Ionic Current measurements

Manual patch clamp assays: Ionic current measurements

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Whole-cell current amplitude and kinetics measurements verify the results of the interaction of a test article with a selected ionic current. (The popular hERG assay is an example of ionic current assay, saimed specifically at the well-known hERG channel.) The assay is generally used to elucidate the mechanism behind various ion-channel-related arrhythmic events, and uncover ion current inhibition, either as a primary or secondary pharmacology manifestation.

A typical patch clamp study involves a pulse protocol whereby a ‘patched’ cell is held at an interpulse low enough to prevent the channels from activating/opening. Generally, activation requires a depolarization to a threshold potential. Increasing voltage pulses are applied to the cell and when the voltage applied approaches, and eventually reaches that threshold potential, the current measures across the cell increases as more and more of the channels are open.

A single-pore channel is generally either open/closed. It can be open, inactive (there may be multiple conformations of inactivity), or closed. From a recording point of view, there are only two states for the current across a membrane: it either flows (open channel) or doesn’t flow (closed channel, inactivated channel). The channels cycle from closed (no current) to open (current) and back to closed (no current) and vice-versa – until the channels take the irreversible path toward inactivation (no current). An inactivated channel has no choice but to go through the ‘closed’ conformation before it can re-open again.

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Whole-cell current levels change because, across the cell, a proportion of the 250,000+ channels are open at any given time, depending on the voltage or pattern of voltages applied.

Highlights:

•Manual patch-clamp
•Primary cardiac cells or transfected cell lines
•Conducted at physiological temperature
•Screens (non-GLP, faster study design) also available
•Currents available:
•Cardiac sodium (INa)
•Cardiac potassium (IKs, IKr, IKur)
•Cardiac calcium  (ICa-L, ICa-T)
•Cardiac exchanger currents (INaCa (NCX1) and INaK)
•CNS voltage-gated sodium (INa)
•CNS voltage-gated potassium (IK)
•Smooth muscle INa, ICa, IK
•Endothelial & epithelial INa, ICa, IK
Current recordings from LNCaP cells transfected with the TRPV6 gene, in order to measure store-activated calcium currents (Icrac). The currents were recorded following the application of voltage ramps.
Current recordings from LNCaP cells transfected with the TRPV6 gene, in order to measure store-activated calcium currents (Icrac). The currents were recorded following the application of voltage ramps.

Ionic current measurement on native or transfected cells

HEK293, LNCaP, CHO or isolated cardiac cells

Manual patch-clamp, whole-cell configuration

Pre-IND (GLP-compliant) or exploratory designs (screens are non-GLP compliant)

Whole-cell current amplitude and kinetics measurements verify the results of the interactions of a test article with a selected ionic current. The assay can be used to elucidate the mechanism behind the block of ion channels by a test article, for instance, or to confirm the nature of a target when ion-channel inhibition is a suspected part of the efficacious mechanism. The experiments are conducted on heterologous transfect ion systems, freshly isolated (primary cultures) cells, or specifically prepared tissues, e.g.: brain slices

Study Outline:

  • Number of cells exposed to the test article: 7 (screens: 3)
  • Number of concentrations of test article: 4 (screens: 3)
  • Method of test article exposure: cumulative concentrations in closed-circuit perfusion, or open-ended perfusion of individual concentrations
  • Voltage pulse protocols customized based on the selected ionic current to measure
  • A study includes positive, negative, and vehicle controls

Study Outcome:

  • A quantitative assessment of ionic current inhibition, type of block, etc.
  • Determination of the IC50 value (if applicable)
  • Analysis of both amplitude and current kinetics as indicators of inhibition
  • FDA-ready hard copy  and e-report for electronic IND submission
  • Holistic interpretation of a positive signal, considering all other data generated

Recommended Reading:

  1. K. Jurkat-Rott and F. Lehmann-Horn, Current Pharmaceutical Biotechnology: The Patch Clamp Technique in Ion Channel Research, 2004, 5, 387-395
  2. Frances M. Ashcroft, From molecule to malady. NATURE Vol 440:23, 2003