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An oily competition: role of {beta} subunit palmitoylation for Ca2+ channel modulation by fatty acids

Striessnig, J. — Mon, 26 Oct 2009 10:01:46 PDT

The Ca2+ channel {beta} subunit determines whether stimulation of Gq-coupled receptors enhances or inhibits N current

Mon, 26 Oct 2009 10:01:46 PDT

In superior cervical ganglion (SCG) neurons, stimulation of M₁ receptors (M₁Rs) produces a distinct pattern of modulation of N-type calcium (N-) channel activity, enhancing currents elicited with negative test potentials and inhibiting currents elicited with positive test potentials. Exogenously applied arachidonic acid (AA) reproduces this profile of modulation, suggesting AA functions as a downstream messenger of M₁Rs. In addition, techniques that diminish AA's concentration during M₁R stimulation minimize N-current modulation. However, other studies suggest depletion of phosphatidylinositol-4,5-bisphosphate during M₁R stimulation suffices to elicit modulation. In this study, we used an expression system to examine the physiological mechanisms regulating modulation. We found the β subunit (Ca_Vβ) acts as a molecular switch regulating whether modulation results in enhancement or inhibition. In human embryonic kidney 293 cells, stimulation of M₁Rs or neurokinin-1 receptors (NK-1Rs) inhibited activity of N channels formed by Ca_V2.2 and coexpressed with Ca_Vβ1b, Ca_Vβ3, or Ca_Vβ4 but enhanced activity of N channels containing Ca_Vβ2a. Exogenously applied AA produced the same pattern of modulation. Coexpression of Ca_Vβ2a, Ca_Vβ3, and Ca_Vβ4 recapitulated the modulatory response previously seen in SCG neurons, implying heterogeneous association of Ca_Vβ with Ca_V2.2. Further experiments with mutated, chimeric Ca_Vβ subunits and free palmitic acid revealed that palmitoylation of Ca_Vβ2a is essential for loss of inhibition. The data presented here fit a model in which Ca_Vβ2a blocks inhibition, thus unmasking enhancement. Our discovery that the presence or absence of palmitoylated Ca_Vβ2a toggles M₁R- or NK-1R–mediated modulation of N current between enhancement and inhibition identifies a novel role for palmitoylation. Moreover, these findings predict that at synapses, modulation of N-channel activity by M₁Rs or NK-1Rs will fluctuate between enhancement and inhibition based on the presence of palmitoylated Ca_Vβ2a.

Orientation of palmitoylated CaV{beta}2a relative to CaV2.2 is critical for slow pathway modulation of N-type Ca2+ current by tachykinin receptor activation

Mitra-Ganguli, T., Vitko, I., Perez-Reyes, E., Rittenhouse, A. R. — Mon, 26 Oct 2009 10:01:46 PDT

The G_q-coupled tachykinin receptor (neurokinin-1 receptor [NK-1R]) modulates N-type Ca²⁺ channel (Ca_V2.2 or N channel) activity at two distinct sites by a pathway involving a lipid metabolite, most likely arachidonic acid (AA). In another study published in this issue (Heneghan et al. 2009. J. Gen Physiol. doi:10.1085/jgp.200910203), we found that the form of modulation observed depends on which Ca_Vβ is coexpressed with Ca_V2.2. When palmitoylated Ca_Vβ2a is coexpressed, activation of NK-1Rs by substance P (SP) enhances N current. In contrast, when Ca_Vβ3 is coexpressed, SP inhibits N current. However, exogenously applied palmitic acid minimizes this inhibition. These findings suggested that the palmitoyl groups of Ca_Vβ2a may occupy an inhibitory site on Ca_V2.2 or prevent AA from interacting with that site, thereby minimizing inhibition. If so, changing the orientation of Ca_Vβ2a relative to Ca_V2.2 may displace the palmitoyl groups and prevent them from antagonizing AA's actions, thereby allowing inhibition even in the presence of Ca_Vβ2a. In this study, we tested this hypothesis by deleting one (Bdel1) or two (Bdel2) amino acids proximal to the interacting domain (AID) of Ca_V2.2's I–II linker. Ca_Vβs bind tightly to the AID, whereas the rigid region proximal to the AID is thought to couple Ca_Vβ's movements to Ca_V2.2 gating. Although Bdel1/β2a currents exhibited more variable enhancement by SP, Bdel2/β2a current enhancement was lost at all voltages. Instead, inhibition was observed that matched the profile of N-current inhibition from Ca_V2.2 coexpressed with Ca_Vβ3. Moreover, adding back exogenous palmitic acid minimized inhibition of Bdel2/β2a currents, suggesting that when palmitoylated Ca_Vβ2a is sufficiently displaced, endogenously released AA can bind to the inhibitory site. These findings support our previous hypothesis that Ca_Vβ2a's palmitoyl groups directly interact with an inhibitory site on Ca_V2.2 to block N-current inhibition by SP.

Activation and desensitization of the olfactory cAMP-gated transduction channel: identification of functional modules

Waldeck, C., Vocke, K., Ungerer, N., Frings, S., Mohrlen, F. — Mon, 26 Oct 2009 10:01:46 PDT

Olfactory receptor neurons respond to odor stimulation with a receptor potential that results from the successive activation of cyclic AMP (cAMP)-gated, Ca²⁺-permeable channels and Ca²⁺-activated chloride channels. The cAMP-gated channels open at micromolar concentrations of their ligand and are subject to a Ca²⁺-dependent feedback inhibition by calmodulin. Attempts to understand the operation of these channels have been hampered by the fact that the channel protein is composed of three different subunits, CNGA2, CNGA4, and CNGB1b. Here, we explore the individual role that each subunit plays in the gating process. Using site-directed mutagenesis and patch clamp analysis, we identify three functional modules that govern channel operation: a module that opens the channel, a module that stabilizes the open state at low cAMP concentrations, and a module that mediates rapid Ca²⁺-dependent feedback inhibition. Each subunit could be assigned to one of these functions that, together, define the gating logic of the olfactory transduction channel.

Closed-channel block of BK potassium channels by bbTBA requires partial activation

Tang, Q.-Y., Zeng, X.-H., Lingle, C. J. — Mon, 26 Oct 2009 10:01:46 PDT

Blockade of large-conductance Ca²⁺-activated K⁺ (BK) channels by the bulky quaternary ammonium compound, N-(4-[benzoyl]benzyl)-N,N,N-tributylammonium (bbTBA), exhibits features consistent with blockade of both closed and open states. Here, we examine block of closed BK channels by bbTBA and how it may differ from block of open channels. Although our observations generally confirm earlier results, we describe three observations that are inconsistent with a model in which closed and open channels are equally accessible to blockade by bbTBA. First, block by bbTBA exhibits Ca²⁺-dependent features that are inconsistent with strictly state-independent block. Second, the steady-state voltage dependence of bbTBA block at negative potentials shows that any block of completely closed states either does not occur or is completely voltage independent. Third, determination of the fractional unblock by bbTBA at either low or high Ca²⁺ reveals deviations from a model in which open- and closed-state block is identical. The results support the view that bbTBA blockade of fully closed channels does not occur. We imagine two general types of explanation. First, a stronger voltage dependence of closed-channel block may minimize the contribution of closed-channel block at negative potentials. Second, voltage-dependent conformational changes among closed-channel states may permit block by bbTBA. The analysis supports the latter view, suggesting that bbTBA blockade of fully closed channels does not occur, but the ability of bbTBA to block a closed channel requires movement of one or more voltage sensors. Models in which block is coupled to voltage sensor movement can qualitatively account for (1) the ability of open-channel block to better fit block of conductance–voltage curves at high Ca²⁺; (2) the voltage dependence of fractional availability; and (3) the fractional unblock at different open probabilities. BK channels appear to undergo voltage-dependent conformational changes among closed states that are permissive for bbTBA block.

Affinity for phosphatidylinositol 4,5-bisphosphate determines muscarinic agonist sensitivity of Kv7 K+ channels

Hernandez, C. C., Falkenburger, B., Shapiro, M. S. — Mon, 26 Oct 2009 10:01:46 PDT

Kv7 K⁺-channel subunits differ in their apparent affinity for PIP₂ and are differentially expressed in nerve, muscle, and epithelia in accord with their physiological roles in those tissues. To investigate how PIP₂ affinity affects the response to physiological stimuli such as receptor stimulation, we exposed homomeric and heteromeric Kv7.2, 7.3, and 7.4 channels to a range of concentrations of the muscarinic receptor agonist oxotremorine-M (oxo-M) in a heterologous expression system. Activation of M₁ receptors by oxo-M leads to PIP₂ depletion through G_q and phospholipase C (PLC). Chinese hamster ovary cells were transiently transfected with Kv7 subunits and M₁ receptors and studied under perforated-patch voltage clamp. For Kv7.2/7.3 heteromers, the EC₅₀ for current suppression was 0.44 ± 0.08 µM, and the maximal inhibition (Inhib_max) was 74 ± 3% (n = 5–7). When tonic PIP₂ abundance was increased by overexpression of PIP 5-kinase, the EC₅₀ was shifted threefold to the right (1.2 ± 0.1 µM), but without a significant change in Inhib_max (73 ± 4%, n = 5). To investigate the muscarinic sensitivity of Kv7.3 homomers, we used the A315T pore mutant (Kv7.3^T) that increases whole-cell currents by 30-fold without any change in apparent PIP₂ affinity. Kv7.3^T currents had a slightly right-shifted EC₅₀ as compared with Kv7.2/7.3 heteromers (1.0 ± 0.8 µM) and a strongly reduced Inhib_max (39 ± 3%). In contrast, the dose–response curve of homomeric Kv7.4 channels was shifted considerably to the left (66 ± 8 nM), and Inhib_max was slightly increased (81 ± 6%, n = 3–4). We then studied several Kv7.2 mutants with altered apparent affinities for PIP₂ by coexpressing them with Kv7.3^T subunits to boost current amplitudes. For the lower affinity (Kv7.2 (R463Q)/Kv7.3^T) or higher affinity (Kv7.2 (R463E)/Kv7.3^T) channels, the EC₅₀ and Inhib_max were similar to Kv7.4 or Kv7.3^T homomers (0.12 ± 0.08 µM and 79 ± 6% [n = 3–4] and 0.58 ± 0.07 µM and 27 ± 3% [n = 3–4], respectively). The very low-affinity Kv7.2 (R452E, R459E, and R461E) triple mutant was also coexpressed with Kv7.3^T. The resulting heteromer displayed a very low EC₅₀ for inhibition (32 ± 8 nM) and a slightly increased Inhib_max (83 ± 3%, n = 3–4). We then constructed a cellular model that incorporates PLC activation by oxo-M, PIP₂ hydrolysis, PIP₂ binding to Kv7-channel subunits, and K⁺ current through Kv7 tetramers. We were able to fully reproduce our data and extract a consistent set of PIP₂ affinities.