes a nonconducting state containing a single Ca(2+) ion in the pore through which Phe-1013 and Tyr-1021 are energetically coupled. The selectivity filter in this energetically coupled high-affinity state is blocked by bound Ca(2+), which is responsible for the high-affinity inhibition of Ca(2+) channels by DHP antagonists.

Allosteric Regulation↗

PubMed2006

Voltage sensor conformations in the open and closed states in ROSETTA structural models of K(+) channels.

Voltage-gated ion channels control generation and propagation of action potentials in excitable cells. Significant progress has been made in understanding structure and function of the voltage-gated ion channels, highlighted by the high-resolution open-state structure of the voltage-gated potassium channel, K(v)1.2. However, because the structure of the closed state is unknown, the gating mechanism remains controversial. We adapted the rosetta membrane method to model the structures of the K(v)1.2 and KvAP channels using homology, de novo, and domain assembly methods and selected the most plausible models using a limited number of experimental constraints. Our model of K(v)1.2 in the open state is very similar in overall topology to the x-ray structure of this channel. Modeling of KvAP in the open state suggests that orientation of the voltage-sensing domain relative to the pore-forming domain is considerably different from the orientation in the K(v)1.2 open state and that the magnitude of the vertical movement of S4 is significantly greater. Structural modeling of closed state of K(v)1.2 suggests gating movement that can be viewed as a sum of two previously suggested mechanisms: translation (2-4 A) plus rotation ( approximately 180 degrees ) of the S4 segment as proposed in the original "sliding helix" or "helical screw" models coupled with a rolling motion of the S1-S3 segments around S4, similar to recent "transporter" models of gating. We propose a unified mechanism of voltage-dependent gating for K(v)1.2 and KvAP in which this major conformational change moves the gating charge across the electric field in an analogous way for both channels.

Cell Membrane↗

PubMed2006

Neuromodulation of Na+ channel slow inactivation via cAMP-dependent protein kinase and protein kinase C.

Neurotransmitters modulate sodium channel availability through activation of G protein-coupled receptors, cAMP-dependent protein kinase (PKA), and protein kinase C (PKC). Voltage-dependent slow inactivation also controls sodium channel availability, synaptic integration, and neuronal firing. Here we show by analysis of sodium channel mutants that neuromodulation via PKA and PKC enhances intrinsic slow inactivation of sodium channels, making them unavailable for activation. Mutations in the S6 segment in domain III (N1466A,D) either enhance or block slow inactivation, implicating S6 segments in the molecular pathway for slow inactivation. Modulation of N1466A channels by PKC or PKA is increased, whereas modulation of N1466D is nearly completely blocked. These results demonstrate that neuromodulation by PKA and PKC is caused by their enhancement of intrinsic slow inactivation gating. Modulation of slow inactivation by neurotransmitters acting through G protein-coupled receptors, PKA, and PKC is a flexible mechanism of cellular plasticity controlling the firing behavior of central neurons.

Amino Acid Sequence↗

PubMed2006

Regulation of sodium and calcium channels by signaling complexes.

Membrane depolarization and intracellular calcium transients generated by activation of voltage-gated sodium and calcium channels are local signals, which initiate physiological processes such as action potential conduction, synaptic transmission, and excitation-contraction coupling. Targeting of effector proteins and regulatory proteins to ion channels is an important mechanism to ensure speed, specificity, and precise regulation of signaling events in response to local stimuli. In this article, we review recent experimental results showing that sodium and calcium channels form local signaling complexes, in which effector proteins, anchoring proteins, and regulatory proteins interact directly with ion channels. The intracellular domains of these channels serve as signaling platforms, mediating their participation in intracellular signaling processes. These protein-protein interactions are important for efficient synaptic transmission and for regulation of ion channels by neurotransmitters and intracellular second messengers. These localized signaling complexes are essential for normal function and regulation of electrical excitability, synaptic transmission, and excitation-contraction coupling.

Animals↗

PubMed2005

Modulation of CaV1.2 channels by Mg2+ acting at an EF-hand motif in the COOH-terminal domain.

Magnesium levels in cardiac myocytes change in cardiovascular diseases. Intracellular free magnesium (Mg(i)) inhibits L-type Ca(2+) currents through Ca(V)1.2 channels in cardiac myocytes, but the mechanism of this effect is unknown. We hypothesized that Mg(i) acts through the COOH-terminal EF-hand of Ca(V)1.2. EF-hand mutants were engineered to have either decreased (D1546A/N/S/K) or increased (K1543D and K1539D) Mg(2+) affinity. In whole-cell patch clamp experiments, increased Mg(i) reduced both Ba(2+) and Ca(2+) currents conducted by wild type (WT) Ca(V)1.2 channels expressed in tsA-201 cells with similar affinity. Exposure of WT Ca(V)1.2 to lower Mg(i) (0.26 mM) increased the amplitudes of Ba(2+) currents 2.6 +/- 0.4-fold without effects on the voltage dependence of activation and inactivation. In contrast, increasing Mg(i) to 2.4 or 7.2 mM reduced current amplitude to 0.5 +/- 0.1 and 0.26 +/- 0.05 of the control level at 0.8 mM Mg(i). The effects of Mg(i) on peak Ba(2+) currents were approximately fit by a single binding site model with an apparent K(d) of 0.65 mM. The apparent K(d) for this effect of Mg(i) was shifted approximately 3.3- to 16.5-fold to higher concentration in D1546A/N/S mutants, with only small effects on the voltage dependence of activation and inactivation. Moreover, mutant D1546K was insensitive to Mg(i) up to 7.2 mM. In contrast to these results, peak Ba(2+) currents through the K1543D mutant were inhibited by lower concentrations of Mg(i) compared with WT, consistent with approximately fourfold reduction in apparent K(d) for Mg(i), and inhibition of mutant K1539D by Mg(i) was also increased comparably. In addition to these effects, voltage-dependent inactivation of K1543D and K1539D was incomplete at positive membrane potentials when Mg(i) was reduced to 0.26 or 0.1 mM, respectively. These results support a novel mechanism linking the COOH-terminal EF-hand with modulation of Ca(V)1.2 channels by Mg(i). Our findings expand the repertoire of modulatory interactions taking place at the COOH terminus of Ca(V)1.2 channels, and reveal a potentially important role of Mg(i) binding to the COOH-terminal EF-hand in regulating Ca(2+) influx in physiological and pathophysiological states.

Amino Acid Sequence↗

PubMed2005

Modulation of CaV2.1 channels by the neuronal calcium-binding protein visinin-like protein-2.

CaV2.1 channels conduct P/Q-type Ca2+ currents that are modulated by calmodulin (CaM) and the structurally related Ca2+-binding protein 1 (CaBP1). Visinin-like protein-2 (VILIP-2) is a CaM-related Ca2+-binding protein expressed in the neocortex and hippocampus. Coexpression of CaV2.1 and VILIP-2 in tsA-201 cells resulted in Ca2+ channel modulation distinct from CaM and CaBP1. CaV2.1 channels with beta2a subunits undergo Ca2+-dependent facilitation and inactivation attributable to association of endogenous Ca2+/CaM. VILIP-2 coexpression does not alter facilitation measured in paired-pulse experiments but slows the rate of inactivation to that seen without Ca2+/CaM binding and reduces inactivation of Ca2+ currents during trains of repetitive depolarizations. CaV2.1 channels with beta1b subunits have rapid voltage-dependent inactivation, and VILIP-2 has no effect on the rate of inactivation or facilitation of the Ca2+ current. In contrast, when Ba2+ replaces Ca2+ as the charge carrier, VILIP-2 slows inactivation. The effects of VILIP-2 are prevented by deletion of the CaM-binding domain (CBD) in the C terminus of CaV2.1 channels. However, both the CBD and an upstream IQ-like domain must be deleted to prevent VILIP-2 binding. Our results indicate that VILIP-2 binds to the CBD and IQ-like domains of CaV2.1 channels like CaM but slows inactivation, which enhances facilitation of CaV2.1 channels during extended trains of stimuli. Comparison of VILIP-2 effects with those of CaBP1 indicates striking differences in modulation of both facilitation and inactivation. Differential regulation of CaV2.1 channels by CaM, VILIP-2, CaBP1, and other neurospecific Ca2+-binding proteins is a potentially important determinant of Ca2+ entry in neurotransmission.

Animals↗

PubMed2005

Differential regulation of CaV2.1 channels by calcium-binding protein 1 and visinin-like protein-2 requires N-terminal myristoylation.

P/Q-type Ca2+ currents through presynaptic CaV2.1 channels initiate neurotransmitter release, and differential modulation of these channels by neuronal calcium-binding proteins (nCaBPs) may contribute to synaptic plasticity. The nCaBPs calcium-binding protein 1 (CaBP1) and visinin-like protein-2 (VILIP-2) differ from calmodulin (CaM) in that they have an N-terminal myristoyl moiety and one EF-hand that is inactive in binding Ca2+. To determine whether myristoylation contributes to their distinctive modulatory properties, we studied the regulation of CaV2.1 channels by the myristoyl-deficient mutants CaBP1/G2A and VILIP-2/G2A. CaBP1 positively shifts the voltage dependence of CaV2.1 activation, accelerates inactivation, and prevents paired-pulse facilitation in a Ca2+-independent manner. Block of myristoylation abolished these effects, leaving regulation that is similar to endogenous CaM. CaBP1/G2A binds to CaV2.1 with reduced stability, but in situ protein cross-linking and immunocytochemical studies revealed that it binds CaV2.1 in situ and is localized to the plasma membrane by coexpression with CaV2.1, indicating that it binds effectively in intact cells. In contrast to CaBP1, coexpression of VILIP-2 slows inactivation in a Ca2+-independent manner, but this effect also requires myristoylation. These results suggest a model in which nonmyristoylated CaBP1 and VILIP-2 bind to CaV2.1 channels and regulate them like CaM, whereas myristoylation allows differential, Ca2+-independent regulation by the inactive EF-hands of CaBP1 and VILIP-2, which differ in their positions in the protein structure. Differential, myristoylation-dependent regulation of presynaptic Ca2+ channels by nCaBPs may provide a flexible mechanism for diverse forms of short-term synaptic plasticity.

Animals↗

PubMed2005

Ion permeation through a voltage- sensitive gating pore in brain sodium channels having voltage sensor mutations.

Voltage-gated sodium channels activate in response to depolarization, but it is unknown whether the voltage-sensing arginines in their S4 segments pivot across the lipid bilayer as voltage sensor paddles or move through the protein in a gating pore. Here we report that mutation of pairs of arginine gating charges to glutamine induces cation permeation through a gating pore in domain II of the Na(V)1.2a channel. Mutation of R850 and R853 induces a K(+)-selective inward cationic current in the resting state that is blocked by activation. Remarkably, mutation of R853 and R856 causes an outward cationic current with the opposite gating polarity. These results support a model in which the IIS4 gating charges move through a narrow constriction in a gating pore in the sodium channel protein during gating. Paired substitutions of glutamine allow cation movement through the constriction when appropriately positioned by the gating movements of the S4 segment.

Amino Acid Substitution↗

PubMed2005

Sodium channel mutation leading to saxitoxin resistance in clams increases risk of PSP.

Bivalve molluscs, the primary vectors of paralytic shellfish poisoning (PSP) in humans, show marked inter-species variation in their capacity to accumulate PSP toxins (PSTs) which has a neural basis. PSTs cause human fatalities by blocking sodium conductance in nerve fibres. Here we identify a molecular basis for inter-population variation in PSP resistance within a species, consistent with genetic adaptation to PSTs. Softshell clams (Mya arenaria) from areas exposed to 'red tides' are more resistant to PSTs, as demonstrated by whole-nerve assays, and accumulate toxins at greater rates than sensitive clams from unexposed areas. PSTs lead to selective mortality of sensitive clams. Resistance is caused by natural mutation of a single amino acid residue, which causes a 1,000-fold decrease in affinity at the saxitoxin-binding site in the sodium channel pore of resistant, but not sensitive, clams. Thus PSTs might act as potent natural selection agents, leading to greater toxin resistance in clam populations and increased risk of PSP in humans. Furthermore, global expansion of PSP to previously unaffected coastal areas might result in long-term changes to communities and ecosystems.

Amino Acid Sequence↗

PubMed2005

Molecular determinants for modulation of persistent sodium current by G-protein betagamma subunits.

Voltage-gated sodium channels are responsible for the upstroke of the action potential in most excitable cells, and their fast inactivation is essential for controlling electrical signaling. In addition, a noninactivating, persistent component of sodium current, I(NaP), has been implicated in integrative functions of neurons including threshold for firing, neuronal bursting, and signal integration. G-protein betagamma subunits increase I(NaP), but the sodium channel subtypes that conduct I(NaP) and the target site(s) on the sodium channel molecule required for modulation by Gbetagamma are poorly defined. Here, we show that I(NaP) conducted by Na(v)1.1 and Na(v)1.2 channels (Na(v)1.1 > Na(v)1.2) is modulated by Gbetagamma; Na(v)1.4 and Na(v)1.5 channels produce smaller I(NaP) that is not regulated by Gbetagamma. These qualitative differences in modulation by Gbetagamma are determined by the transmembrane body of the sodium channels rather than their cytoplasmic C-terminal domains, which have been implicated previously in modulation by Gbetagamma. However, the C-terminal domains determine the quantitative extent of modulation of Na(v)1.2 channels by Gbetagamma. Studies of chimeric and truncated Na(v)1.2 channels identify molecular determinants that affect modulation of I(NaP) located between amino acid residue 1890 and the C terminus at residue 2005. The last 28 amino acid residues of the C terminus are sufficient to support modulation by Gbetagamma when attached to the proximal C-terminal domain. Our results further define the sodium channel subtypes that generate I(NaP) and identify crucial molecular determinants in the C-terminal domain required for modulation by Gbetagamma when attached to the transmembrane body of a responsive sodium channel.

Amino Acid Sequence↗

PubMed2005

Sites of proteolytic processing and noncovalent association of the distal C-terminal domain of CaV1.1 channels in skeletal muscle.

In skeletal muscle cells, voltage-dependent potentiation of Ca2+ channel activity requires phosphorylation by cAMP-dependent protein kinase (PKA) anchored via an A-kinase anchoring protein (AKAP15), and the most rapid sites of phosphorylation are located in the C-terminal domain. Surprisingly, the site of interaction of the complex of PKA and AKAP15 with the alpha1-subunit of Ca(V)1.1 channels lies in the distal C terminus, which is cleaved from the remainder of the channel by in vivo proteolytic processing. Here we report that the distal C terminus is noncovalently associated with the remainder of the channel via an interaction with a site in the proximal C-terminal domain when expressed as a separate protein in mammalian nonmuscle cells. Deletion mapping of the C terminus of the alpha1-subunit using the yeast two-hybrid assay revealed that a distal C-terminal peptide containing amino acids 1802-1841 specifically interacts with a region in the proximal C terminus containing amino acid residues 1556-1612. Analysis of the purified alpha1-subunit of Ca(V)1.1 channels from skeletal muscle by saturation sequencing of the intracellular peptides by tandem mass spectrometry identified the site of proteolytic processing as alanine 1664. Our results support the conclusion that a noncovalently associated complex of the alpha1-subunit truncated at A1664 with the proteolytically cleaved distal C-terminal domain, AKAP15, and PKA is the primary physiological form of Ca(V)1.1 channels in skeletal muscle cells.

Amino Acid Sequence↗

PubMed2005

Convergent regulation of skeletal muscle Ca2+ channels by dystrophin, the actin cytoskeleton, and cAMP-dependent protein kinase.

The skeletal muscle L-type Ca2+ channel (Ca(V)1.1), which is responsible for initiating muscle contraction, is regulated by phosphorylation by cAMP-dependent protein kinase (PKA) in a voltage-dependent manner that requires direct physical association between the channel and the kinase mediated through A-kinase anchoring proteins (AKAPs). The role of the actin cytoskeleton in channel regulation was investigated in skeletal myocytes cultured from wild-type mice, mdx mice that lack the cytoskeletal linkage protein dystrophin, and a skeletal muscle cell line, 129 CB3. Voltage dependence of channel activation was shifted positively, and potentiation was greatly diminished in mdx myocytes and in 129 CB3 cells treated with the microfilament stabilizer phalloidin. Voltage-dependent potentiation by strong depolarizing prepulses was reduced in mdx myocytes but could be restored by positively shifting the stimulus potentials to compensate for the positive shift in the voltage dependence of gating. Inclusion of PKA in the pipette caused a negative shift in the voltage dependence of activation and restored voltage-dependent potentiation in mdx myocytes. These results show that skeletal muscle Ca2+ channel activity and voltage-dependent potentiation are controlled by PKA and microfilaments in a convergent manner. Regulation of Ca2+ channel activity by hormones and neurotransmitters that use the PKA signal transduction pathway may interact in a critical way with the cytoskeleton and may be impaired by deletion of dystrophin, contributing to abnormal regulation of intracellular calcium concentrations in dystrophic muscle.

Actins↗