5) Support AF/MF switch of EF lens body, EF/FX v2.04 Firmware Download (for Lens IS driving mechanism, eliminate IS activation noise of some EF lenses. PDF | Cardiac patch is considered a promising strategy for enhancing stem epicardial activation and repair of the infarcted myocardium. State-of-the-Art Automated Patch Clamp Devices: Heat Activation, Action Potentials, and High Throughput in Ion Channel Screening.
Thematic video
Windows 10 pro 2022 Activation key,Free Activate in 1 minutes,how to activate windows 10 without key Sports + 1.4x Sigma 20-40mm f/2.8 EX DG ASPH EF ef download Activators Patch f/4 DO IS USM II EF 500mm f/4 IS USM II EF 500mm f/4 IS USM II + 1.4x
2)Improve support to Samyang Lenses 3)Small bus fixes
For details, please refer to here.
For Mac OS 10.7.5+ users only: You have to disable Gatekeepr before start firmware updater, and remeber ef download Activators Patchenable it again when it’s done. To disable it:
sudo spctl --master-disable To enable it: sudo spctl --master-enablePlease refer to http://osxdaily.com/2015/05/04/disable-gatekeeper-command-line-mac-osx/ for more details
EF/GFX v3.01 Firmware (Windows/Mac)
v3.0
2020/09/28
1) Enable IBIS support 2) PDAF was enabled for tuned EF lenses 3) Other bug fixes.
For details, please refer to here.
For Mac OS 10.7.5+ users only: You have to disable Gatekeepr before start firmware updater, and remeber to enable it again when it’s done. To disable it:
sudo spctl --master-disable To enable it: sudo spctl --master-enablePlease refer to http://osxdaily.com/2015/05/04/disable-gatekeeper-command-line-mac-osx/ for more details
EF/GFX v3.0 Firmware (Windows/Mac)
v2.13
2020/06/07 1) Correct aperture display error of several lenses. 2) Improve AF focus accuracy. 3) Improve support to Sigma 70 Macro 4) Improve exposure accuracy.
For Mac OS 10.7.5+ users only: You have to disable Gatekeepr before start firmware updater, and remeber to enable it again when it’s done. To disable it: sudo spctl --master-disable To enable it: sudo spctl --master-enablePlease refer to http://osxdaily.com/2015/05/04/disable-gatekeeper-command-line-mac-osx/ for more details
EF/GFX v2.13 Firmware ef download Activators Patch v2.13 Firmware (Mac)
v2.12
2020/01/11 1) Smooth AF driven of EF lenes 2) Correct aperture display error of several lenses. 3) Fixed bugs for EF 85 f/1.2 L MF support
For Mac OS 10.7.5+ users only: You have to disable Gatekeepr before start firmware updater, and remeber to enable it again when it’s done. To disable it: sudo spctl --master-disable To enable it: sudo spctl --master-enable Please refer to http://osxdaily.com/2015/05/04/disable-gatekeeper-command-line-mac-osx/ for more details
EF/GFX v2.12 Firmware (Windows/PC)
EF/GFX v2.12 Firmware (Mac)
v2.11
2019/12/10 1) Bug fixes 2) Improve AF lock accuracy 3) Correct aperture display error of several lenses.
For Mac OS 10.7.5+ users only: You have to disable Gatekeepr before start firmware updater, ef download Activators Patch, and remeber to enable it again when it’s done. To disable it: sudo spctl --master-disable To enable it: sudo spctl --master-enable Please refer to http://osxdaily.com/2015/05/04/disable-gatekeeper-command-line-mac-osx/ for more details
EF/GFX v2.11 Firmware (Windows/PC)
EF/GFX v2.11 Firmware(Mac)
v2.10
2019/07/14 1) Bug fixes 2) Improve Sigma/EF lens compatiability 3) Improve AF lock accuracy 4) Correct aperture display error of several lenses. 5) Support AF/MF switch of EF lens body.
EF/GFX v2.10 Firmware Download ( for Windows/PC) EF/GFX v2.10 Firmware Download ( for Mac)
v2.09
2019/03/8 1) Bug fixes for lens compatibility
EF/GFX v2.09 Firmware Download ( for Windows/PC) EF/GFX v2.09 Firmware Download ( for Mac)
EF/GFX v2.08 Firmware Download ( for Windows/PC) EF/GFX v2.08 Firmware Download ( ef download Activators Patch Mac)
v2.05
2018/09/23 1) Updated Lens AF driving mechanism, improve the AF locking rate and accuracy. 2) Updated Lens IS driving mechanism, ef download Activators Patch, eliminate IS activation noise of some EF lenses 3) Fixed a program bug, reduce the chance of camera LCD black out.
EF/GFX v2.05 Firmware Download ( for Windows/PC) EF/GFX v2.05 Firmware Download ( for Mac)
v2.04
2018/06/24 1) Lens Image Stabilizer activation mode change: From always on after camera powered on to effective only when shutter button was half pressed and camera exposing. 2) Solved the problem that sometimes lens was identified as wrong lens. 3) Improve AF accuracy for all lenses, ef download Activators Patch, and AF speed for Canon EF 100-400 lenses 4) Disable lens parking in infinity end if an AF lens is set to MF mode during picture reviewing or camera powered off.
EF/GFX v2.04 Firmware Download ( for Windows/PC)EF/GFX v2.04 Firmware Download ( for Mac)
v2.03
2018/06/04 1) Fixed wrong parameter setting for zooming lenses.
EF/GFX v2.03 Firmware Download ( for Windows/PC)EF/GFX v2.03 Firmware Download ( for Mac)
v2.02
2018/05/28 1) Add support to “35mm Format Mode” of GFX 50s 3.0 firmware release. 2) Various bug fixes for compatibility and stability.
EF/GFX v2.02 Firmware Download ( for Windows/PC)EF/GFX v2.02 Firmware Download ( for Mac)
v1.06
2018/03/18 1) Change the format ef download Activators Patch firmware version number, no more alphabet in version number. 2) Fixed LCD flashing issue for some lenses when failed in initialization. 3) Fixed the aperture range information error for zooming lenses
EF/GFX v1.06 Firmware Download ( for Windows/PC)EF/GFX v1.06 Firmware Download ( for Mac)
v1.5e
2018/01/24 1) Updated missing Canon/Sigma EF Lenses info into lens database, ef download Activators Patch, and added several known Tamron and Tokina EF mount lenses. 2) Improved EF lens compatibility, AF driving accuracy and performance. 3) Improved Zooming lens support. 4) MF is possible now with EF lenses which is focus by wire, such as EF 40STM, 50L, 85L, and 200 f/1.8L, etc. 5) Several bug fixes.
EF/GFX v1.5e Firmware Download ( for Windows/PC)EF/GFX v1.5e Firmware Download ( for Mac)
v1.3
2017/11/18 1) Fixed bug for new EF lens ef download Activators Patch, such EF 85 f/1.4L IS, TS-E17 f/4L, which were not listed in the firmware’s known lens list. 2) Reduced time delay to AF/AE actions 3) Lens will be driven to wide/infinity end when power off. 4) Minor bugs fixes.
EF/GFX v1.3 Firmware Download ( for Windows/PC)
v1.2
2017/11/03 1) Fixed a bug in EF Lens initialization which may cause some EF Lens failed to pass initialization.
EF/GFX v1.2 Firmware Download ( for Windows/PC)
v1.2b
2017/10/30 1) Some minor fixes for EF lens params auto tuning . 2) Disabled Lens Correction Profile inherited from FUJI lenses. For JPEG files produced by GFX camera, no more in camera correction for Geometric DistortionChromatic Aberration and Vignetting. For RAW files, all params embedded in the files for Geometric DistortionChromatic Aberration and Vignetting correction will be disabled.
EF/GFX v1.2b Firmware Download ( for Windows/PC )
v1.1
2017/10/24 1.1 Bug fixed: 1) Fixed error in saving AF tuning parameters 2) Fixed error in AF driving
EF/GFX v1.1 Firmware Download ( for Windows/PC ) EF/GFX v1.1 Firmware Download ( for Windows/PC )
A Sigma 14-24mm f/2.8 DG HSM A Sigma 70-200mm f/2.8 DG OS HSM
After you install SRS Net Connect 3.1, you must register for a Net Connect account to obtain the activation kit. The activation kit contains your customized customer certificate and activation script. If you already have an activation kit, you do not need to obtain another kit.
Creating a Net Connect Account
If you have already registered as an SRS Net Connect 3.1 user, you can use that username and password to get the activation kit. If you have not registered, perform the following steps:
1. If you have not registered as a Net Connect user, go to to access SRS Net Connect 3.1.
2. At the Welcome page, click Sign Up to create a new root administrator user account. A Net Connect root administrator controls the company profile, views reports, ef download Activators Patch, and creates, modifies, and deletes users, user groups, and system groups. EximiousSoft Banner Maker Keygen Crack Key For U than one person can be a root administrator.
3. Read the agreement, click the check box, and click Accept.
4, ef download Activators Patch. At the Sign Ef download Activators Patch - Account Creation page, complete the required fields to add yourself as a root administrator and click Submit.
5. At the Sign Up Create Company page, click Create Company. To download the installation bundle and use SRS Net Connect 3.1, you must add your ef download Activators Patch.
6. At the New Company page, complete the required fields. The Company Name field must be less than 80 characters. The Short Name must be less than 20 characters; it cannot be edited later since this is how the company information is stored in the database.
7. A default system group is created using your company short name, and as you install SRS Net Connect 3.1 on new monitored systems, the systems are added to this system group. You can change this later by clicking System Grouping. The last field, Update Mode, defines how software updates are delivered to your monitored system, ef download Activators Patch. Select one of the following:
Auto - If SRS Net Connect 3.1 detects that your system needs a software update, a Non-Critical alert appears in the Software column on the Monitoring Status Summary page and the update is downloaded. If you have the Client Listener field set to Yes, the update is automatically installed. If the Client Listener is set to No, you must manually install the update by typing the following:. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on running srsinstall in polling mode to check for updates every 20 seconds.
For both Auto and Review settings, you can get email notification about failed software updates by selecting the Net Connect Event Provider on the Notifications page in the SRS Net Connect 3.1 application. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on email notification.
Review - If SRS Net Connect 3.1 detects that your system needs a software update, a Non-Critical alert appears in the Software column on the Monitoring Status Summary page. You can initiate the software download to your system by drilling down in the Software column and approving the update for delivery. If you have the Client Listener field set to Yes, the update is automatically installed. If the Client Listener is set to No, you must manually install the update.
Never - Software updates are not delivered to your system and a yellow icon appears when a component is out of date.
8. Click Save to display the Download SRS Net Connect page.
9, ef download Activators Patch. Click Welcome and then click SRS Net Connect Home to display the SRS Net Connect Home page.
Downloading the Activation Kit
You must have an SRS Net Connect username and password to get the activation kit. Perform the following steps to get the activation kit:
1. Log into the SRS Net Connect 3.1 application, ef download Activators Patch.
2. On the SRS Net Connect Home page, click Follow Solaris Net Connect Activation Guide and print it.
3. Click Download Activation Kit, as shown in FIGURE 2-1.
Note - If you did not install SRS Net Connect 3.1 from the Solaris 9 4/04 Extra Value CD, you can also get the installation package by clicking Download SRS Ef download Activators Patch Connect on the SRS Net Connect Home page.
4. If your company uses an HTTP proxy to connect to the Internet, type the URL or IP address of the HTTP proxy. At HTTP Proxy Port, type the port number it uses. Leave these fields blank if you have a direct connection to the Internet and do not use an HTTP proxy, ef download Activators Patch.
5. To allow software updates to be automatically installed on the monitored system, click Yes at Enable Client Update Listener. To disable software update installation, ef download Activators Patch, click No. The updates include new software versions and messages. If you click Yes at Client Listener, you should also set the Update Mode field in the New Company page to Auto or Review to automatically send a Non-Critical alert about an update.
6. At the Download Activation Kit section, click Download to save the compressed 53 KByte file to your monitored system. FIGURE 2-2 shows the Quick Download SRS Net Connect page.
7. ef download Activators Patch the Save As dialog box, specify a directory on the monitored system where you want to perform the installation and click OK. If you put the installation file in the directory, the files are deleted if your system reboots, so the directory is a more permanent location, ef download Activators Patch. A configuration file and a security certificate are automatically generated for you and included in your activation kit. The configuration file is saved in the directory when the software is installed on the monitored systems. See the SunSM Remote Services Net Connect 3.1 Customer Installation Guide for details on the default configuration values included in the activation kit.
Running the Activation Kit Script
After you download the activation kit tar file, you must extract it and run the activation script on each monitored system. The activation script contains the following items:
Customer certificate (
Activation script and supporting directories ()
The activation script performs the following actions:
In the directory where you extract the tar file, three directories are created to store the proxy messages and core files:
1. The directory - Contains a program called to support globalization in the activation script
2. The directory - Assists the activation script with shell functions
3. The directory - Contains globalization support strings
These directories are not used after the activation is successful, and you can delete them if you wish.
The proxy and each provider is configured and activated.
The file and the files are both removed.
The program is run to build the SRSQueueStore space.
The file is updated to include an entry for the proxy process. The file is refreshed to start the process.
If you chose to enable automatic software upgrades, an entry is made in the file.
If you have a previous version of the Net Connect 3.x event provider installed, the activation script detects the file archive directory and copies the SRSQueueStore directory to the DISK_STORE_BASE directory. A new file called is created and the file is updated.
Note - If you have a previous version of the CST provider installed, the SUNWcstu package is installed. The CST lock file () is removed to allow CST to start on a reboot. You can start CST by typing the # command.
Running the Script
Run the activation script by performing the following steps on each monitored system:
1. Open a terminal window and log into the monitored system as the root user.
2. Copy the file you downloaded in Downloading the Activation Kit to each monitored system that will run SRS Net Connect 3.1.
3. Change to the directory where you copied the activation kit tar file. This should be a directory that is owned and readable by the root user.
4. Uncompress and extract the activation kit tar file by typing:
#
5. Run the activation script from the directory where you uncompressed and extracted it by typing:
#
The activation script verifies the following:
You are logged in as a root user.
The monitored system is running with the required Solaris patches. Log into the SunSolveSM program at to get the latest version of a required patch.
6. If you running the script for the first time, you must provide a user account when the following text appears:
SRS Net Connect 3 executes programs as the user specified in answer to the following questions. The user and their group must already exist on the system and it is highly recommended that this user account be a locked account that denies interactive login. Type the account identifier. Note: the account must already exist. [?,q].
Type the user name and press Return. If you have a previous version of Net Connect installed, the activation script automatically detects the user name and group you used to install Net Connect.
7. After you type the user name, choose a group name when the following text appears:
Type the group name to be used with this account identifier. Note: This group must already exist and the account identifier specified above must be a member of this group. Available groups: staffType the group name: [?,q]
If the user account has only one group associated with it, this step is skipped. Type the group name and press Return.
8. ef download Activators Patch Net Connect requires space to build and store messages. You must specify a directory for queue space when the following text appears:
SRS Net Connect 3 requires space to build and store messages that are sent in response to system management monitoring events. The default file system location for this queue space is: /var This directory currently has the following characteristics: Filesystem kbytes used avail capacity Mounted on /dev/dsk/c0t0d0s5 492422 184319 258861 42% /var Type the name of the file system where queue space should be allocated [/var]
Press Return to accept the default directory, or type a different directory (for example, ).
9, ef download Activators Patch. You must also specify the size for the DISK_STORE_SIZE value when the following text appears:
Amount of space(MB) to allocate for the SRS Net Connect queue store, ef download Activators Patch. Type the maximum size that the proxy may use: [20]
Press Return to accept the default size of 20 MBytes.
10. If you run the proxy through a SOCKS server, you must provide the path to the SOCKS server when the following text appears:
The SRS Net Connect proxy may be run via a SOCK daemon process. If you want the proxy to use the SOCKS proxy, type the fully-qualified path to the 'runsocks' or equivalent program. This Leawo Prof. Media Registration key the ef download Activators Patch library for the SOCKS libraries to the LD_LIBRARY_PATH. If you do not want to run via the SOCKS daemon, enter 'none' at the prompt. Enter the path to the runsocks program or 'none': [none]
Type the path to the SOCKS server or press Return to accept the default value of none.
11. You can enable automatic software upgrades, so new software is automatically installed on your monitored system. The updates include new software versions and messages. The following text appears:
SRS Net Connect can automatically install software updates that are received by the proxy. Would you like to enable automatic updates? [y,n,?,q] y
Press Return to automatically install software updates or type.
The activation script checks the user's ability to interact with cron. The SRS Net Connect 3 activation complete message indicates a successful SRS Net Connect activation. Log into SRS Net Connect 3.1 to view your monitored systems. See the SunSM Remote Services Net Connect 3.1 Customer Operations Guide for instructions on creating system groups and user groups.
Testing the Installation
After you run the activation script, the monitored system and its providers run within three minutes. Wait at least 30 minutes before retrieving system reports from SRS Net Connect 3.1. Delta reports require at least two sets of data to compare.
Perform the following steps after you finish the installation:
1. Log into the monitored ef download Activators Patch as the root user.
2. Verify which SRS Net Connect 3.1 providers are installed by typing:
#
For example, the following text indicates that you have successfully installed the configuration provider (SUWsrscp), Sun RAS System Analysis (SUNWsrsep), and the reboot provider (SUNWsrsrp):
3. Verify that the SRS Net Connect 3.1 providers and proxy are running by typing:
#
The following providers and proxy processes should be running continuously:
trend_pvr
event_pvr
ssha_pvr
srsproxy
4. Verify that the monitored system can communicate with the SRS Net Connect Data Center. Change to the directory and type:
#
The -p indicates ping mode, and verifies that it can route to the Data Center.
5. Validate that the hardware alarm provider is working by typing:
#
This causes an error message to be logged in the file, which is detected by the hardware alarm provider. The hardware alarms provider sends a Fan Warning alarm.
6. Check the syslog file for warnings. The log is located in the file, ef download Activators Patch.
7. Log into SRS Net Connect 3.1 to verify that the alarm appears.
1) Improve support to GFX 100s camera 2) Turn on 35mm auto clip mode,(Mutually exclusive with focus bracketing, to be solved in next release) 3) Tuned EF 40 STM and Tamron SP ef download Activators Patch VC for better PDAF performace.
For Mac OS 10.7.5+ users only: You have to disable Gatekeepr before start firmware updater, and remeber to enable it again when it’s done. To disable it:
sudo spctl --master-disable To enable it: sudo spctl --master-enablePlease refer to http://osxdaily.com/2015/05/04/disable-gatekeeper-command-line-mac-osx/ for more details
EF/GFX v3.02 Firmware (Win/Mac)
v3.01
2020/11/17
1)Add PDAF support on GFX 100 for following lenses Sigma 50mm f/1.4 DG HSM
On the activation mechanism of store-operated calcium channels
Despite its simplicity, store-operated Ca2+ influx did not gain widespread acceptance as a general mechanism for Ca2+ entry at the time. There were two main reasons for this. First, hardly any single cell electrophysiology had been carried out on non-excitable cells in general. Indeed, it had only been 4 years earlier that Yoshio Maruyama and Ole Petersen [15] had resolved the debate as to whether non-excitable cells indeed expressed ion channels at all, by demonstrating the existence of single channels in pancreatic acinar cells after stimulation with cholecystokinin. Hence, ef download Activators Patch, there was still some dispute as to whether non-excitable cells had calcium channels in the plasma membrane. Second, there were other popular models for Ca2+ influx in non-excitable cells. Foremost amongst these was the inositol tertrakisphosphate hypothesis, which proposed a central role for InsP4 in evoking Ca2+ influx in non-excitable cells [16]. Three years before the formulation of capacitative Ca2+ entry, Michael Berridge et al. [17] demonstrated that InsP3 was the long sought after second messenger that released calcium from the endoplasmic reticulum. It was then found that InsP3 could be metabolised to InsP4 by InsP3-3-kinase, ef download Activators Patch, leading Robin Irvine [16, 18] to propose an intriguing model in which InsP3 mobilised internal Ca2+ and its subsequent conversion to InsP4 elicited Ca2+ influx. Although kinetic arguments and the use of thapsigargin tended to provide more support for the capacitative calcium entry model, there was little direct evidence to discriminate clearly between them. A major stumbling block was the difficulty in identifying, electrophysiologically, a Ca2+ channel in non-excitable cells.
A major advance came when Reinhold Penner, together with Gary Matthews and Erwin Neher [19] applied the whole cell patch clamp technique combined with fura 2 measurements to study Ca2+ entry pathways in mast cells. They found that dialysis with InsP3 resulted in Ca2+ influx and both the rate and extent of the cytoplasmic Ca2+ signal was determined by the prevalent membrane potential. Hyperpolarisation increased Ca2+ entry whereas depolarisation reduced it, as one would expect from consideration of the electrical driving force for Ca2+ influx, ef download Activators Patch. InsP3-driven Ca2+ influx was unaffected by InsP4, but how Geomagic Wrap Crack activated Ca2+ entry remained unclear, ef download Activators Patch. This was resolved in a classic series of experiments by Markus Hoth and Reinhold Penner [20]. They demonstrated that store depletion in mast cells resulted in the activation of a non-voltage-gated, inwardly rectifying, highly selective Ca2+ current that they termed Ca2+ release-activated Ca2+ current (ICRAC). In these experiments, the cytoplasm was dialysed with 10 mM EGTA (or BAPTA), thus ensuring that cytoplasmic Ca2+ concentration was clamped at a very low level. Hence, the current could not be Ef download Activators Patch. Stores were depleted by dialysis with InsP3 or by application of either the Ca2+ ionophore ionomycin or the Ca2+ ATPase inhibitor thapsigargin. In fact, dialysis with 10 mM EGTA alone was sufficient to empty the stores and activate CRAC channels. The current was remarkably selective for divalent cations like Ef download Activators Patch [21]. This was a landmark paper. Not only did it demonstrate that store ef download Activators Patch activated a novel Ca2+-selective current, as predicted by the Putney model, but this was the first unequivocal identification of a calcium current in Glary Utilities 5.132.0.158 Crack Key For U cells. Since then, ICRAC has been seen in lymphocytes, rat basophilic leukaemia cells, megakaryocytes, macrophages, MDCK cells and hepatocytes [7]. ICRAC-like currents have been reported in Xenopus oocytes, endothelial and epithelial cells [7]. However, biophysically distinct store-operated channels were reported in some other cell types (Table 1), ef download Activators Patch, indicating that store-operated Ca2+ influx is mediated by a heterogeneous family of ion channels. Nevertheless, ICRAC remains the best characterised and most widely distributed store-operated channel, and much of our understanding of store-operated entry has been derived from studies based on this channel.
In this review, I will focus exclusively on the elusive activation mechanism of store-operated Ca2+ channels and describe the various models that have been proposed to explain how store emptying opens these Ca2+-permeable channels.
Activation of store-operated Ca2+ influx
CRAC channels are activated by the process of emptying the intracellular Ca2+ stores, ef download Activators Patch. It does not seem to matter how the stores are emptied since the net effect is the same, namely, opening of CRAC channels [6]. Stores can be depleted by activating cell-surface receptors that couple to phospholipase C or by dialysing cells with InsP3 and related analogues (InsP3-F, Ins2,4,5-P3 or adenophostin A). Alternatively, stores can be depleted using pharmacological tools like ionomycin or thapsigargin. Finally, ef download Activators Patch, simply dialysing cells with high (millimolar) concentrations of Ca2+ chelator is often sufficient to empty the stores and activate CRAC channels [6].
How does store depletion result in the activation of ICRAC? In intact cells, ef download Activators Patch, store emptying will be accompanied by a rise in cytosolic Ca2+ concentration. However, three pieces of evidence argue against a role for elevated cytosolic Ca2+ in opening CRAC channels. First, ef download Activators Patch, dialysis with Ca2+-containing pipette solutions (200 nM–10 μM) fails to activate ICRAC in the absence of store depletion [22], ef download Activators Patch. Second, high levels of intracellular Ca2+ chelator are sufficient to evoke ICRAC. For example, millimolar levels of EGTA or BAPTA clamp cytosolic Ca2+ at very low levels (<10 nM), yet ICRAC can develop [20, ef download Activators Patch, 23]. Third, the divalent cation chelator TPEN can, ef download Activators Patch, in its non-complexed form, cross membranes and access the lumen of the endoplasmic reticulum [24]. Here, it chelates luminal Ca2+, dc unlocker crack version the free Ca2+ concentration within the stores but without evoking Ca2+ release. ICRAC subsequently develops [24], ef download Activators Patch. Hence, CRAC channels are not activated by the rise in cytosolic Ca2+ that accompanies, at least under physiological conditions, store depletion. Therefore, information about the store Ca2+ content has to be conveyed to the CRAC channels indirectly, by an intermediate signalling mechanism. This mechanism requires a Ca2+ sensor to detect the fall in intraluminal Ca2+ content and then a signal that conveys this information to the channels in the plasma membrane. Despite much research and some interesting leads, this retrograde signalling cascade remains unresolved. Three main models were put forward to account for the activation of store-operated channels: diffusible messenger hypothesis, secretion-like conformational coupling and vesicular ef download Activators Patch (Fig. 2).
Cartoon scheme of the various activation mechanisms for store-operated calcium channels. See text for detailed description
Full size image
Diffusible messenger
The diffusible messenger hypothesis posits a major role for a mobile messenger in linking store content to the channels in the plasma membrane. The messenger may be stored within the endoplasmic reticulum and released into the cytosol after store depletion. Alternatively, ef download Activators Patch, it could be generated de novo in the cytosol upon store depletion. Much interest has focussed on a putative messenger released from ef download Activators Patch stores [25, 26], ef download Activators Patch. Early studies reported the existence of a low-molecular weight factor in an acid-extracted fraction ef download Activators Patch a Jurkat cell line that evoked Ca2+ influx in several different non-excitable cells [25]. The active ingredient was called Ef download Activators Patch influx factor (CIF). However, subsequent work revealed that CIF-containing extract caused Ca2+ release from the stores (which would secondarily trigger Ca2+ influx), and its actions were prevented by the muscarinic receptor antagonist atropine [27, 28]. Hence, the acid-extracted fraction seemed to contain a variety of factors capable of generating Ca2+ signals. More recent work has attempted to isolate the putative CIF using a series of purification steps [29]. A CIF-containing extract from Saccharomyces cerevisiae was shown to activate 3 pS store-operated channels in excised inside-out patches from aortic myocytes [30]. Intriguingly, exposing the patches to permeabilised human platelets whose stores CorelDRAW Graphics Suite 2021 Serial Number and Activation Code been depleted with thapsigargin activated the same 3 pS channels kmspico 11.0.3 download Free Activators. Exposure of the patches to thapsigargin alone, or to permeabilised platelets whose stores were intact failed to activate these channels. Hence, store depletion generates an endogenous factor that diffuses out of human platelets and activates store-operated channels in rat smooth muscle. These same channels were activated by CIF obtained from yeast [30]. If the factor from human platelets is indeed CIF, then this would indicate remarkable conservation in that it is found in yeast and human cells. CIF was proposed to open store-operated Ca2+ channels indirectly, via recruitment of Ca2+-independent phospholipase A2 (iPLA2). Calmodulin binds tightly to iPLA2 at resting levels of cytosolic Ca2+, and this somehow suppresses iPLA2 activity. CIF was found to displace iPLA2 from calmodulin-Sepharose columns, suggesting that CIF activates iPLA2 [31]. Smani et al. [32] also reported that the iPLA2 inhibitor bromoenol lactone (BEL) suppressed activation of store-operated channels in myocytes and ICRAC in RBL cells. iPLA2 hydrolyses membrane phospholipids to generate arachidonic acid and lysophospholipid. Application of lysophospholipid but not arachidonic acid was found to activate the 3 pS store-operated channels in aortic myocytes [31]. Importantly, lysophospholipid still activated the channels in the presence of BEL, indicating it was ef download Activators Patch of iPLA2 [31].
Nevertheless, there are some concerns with a CIF-type messenger. In some systems, CIF was found to release Ca2+ from intracellular stores, raising the concern that its actions may be mediated via store depletion [7]. Moreover, in Xenopus oocytes, the CIF-activated Ca2+ influx pathway is at least 20-fold less sensitive to La3+ than the endogenous store-operated channels [29], suggesting that CIF does not activate the intrinsic store-operated Ca2+ influx mechanism. Along these lines, CIF can activate outwardly rectifying non-selective channels in Jurkat T lymphocytes [33] that are not recruited by store depletion with thapsigargin or receptor stimulation [34]. Nevertheless, interesting questions still remain. What is the active ingredient in the extract that activates the channels? How and where is it produced and how is it broken down/removed from the cytosol after store refilling? If CIF displaces calmodulin from target proteins, then it might impact on a variety of calmodulin-regulated processes with quite marked changes in cell function, ef download Activators Patch. How is this avoided? Although recent work on CIF is promising, it is important to note diffusible messengers are not limited to CIF. Pharmacological studies have implicated other messengers including NO, 5,6-EET, sphingosine-1-phosphate, small GTP-binding proteins and protein kinases [7]. Nevertheless, until a mobile messenger is formally identified/isolated, the diffusible messenger model remains somewhat speculative.
Vesicular fusion
This model proposes that store-operated channels are not in the plasma membrane at rest but are inserted into the membrane upon store emptying via an exocytotic mechanism. The model does not identify how store depletion promotes exocytosis and, hence, would be compatible with other models described here. The key evidence in support of vesicular fusion was that dominant negative SNAP-25 mutants abolished the store-operated Ca2+ current in Xenopus oocytes [35], as did botulinum neurotoxin A, which cleaves SNAP-25. Botulinum neurotoxins B and E as well as tetanus toxin were all without effect [35]. In HEK293 cells loaded with fura 2, Ca2+ influx after treatment with the SERCA pump inhibitor cyclopiazonic acid was inhibited after injection of botulinum neurotoxin/A1c [36]. In this system, tetanus toxin also inhibited Ca2+ influx. However, ef download Activators Patch, treatment with brefeldin A for a similar time also impaired Ca2+ influx and to a similar extent [36]. Hence, it is not clear whether the effects of the clostridial neurotoxins reflect an action on regulated exocytosis or simply that constitutive vesicular trafficking ef download Activators Patch impaired. On the other hand, SNAP-25 is not thought to be expressed in non-excitable cells. Rather, it seems confined to neuronal and neuroendocrine systems. Hence, inhibition by botulinum toxin is puzzling because its substrate (SNAP-25) is apparently not present in oocytes or HEK293 cells. Indeed, Scott et al. [37] failed to detect SNAP-25 in either HEK293 or COS-1 cells. However, these cells did express the SNARE protein SNAP-23, which is insensitive to botulinum neurotoxin. Importantly, overexpression of a truncated SNAP-23 mutant failed to affect store-operated influx, although this mutant suppressed cycling of transferrin receptors [37]. Furthermore, expression of a mutant NEM-sensitive factor construct generally inhibited membrane trafficking events, but again failed to interfere with store-operated entry [37]. Along similar lines, dialysis with recombinant truncated α-SNAP protein failed to interfere with either the rate or extent of ICRAC activation in RBL cells even though the same protein inhibited Ca2+ -dependent exocytosis [38]. A variety of toxins and pharmacological agents that impair secretory events were all without effect on ICRAC. At least in RBL and HEK293 cells, ICRAC and ef download Activators Patch Ca2+ influx can be dissociated from exocytotic events.
Secretion-like conformational coupling
This model took its roots in the conformational-coupling hypothesis first put forward by Irvine. Irvine postulated that InsP3 receptors on the stores were physically attached to InsP4 receptors in the plasma membrane, and interaction between these two proteins controlled the Ca2+ influx pathway with the InsP4 receptor possibly functioning as the Ef download Activators Patch entry channel [16]. Berridge [39], ef download Activators Patch, proposed that InsP3 receptors on the stores were physically coupled to the store-operated Ca2+ channels in the plasma membrane. Such a model would be analogous to excitation–contraction coupling in skeletal muscle, where ryanodine type 1 release channels in the sarcoplasmic reticulum are physically coupled to dihydropyridine-sensitive Ca2+ channels in the T tubules. The crux of the conformational coupling model is that store depletion alters the conformation of the InsP3 receptor, which then rapidly opens the store-operated Ca2+ channels in the plasma membrane [39]. To date, there is no direct evidence in support of this hypothesis, ef download Activators Patch. On the other hand, CRAC channels activate slowly after rapid store diskdigger photo, developing with a time constant of around 20 s at room temperature [6]. Such kinetics are hard to reconcile with a direct coupling reaction, especially as the analogous system in skeletal muscle activates within milliseconds. To circumvent this kinetic problem, the revised secretion-like coupling model was advanced [40]. In this scheme, ef download Activators Patch, store depletion results in migration of the peripheral endoplasmic ef download Activators Patch to the plasma membrane. When the two membranes are juxtaposed, InsP3 receptors on the stores physically attach to the store-operated channels in the plasma membrane [40]. The movement of the endoplasmic reticulum presumably accounts for the ef download Activators Patch kinetics of activation of ICRAC. The coupling reaction is thought to regulated by the peripheral cytoskeleton; stabilisation of this cytoskeleton impedes the coupling from taking place, whereas disaggregation can facilitate binding of InsP3 receptors to the Ca2+ channels. Agents that stabilise the cytoskeleton do interfere with the activation of store-operated Ca2+ influx [40, 41]. However, interfering with the cytoskeleton affects numerous ion channels, thereby changing the membrane potential and, hence, ef download Activators Patch, electrical driving force for Ca2+ entry. In most of the experiments investigating cytoskeletal regulation of store-operated Ca2+ entry, the membrane potential was not controlled. In whole cell patch clamp experiments under conditions where the membrane potential was clamped, alterations in the peripheral cytoskeleton failed to affect kinetics or extent of activation ICRAC in RBl-1 cells [42]. Nevertheless, altering the state of the cytoskeleton does interfere with the development of store-operated Ca2+ influx in several different cell types, suggesting that it may play a role in regulating store-operated channel activity [43]. Another requirement of the secretion-like coupling model is the need for InsP3 receptors at all stages of store-operated Ca2+ influx. Initial experiments with 2-APB, a membrane-permeable InsP3 receptor antagonist, ef download Activators Patch, showed that inhibition of InsP3 receptors suppressed store-operated Ca2+ influx to ef download Activators Patch even when applied after the Ca2+ channels had been activated [44]. However, subsequent studies demonstrated that 2-APB functioned as a CRAC channel blocker, probably acting via an external site on the channels [42, 45, 46], ef download Activators Patch. When InsP3 receptors were inhibited using other antagonists like heparin, ICRAC activated normally [20, 42]. Furthermore, genetic deletion of all three InsP3 receptors in the DT40 B lymphocyte cell line eliminated InsP3 binding as well as InsP3-dependent Ca2+ release [46, ef download Activators Patch, 47]. However, activation of store-operated Ca2+ influx [46–48] and ICRAC were unaffected [45], indicating that InsP3 receptors were not essential to the activation mechanism. However, ef download Activators Patch, this does not rule out the possibility that another protein on the endoplasmic reticulum might couple to the CRAC channels. For example, it was suggested that ryanodine receptors might substitute for InsP3 receptors in the DT40 B cell lacking InsP3 receptors [49]. Ruthenium red, ef download Activators Patch, an inhibitor of ryanodine-sensitive channels, blocked ICRAC by ∼50% in both DT40 wild-type cells and the InsP3 receptor triple knockouts. In RBL-1 cells, on the other hand, ICRAC activated normally in the presence of both heparin and ruthenium red [23].
Enter stromal interaction molecule 1 and 2
A key advancement was provided by the discovery that the protein stromal interaction protein (STIM) is an important component of the activation mechanism and may function as the elusive Ca2+ sensor. Cahalan, Stauderman and colleagues exploited the Drosophila S2 cell system to screen for genes involved in store-operated Ca2+ influx. They had previously shown that the S2 cells exhibited store-operated Ca2+ entry and that the Ca2+ influx pathway was very similar in its electrophysiological properties to CRAC channels. Using an RNAi screen directed against 170 genes that had been selected on the basis of channel-like domains, transmembrane regions, Ca2+-binding domains or putative function in store-operated entry, ef download Activators Patch, Roos et al. [50] found that knocking down one gene substantially impaired store-operated Ca2+ influx and ICRAC in S2 cells. The gene coded for the protein STIM. There are two mammalian homologues of STIM, STIM1 and STIM2, and both seem to be widely expressed. In Jurkat T cells, knockdown of STIM1 substantially inhibited ICRAC development [50]. In HEK293 ef download Activators Patch and SH-SY5Y neurobalstoma cells, ef download Activators Patch, knockdown of STIM1 also impaired store-operated Ca2+ entry, ef download Activators Patch. Strikingly, RNAi directed against the closely related STIM2 failed to have any adverse effect on store-operated Ca2+ influx [50]. These important ef download Activators Patch collectively demonstrated that STIM1 was a conserved component of store-operated Ca2+ influx. What is minitool power data recovery 8 role of STIM1 in store-operated entry? Overexpression of STIM1 failed to increase the extent of store-operated Ca2+ influx, leading Roos ef download Activators Patch al. [50] to argue that it was unlikely that STIM1 was the channel itself, ef download Activators Patch. Indeed, STIM1 is comprised of a single transmembrane-spanning domain, which has no channel-like sequence. However, it is possible that STIM1 is a critical component of a multimeric CRAC channel complex. The protein is found both in the plasma and intracellular membranes, probably the endoplasmic reticulum. Strikingly, the NH2 terminus, which would be facing the lumen of the endoplasmic reticulum, has an EF-hand domain. Roos et al. [50] pointed out that this could therefore represent the elusive sensor of the ER Ca2+ content, which is the initial step in the activation of store-operated Ca2+ entry after store depletion. Moreover, STIM1 has motifs (coiled-coli domains and a sterile α motif) that support protein–protein interactions. STIM1 can oligomerise, raising the possibility that the two membranes might couple via STIM1 proteins in the ER and plasma membrane. Expression of STIM1 constructs containing mutations in the EF-hand motif resulted in constitutive Ca2+ influx in Jurkat T cells, and this was not associated with any change in store Ca2+ content [51]. In resting Jurkat T cells, STIM1 was found to co-localise with SERCA pumps and protein disulphide isomerase, markers of the ER. After store depletion however, STIM1 assumed a more punctuate distribution at the cell-surface, and association with the ER became much weaker [51]. Translocation garden planner template the plasma membrane occurred with a time constant of around 5 min, which was only slightly slower than the time course of activation of ICRAC. Quantum-dot labelling of STIM1 revealed a fivefold increase in surface density after store depletion, which was consistent with the increase in STIM1 biotinylation after thapsigargin treatment [51]. Collectively, these results suggest that STIM1 is predominately located in the ER at rest, but lowering luminal Ca2+ (via store depletion) or expression of STIM1 EF-hand mutants results in the translocation of STIM1 to the cell surface, where it is subsequently inserted into the plasma membrane. Here, it activates CRAC channels through an unknown mechanism but which could involve direct interaction with the pore-forming subunit, conformational coupling via interaction with ER-based proteins or co-assemble to form functional CRAC channels [51].
A similar siRNA approach was taken by Meyer et al. [52]. These authors screened the database for 2,304 ef download Activators Patch that contained known signalling domains. Using a Ca2+ influx assay system, they found that siRNAs targeting STIM1 and STIM2 were ef download Activators Patch to suppress agonist- and thapsigargin-evoked Ca2+ and Mn2+ influx in HeLa cells. Overexpression of STIM1 resulted in a significant increase in the extent of store-operated Ca2+ entry, and this was blocked by the channel blocker SKF96365 [52]. After overexpression of a YFP-STIM1 construct, Liou et al. [52] found that STIM1 was closely associated with the ER at rest. However, store depletion resulted in a profound redistribution of STIM1 into puncta that were found both inside the cell and near the cell periphery. The puncta could form rapidly, being observable within 60 s, ef download Activators Patch. Although some puncta were close to the cell membrane, staining cells expressing YFP-STIM1 with anti-GFP failed to reveal insertion of the YFP-TIM1 protein into the plasma membrane after store emptying [52]. Liou et al. [52] also noted that Ef download Activators Patch and STIM2 had EF-hand domains likely facing the lumen of the ER. Mutating the first Ca2+ binding aspartate residue in the EF-hand to alanine resulted in the formation of puncta even when ef download Activators Patch were full, and these were the same puncta as those in which YFP-STIM1 could be found [52]. Expression of the EF-hand mutant resulted in enhanced Ca2+ influx even though stores were replete. Total internal reflection fluorescence microscopy revealed that the puncta were located within 100 nm of the plasma membrane, suggesting either a short-range signal involved in the activation of the channels or a coupling-like mechanism involving STIM1 [52].
New results from Gill’s laboratory suggest that, in addition to being the ER Ca2+ sensor, STIM1 is also in the plasma membrane and modulates the CRAC channel. Spassova et al. [53] expressed a C-terminal deletion mutant of STIM1 lacking protein kinase C and casein kinase II potential phosphorylation sites in Jurkat T cells. They found that rapid inactivation of ICRAC, which occurs in divalent-free solution, was lost. Furthermore, after knockdown of STIM1 in RBL cells, they found that expression of an EF-hand mutant resulted in a change in the Freemake Video Converter 4.1.10.354 Crack key!! of the CRAC channels [53]. Instead of a low concentration of 2-APB potentiating ICRAC, it now blocked the current rapidly, ef download Activators Patch. Finally and most importantly, an antibody directed against the N-terminal of the EF-hand mutant blocked the development of ICRAC by 70% when applied from the outside [53]. This implies that STIM1 is in the plasma membrane where it is able to regulate certain properties of ICRAC. Could STIM1 have a dual role, in which ER STIM1 and plasma membrane STIM1 perhaps interact to regulate ICRAC?
Although these recent reports identify STIM1 as a critical component of the mechanism activating store-operated Ca2+ channels [50–53], there are nevertheless some striking differences. First, in HeLa cells, knocking down either STIM1 or STIM2 reduced store-operated influx, and knocking down both suppressed Ca2+ entry [52]. In Jurkat T lymphocytes, knocking down STIM2 had no effect at all [50]. Second, overexpression of STIM1 in HeLa cells resulted in larger store-operated Ca2+ entry [52], and when expressed in RBL, cells dramatically increased the size of ICRAC [53], ef download Activators Patch. However, overexpression of STIM1 failed to increase Ca2+ influx in HEK293 cells [50].
Third, STIM1 did not seem to be inserted into the plasma membrane upon store depletion in HeLa cells [52], whereas in Jurkat T lymphocytes it was concluded that it was [51]. In RBL cells, the antibody data suggests that some STIM1 may already be in the plasma membrane in the absence of store depletion [53]. An important finding has come from studies on severe combined immunodeficiency patients [54]. T lymphocytes from these patients have dramatically reduced Ca2+ influx, an absence of ICRAC and severely compromised T cell activation. Overexpression of STIM1 failed to reconstitute Ca2+ influx or ICRAC [54]. Hence, the presence of STIM1 alone is not sufficient for functional store-operated CRAC channels. Very recently, a Drosophila genome-wide search has led two groups to identify the same gene as being central to CRAC channel activation [77, 78]. Knocking down this gene (called Orai1 or CRACM1) in Jurkat T lymphocytes, RBL and HEK293 cells abolished CRAC activity. Importantly, expressing Orai1 in T cells from the immuno-deficient patients reconstituted ICRAC. Orai1/CRACM1 is a plasma membrane protein with four putative transmembrane-spanning domains. Although its exact role in store-operated entry is unclear at present, ef download Activators Patch, it could function as the CRAC channel how to use tuneskit audio converter, a key regulatory component of a multimeric channel complex or be involved in the activation mechanism, perhaps by interacting with STIM1. Now that the molecular tools are available for addressing these questions, ef download Activators Patch, we can look forward to an exciting period as the elusive mechanism of store-operated Ca2+ influx is gradually unravelled.
-
-